WO2008156725A2

WO2008156725A2 - Methods for inhibiting cartilage mineralization

Info

Publication number: WO2008156725A2
Application number: PCT/US2008/007485
Authority: WO
Inventors: Helen Lu; Jie Jiang
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2007-06-12
Filing date: 2008-06-16
Publication date: 2008-12-24
Also published as: WO2008156725A3

Abstract

Methods and apparatuses are provided for inhibiting articular cartilage mineralization in a subject comprising administering to articular cartilage of the subject an amount of a functional domain of parathyroid hormone- related peptide (PTHrP). In addition, methods and apparatuses for preventing or treating osteoarthritis in a subject comprising administering to articular cartilage of the subject a prophylactically or therapeutically effective amount of a functional domain of parathyroid hormone- related peptide (PTHrP), respectively, are provided. Also, methods and apparatuses for promoting articular cartilage repair in a subject comprising comprising administering to articular cartilage of the subject an effective amount functional domain of parathyroid hormone-related peptide (PTHrP) are provided. Further, methods for diminishing damaging effects of physical activity on a joint in a subject comprising administering to articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) are provided.

Description

METHODS FOR INHIBITING CARTILAGE MINERALIZATION

Throughout this application, certain publications are referenced by Arabic numerals. Full citations for these publications, as well as additional related references, may be found immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art as of the date of the methods and apparatuses described and claimed herein.

Background

Arthritis is the leading cause of disability among Americans¹. The most common form of arthritis is osteoarthritis (OA) , with 21 million Americans suffering from this degenerative condition¹. Articular cartilage provides smooth gliding surfaces for joint articulation and its functional properties are derived from its structural organization and composition. Articular cartilage can be divided into three regions: the tangential (surface) zone, the transitional (middle) zone and the radial (deep) zone, with each region exhibiting characteristic cellular phenotype and matrix properties^2"8. Below the radial zone lies the tidemark, separating articular cartilage from the calcified cartilage region^9"14. The tidemark and the calcified cartilage collectively constitute the osteochondral interface, which functions as a physical barrier for vascularization and facilitates the pressurization and physiological loading of articular cartilage^15"17. Advancement of the calcified region and tidemark duplication are observed with age¹³'^18"21, and has been associated with OA^22"28. Elucidation of the biochemical processes responsible for the regulation of articular chondrocyte mineralization is critical in the treatment and eventual prevention of osteoarthritis.

Current understanding of cartilage mineralization derives largely from the knowledge of endochondral ossification during embryonic limb development^29'33, fracture healing^34"36, and from studies of pathological mineralization associated with crystal deposition arthropathies²⁷'³⁷. Systemic factors such as thyroid hormone promote hypertrophy of growth plate chondrocytes and the formation of mineralized cartilage, which is later remodeled into bone during endochondral ossification²⁰'³²'^38"41. Articular cartilage in immature animals provides the matrix source for the growth of the epiphyseal nucleus⁴². Chondrocytes near the articular surface proliferate and form new cartilage, the matrix compartment then calcifies and the mineralization front advances toward the articular . surface²⁰'²¹'³²'³⁸'³⁹'⁴². This process, however, slows down following puberty and becomes dormant upon reaching skeletal maturity²⁰'²¹'³⁸.

Multiple theories have been proposed regarding the mechanisms underlying the observed age-¹³'^18"21 and disease- related²⁰'^22'28 advancement of the calcification front in articular cartilage. Carter and Wong postulated that upward advancement is modulated by intermittent hydrostatic pressure generated in the deep layers of cartilage during physiological loading⁴³. Another hypothesis suggests that cells residing near the tidemark exert an active suppression of the mineralization processes in this area⁴⁴. It has also been postulated that cellular activity and mineralization are quiescent at or near the transition region, and that only after trauma or during the early phases of osteoarthritis, the chondrocytes near the tidemark become active and begin to mineralize⁴⁴'⁴⁵. To date, direct experimental validation of these hypotheses has been limited and the mechanisms controlling chondrocyte mineralization remain elusive.

Summary

This application describes methods and apparatuses for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP)

This application further provides methods and apparatuses for preventing or treating osteoarthritis in a subject comprising administering to a deep zone of articular cartilage of the subject a prophylactically or therapeutically effective amount of a functional domain of parathyroid hormone-related peptide (PTHrP), respectively.

This application further provides methods and apparatuses for promoting articular cartilage repair in a subject comprising comprising administering to a deep zone of articular cartilage of the subject an effective amount functional domain of parathyroid hormone-related peptide (PTHrP) .

This application further provides methods for diminishing damaging effects of physical activity on a joint in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) .

Brief Description of the Figures

Figure 1 : Effects of T3 treatment on the ALP activities of single- and co-cultured chondrocytes derived from different cartilage zones (n=6, *:p<0.05). Minimal ALP activity was measured in the SZC and MZC groups (not shown) , while ALP activity for DZC (A) exhibited a significant increase when stimulated with T3. In contrast, co-culture of DZC with SZC (B) suppressed this increase in ALP activity, and a similar response was found in the full thickness (FC) culture (C) which consisted of chondrocytes derived from full thickness articular cartilage.

Figure 2 : Effects of cellular interactions on the ALP activity of T3-stimulated chondrocytes cultures (Day 4, n=6, *:p<0.05). A) T3-stimulation of DZC led to a significant increase in ALP activity over the DZC control. The co-culture of DZC with MZC measured a significant increase in ALP activity after stimulation with T3. In contrast, co-culture of DZC with SZC under T3 stimulation did not result in a significant increase in ALP activity when compared to un-stimulated control. B) Histological staining corroborated the quantitative findings, as no difference in ALP staining intensity was seen between T3- stimulated (SZC+DZC, 1OnM) group and the un-stimulated co- culture control (Fast Blue stain, day 4) .

Figure 3 : Effects of cellular interactions on mineralization (n=5, *:p<0.05). A) Calcium deposition in the DZC cultures stimulated with T3 increased significantly at day 7 compare to DZC control, while no such increase was found in co-culture regardless of T3 stimulation. B) Histological staining corroborated the quantitative findings (Alizarin Red-S, Day 8).

Figure 4 : Effects of conditioned media treatment on the ALP activity of DZC cultures (Day 4, n=6, *:p<0.05). Addition of the DZC-conditioned media resulted in a significant increase in DZC ALP activity, while no such increase was found in DZC cultures treated with conditioned media from either the co-cultured (SZC+DZC) or SZC-only groups. ( — indicate addition of conditioned media) .

Figure 5 : Effects of cellular interactions on the relative gene expression of PTHrP (Day 4, n=3, *:p<0.05). Gene expression for PTHrP increased significantly when DZC was co-cultured with SZC. The co-culture of DZC with MZC did not increase PTHrP gene expression.

Figure 6 : Effects of PTHrP on the ALP activity of T3- stimulated DZC Cultures (n=6, *:p<0.05). A) All samples treated with PTHrP measured similar ALP activity level as the non-stimulated DZC control. B) Fast Blue staining confirmed that treating DZC cultures with PTHrP prevented the increase in ALP activity normally associated with T3 stimulation. This regulatory effect of PTHrP is similar to that found when DZC is co-cultured with SZC.

Figure 7 : Effects of PTHrP treatment on the relative gene expression of chondrocyte hypertrophy markers (Day 4, p<0.05, n=6) . Treatment with PTHrP suppressed the gene expression of Ihh, type X collagen, MMP13 and ALP. All gene expressions were normalized to expression of the housekeeping gene β-actin. * : significantly higher than OnM T3, OnM T3 w/PTHrP, 1OnM T3 w/PTHrP, and 10OnM T3 w/PTHrP

**: significantly lower OnM T3, 1OnM T3, 10OnM T3, and 10OnM T3 w/PTHrP

# : significantly higher than OnM T3, OnM T3 w/PTHrP and 1OnM T3 w/PTHrP

sc: significantly lower than 1OnM T3, 10OnM T3, 1OnM T3 w/PTHrP, 10OnM T3 w/PTHrP

♦ : significantly higher than OnM T3, OnM T3 w/PTHrP, 1OnM w/PTHrP, 100 nM w/PTHrP

♦♦: significantly lower than 1OnM T3, 10OnM T3, 1OnM T3 w/PTHrP, 10OnM T3 w/PTHrP

Figure 8: Effects of PTHrP antagonist on the ALP activity of T3-stimulated SZC+DZC co-culture (Day 4, n=β, *:p<0.05). In the presence of T3, blocking of PTHrP in SZC+DZC co-culture inhibited the suppressive effect of SZC on DZC mineralization potential. Samples treated with 100OnM of PTHrP antagonist and 5OnM T3 measured significantly higher ALP activity compared to all other groups.

Figure 9 : Effects of cellular interactions on DZC ALP activity in a 3-D co-culture model (n=6, * :p<0.05) . In the 3-D model, deep zone chondrocytes were first encapsulated in a hydrogel and then co-cultured with a monolayer of surface zone chondrocytes. An acellular hydrogel disc separated the DZC disc from the SZC monolayer. As in the 2-D co-culture models, SZC-DZC co-culture decreased the ALP activity of DZC embedded in the hydrogel. A significant decrease in DZC ALP activity was found in co-culture when compared to the DZC- only group at day 7.

Figure 10: Schematic of 3-D co-culture model. SZC monolayer and DZC agarose disc is separated by a 1.5% agarose gel disc .

Figure 11: ALP activity of DZC at day 4. ALP activity of DZC increased significantly when stimulated with T3 (first 2 columns on the left) . SZC+DZC co-culture (third, fourth and fifth columns from the left) ) showed a significant decrease in ALP activity compared to DZC single culture in the presence of T3. MZC+DZC co-culture (first 2 columns from the right) in the presence of T3 had similar ALP activity compare to DZC single culture treated with T3 ALP. Blocking of PTHrP eliminated the effect of co-culture. (*,# significant compare to all others).

Figure 12 : ALP gene expression of DZC cultures at day 4. A significant increase in ALP activity was seen in cultures treated with T3. Both co-culture with SZC and PTHrP treatment significantly decreased ALP activity. (All groups are significantly different from each other (p<0.05)) .

Figure 13: Gene expression of DZC cultures at Day 4. Co- culture and PTHrP significantly decreased Ihh, Col X and PTHrP/PTH receptor expression. Co-culture significantly increased MMP13 expression and PTHrP significantly decreased Runx2 gene expression.

Figure 14 : Gene expression of SZC. A significant increase in PTHrP expression and TGF-β3 was seen in the co-culture group while no differences were seen in TGF-βl and TGF-β2 gene expression.

Figure 15: ALP activity of DZC in agarose. (a) A significant increase in ALP activity was seen in the presence of T3 (**), and when culture were treated with PTHrP, a significant decrease was seen in both OnM and 5OnM T3 groups (*) . (b) ALP stain of cross section of DZC agarose disc .

Figure 16: A) Effects of Co-Culture on DZC ALP activity (*p<0.05). Co-culture significantly suppressed ALP activity of DZC stimulated by T3. B) PTHrP gene expression by SZC. Co-culture induced a significantly higher PTHrP expression by SZC (p<0.05).

Figure 17: Effects of PTHrP treatment on DZC response. A) Expression of hypertrophic markers. B) ALP activity. C) ALP deposition.

Figure 18: (A) Bovine chondrocyte growth on 25% PLAGE-BG composite scaffolds. (B) Effects of BG content on alkaline phosphatase (ALP) activity of chondrocytes.

Figure 19: Osteochondral Composite (G = Gel, I = Interface, M = Microsphere)

Figure 20: Matrix organization on the osteochondral construct after 10 days of culture. (A) GAG deposition (blue) . (B) Collagen (red) . (C) Mineralization (red) . (Co = Collagen, CH = Chondrocyte, M = Microsphere, G = Gel, 2Ox) Figure 21: (Left) Micro-CT scan of the osteochondral construct, and (Right) EDAX spectrum of the Interface region indicate that mineralization was limited to the Interface (I) and Microsphere (M) regions.

Figure 22: Preparation of sample using a water-oil-water emulsion method.

Figure 23: Effects of BG% on chondrocyte growth.

Figure 24: Media pH measurements for 25% BG composites.

Figure 25: ALP activity for 25% BG composites and 0% BG composites .

Figure 26: GAG content for 25% BG composites and 0% BG composites .

Figure 27: Histological stains of day 28 scaffolds (A) Trichrome of PLAGA-BG (10X), (B) Von Kossa of PLAGA-BG (10X) .

Detailed Description

In order to facilitate an understanding of the material which follows, one may refer to Freshney, R. Ian. Culture of Animal Cells - A Manual of Basic Technique (New York: Wiley-Liss, 2000) for certain frequently occurring methodologies and/or terms which are described therein.

However, except as otherwise expressly provided herein, each of the following terms, as used in this application, shall have the meaning set forth below.

"Administering" an agent can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intralesionally, intravenously, intraperitoneally, intramuscularly, orally, nasally, via the cerebrospinal fluid, via implant, via liposome-mediated delivery, anally, ocularly, topically, otically transmucosally, transdermally, and subcutaneously . The following delivery systems, which employ a number of routinely used pharmaceutically acceptable carriers, are only representative of the many embodiments envisioned for administering compositions according to the instant methods .

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone . Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids) , and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine) , preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid) , anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

As used herein, "administering to a deep zone" shall mean administering the material targeted to the deep zone of the articular cartilage of a subject.

As used herein, "ALP activity" shall mean alkaline phosphatase activity.

As used herein, "autologus chondrocyte implantation" shall mean a surgical procedure where cartilage cells are harvested from the knee, grown in the lab, and implanted into the area of cartilage damage.

As used herein, "chondrocyte" shall mean a differentiated cell responsible for secretion of extracellular matrix of cartilage .

As used herein, "functional domain" of a protein shall mean the segment of peptite with desired functions.

As used herein, "pharmaceutically acceptable carrier" shall mean a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

As used herein, "PTHrP" shall mean Parathyroid hormone- related protein (or PTHrP) , a protein member of the parathyroid hormone family, which includes all isoforms of the protein.

As used herein, "subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being.

As used herein, "surface zone chondrocyte", "middle zone chondrocyte", and "deep zone chondrocyte," shall mean chondrocytes found in the three zones of articular cartilage each with distinct matrix composition, organization, and size and shape of chondrocytes, where the surface zone is located at the articular surface representing 10% to 20% of the total thickness of articular cartilage, the middle zone is located in the middle of the articular cartilage comprising 40% to 60% of the total thickness of articular cartilage, and the deep zone is located adjacent to calcified cartilage, representing about 30% of the total thickness of articular cartilage .

As used herein, "sustained presence" shall mean continued presence over a period of time

As used herein, "therapeutically effective amount" and "prophylactically effective amount" shall mean an amount sufficient to treat a subject afflicted with, or inhibit the onset of, respectively, a disorder or a complication associated with a disorder. The therapeutically or prophylactically effective amount will vary with the subject, the condition, the agent delivered and the route of delivery. A person of ordinary skill in the art can perform routine titration experiments to determine such an amount. Depending upon the agent delivered, the therapeutically or prophylactically effective amount of agent can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions) . Desired time intervals of multiple amounts of a particular agent can be determined without undue experimentation by one skilled in the art. In one embodiment, the therapeutically or prophylactically effective amount is from about 1 mg of agent/subject to about 1 g of agent/subject per dosing. In another embodiment, the therapeutically or prophylactically effective amount is from about 10 mg of agent/subject to 500 mg of agent/subject . In a further embodiment, the therapeutically or prophylactically effective amount is from about 50 mg of agent/subject to 200 mg of agent/subject. In a further embodiment, the therapeutically or prophylactically effective amount is about 100 mg of agent/subject. In still a further embodiment, the therapeutically or prophylactically effective amount is selected from 50 mg of agent/subject, 100 mg of agent/subject, 150 mg of agent/subject, 200 mg of agent/subject, 250 mg of agent/subject, 300 mg of agent/subject, 400 mg of agent/subject and 500 mg of agent/subject .

As used herein, "treating" a subject afflicted with a disorder shall mean causing the subject to experience a reduction, remission or regression of the disorder and/or its symptoms. In one embodiment, recurrence of the disorder and/or its symptoms is prevented. In the preferred embodiment, the subject is cured of the disorder and/or its symptoms.

This application provides a method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit alkali phosphtase (ALP) activity in the subject's deep zone chondrocytes .

In one embodiment, the articular cartilage includes the said deep zone and a surface zone, and the PTHrP is administered to the deep zone. In another embodiment, the administered PTHrP has a sustained presence in the deep zone of the articular cartilage of the subject.

In one embodiment, the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

In one embodiment, the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

In another embodiment, the PTHrP is administered via local injection into the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered via autologous chondrocyte implantation.

In another embodiment, the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP. In yet another embodiment, the PTHrP is administered by transporting the PTHrP from the surface zone of the articular cartilage to the deep zone of the articular cartilage via mechanical loading.

In another embodiment, the PTHrP is administered to limit chondrocyte mineralization in the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of the subject.

In another embodiment, the amount of PTHrP administered into the deep zone of the articular cartilage of the subject is in a range of 0.1-15 nM.

In another embodiment, the PTHrP is administered into the deep zone of the articular cartilage of the subject in a plurality of doses over a period of time.

In one embodiment, the PTHrP administered is not more than 100 nm in size. In another embodiment, the functional domain of the PTHrP is comprised in PTHrP. In yet another embodiment, the functional domain of PTHrP comprises peptides 1-36.

In another embodiment, PTHrP administered is synthesized from peptides of the entire PTHrP molecule (in humans it is either composed of 141, 139 or 173 amino acids), or the respective functional domains of the structure such as PTHrP(l-36), PTHrP(l-34), PTHrP(l-7), PTHrP (37-107 ), PTHrP (107-111) , or a combinations of these PTHrP functional domains (sometimes more than domain are needed for protein binding or activation) . These peptides can be delivered alone or encapsulated in hydrogel-based delivery vehicles of lOOnm or less and then delivered into the joint and deep zone of the cartilage.

In another embodiment, the subject is a mammal. In the preferred embodiment, the subject is human.

This application further provides method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of type X collagen gene in the subject's deep zone chondrocytes.

In another embodiment, the PTHrP is administered via local injection into the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered via autologous chondrocyte implantation. In another embodiment, the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP. In yet another embodiment, the PTHrP is administered by transporting the PTHrP from the surface zone of the articular cartilage to the deep zone of the articular cartilage via mechanical loading.

In another embodiment, PTHrP administered is synthesized from peptides of the entire PTHrP molecule (in humans it is either composed of 141, 139 or 173 amino acids) , or the respective functional domains of the structure such as PTHrP(l-36), PTHrP(l-34), PTHrP(l-7), PTHrP (37-107) , PTHrP(107-lll) , or a combinations of these PTHrP functional domains (sometimes more than domain are needed for protein binding or activation) . These peptides can be delivered alone or encapsulated in hydrogel-based delivery vehicles of lOOnm or less and then delivered into the joint and deep zone of the cartilage.

This application further provides a method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the Indian Hedgehog (Ihh) gene in the subject's deep zone chondrocytes and thereby inhibit mineralization into calcium phosphate.

In one embodiment, the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers. In another embodiment, the PTHrP is administered via local injection into the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered via autologous chondrocyte implantation.

In one embodiment, the PTHrP administered is not more than 100 nm in size. In another embodiment, the functional domain of the PTHrP is comprised in PTHrP. In yet another embodiment, the functional domain of PTHrP comprises peptides 1-36. In another embodiment, PTHrP administered is synthesized from peptides of the entire PTHrP molecule (in humans it is either composed of 141, 139 or 173 amino acids) , or the respective functional domains of the structure such as PTHrP(l-3β), PTHrP(l-34), PTHrP(l-7), PTHrP (37-107 ), PTHrP(107-lll) , or a combinations of these PTHrP functional domains (sometimes more than domain are needed for protein binding or activation) . These peptides can be delivered alone or encapsulated in hydrogel-based delivery vehicles of lOOnm or less and then delivered into the joint and deep zone of the cartilage.

This application further provides a method for preventing osteoarthritis in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) .

In one embodiment, the PTHrP administered is not more than 100 nm in size. In another embodiment, the functional domain of the PTHrP is comprised in PTHrP. In yet another embodiment, the functional domain of PTHrP comprises peptides 1-36. In another embodiment, PTHrP administered is synthesized from peptides of the entire PTHrP molecule (in humans it is either composed of 141, 139 or 173 amino acids), or the respective functional domains of the structure such as PTHrP(l-36), PTHrP(l-34), PTHrP(l-7), PTHrP (37-107 ), PTHrP(107-lll) , or a combinations of these PTHrP functional domains (sometimes more than domain are needed for protein binding or activation) . These peptides can be delivered alone or encapsulated in hydrogel-based delivery vehicles of lOOnm or less and then delivered into the joint and deep zone of the cartilage.

This application further provides a method for treating osteoarthritis in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP), thereby preventing osteoarthritis in the subject .

In one embodiment, the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier. In one embodiment, the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

This application further provides a method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit alkaline phosphatase (ALP) activity in the subject's chondrocytes .

In one embodiment, the articular cartilage includes the said deep zone and a surface zone, and the PTHrP is administered to the deep zone. In another embodiment, the administered PTHrP has a sustained presence in the deep zone of the articular cartilage of the subject. In one embodiment, the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

In another embodiment, the amount of PTHrP administered into the deep zone of the articular cartilage of the subject is in a range of 0.1-15 nM. In another embodiment, the PTHrP is administered into the deep zone of the articular cartilage of the subject in a plurality of doses over a period of time.

This application further provides a method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the type X collagen gene in the subject's chondrocytes . In one embodiment, the articular cartilage includes the said deep zone and a surface zone, and the PTHrP is administered to the deep zone. In another embodiment, the administered PTHrP has a sustained presence in the deep zone of the articular cartilage of the subject.

In another embodiment, the PTHrP is administered to limit chondrocyte mineralization in the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of the subject. In another embodiment, the amount of PTHrP administered into the deep zone of the articular cartilage of the subject is in a range of 0.1-15 nM.

This invention further provides a method method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the Ihh gene in the subject's chondrocytes and thereby inhibit mineralization into calcium phosphate.

In another embodiment, the subject is a mammal. In the preferred embodiment, the subject is human. This application further provides a method for diminishing damaging effects of physical activity on a joint in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) .

In another embodiment, the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP. In yet another embodiment, the PTHrP is administered by transporting the PTHrP from the surface zone of the articular cartilage to the deep zone of the articular cartilage via mechanical loading. In another embodiment, the PTHrP is administered to limit chondrocyte mineralization in the deep zone of the articular cartilage of the subject. In yet another embodiment, the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of the subject.

In another embodiment, PTHrP administered is synthesized from peptides of the entire PTHrP molecule (in humans it is either composed of 141, 139 or 173 amino acids) , or the respective functional domains of the structure such as PTHrP(l-36), PTHrP(l-34), PTHrP(l-7), PTHrP (37-107 ), PTHrP (107-111) , or a combinations of these PTHrP functional domains (sometimes more than domain are needed for protein binding or activation) . These peptides can be delivered alone or encapsulated in hydrogel-based delivery vehicles of lOOnm or less and then delivered into the joint and deep zone of the cartilage. In another embodiment, the subject is a mammal. In the preferred embodiment, the subject is human.

This application further provides a scaffold apparatus for inhibiting articular cartilage mineralization in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

In one embodiment, at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes.

In another embodiment, one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impreganted with PTHrP.

In one embodiment, the subject is a mammal. In the preferred embodiment, the subject is human.

This application further provides a scaffold apparatus for preventing osteoarthritis in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

This application further provides a scaffold apparatus for treating osteoarthritis in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

This application further provides a scaffold apparatus for promoting articular cartilage repair in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

In one embodiment, at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes . In another embodiment, one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impreganted with PTHrP.

The specific embodiments and examples described herein are illustrative, and many variations can be introduced on these embodiments without departing from the spirit of the disclosure or from the scope of the appended claims. Elements and/or features of different illustrative embodiments and/or examples may be combined with each other and/or substituted for each other within the scope of this disclosure and appended claims.

Further non-limiting details are described in the following Experimental Details section which is set forth to aid in an understanding of the subject matter but is not intended to, and should not be construed to, limit in any way the claims which follow thereafter.

Review of PTH and PTHrP:

Parathyroid hormone (PTH) is a peptide hormone that is secreted from the parathyroid glands. It is involved in systemic calcium homeostasis, with bone and kidney as main target organs. In most mammalian species investigated so far, PTH is an 84-amino acid-long peptide with only a few interspecies, differences, specifically in the NH2-terminal part of the peptide (8) . The classical target cells of PTH in bone and kidney, namely, chondrocytes, osteoblasts, and osteoclasts from bone tissue and tubule cells from kidney, respond commonly to PTH with an intracellular accumulation of adenosine 3 ',5 '-cyclic monophosphate (cAMP) . However, PTH also activates adenylate cyclase on other target cells, named nonclassical target cells for PTH. Examples are blood cells (erythrocytes and lymphocytes) , liver cells, smooth muscle cells, pacemaker cells of the heart, and heart muscle cells. During the last two decades, this peptide hormone has become a subject of increasing interest. It shares its function with a groμp of structurally related peptides that are now named PTH-related peptide (PTHrP). PTHrP, however, differs from PTH in three important aspects: first, it is synthesized and released by various normal and malignant tissues; second, in comparison with PTH, itis elongated at its COOH-terminal end; and third, it acts in many tissues as a paracrine or autocrine factor rather than as a classical hormone. It is now clear that PTH and PTHrP, although structurally and sometimes functionally similar, exert different effects because of differences in targets and structure- function relationships. This review summarizes the present body of knowledge dealing with second messenger pathways and physiological responses of cells to PTH and PTHrP that are distinct from the classical actions previously identified on bone and kidney cells.

Parathyroid hormone : structure and signal transduction pathways

The identification of the primary structure of bovine PTH in 1970 was the starting point for the characterization of the structure-function relationship of PTH. Knowledge of the exact location of those parts of the PTH molecule that cause physiological responses is a prerequisite for the design of pharmacological antagonists that may be used in patients with excessive serum PTH levels. Because of the multiplicity of target cells of PTH, high serum levels of PTH may be pathogenetically related to a multitude of disorders, e.g., of bone, kidney, blood, or the cardiovascular system. PTH consists of 84 amino acids. The NH2-terminal portion of the molecule exhibits the greatest homology between different species. The first 34 amino acids seem to be a full biological agonist on classical target cells. In this part of the molecule, PTH consists of two interacting D-helixes. As mentioned in the introduction, all classical target cells of PTH respond to PTH by an activation of adenylate cyclase. The responsible functional domain is located on the first two amino acids of the native molecule. Bone- or kidney-derived cells show an intracellular.^" accumulation of cAMP when treated with PTH(l-84), PTH(l-34), and, to a lesser extent, PTH(2-34) but not after treatment with PTH fragments missing the first two amino acids. Elevation of intracellular cAMP levels leads subsequently to an activation of the cAMP- dependent protein kinase (PKA) . Amino acids 3-6 play a small but still significant role in the activation of the PKA dependent pathway. It was found in vivo thatdeletion of only the first two amino acids is insufficient to totally abolish PKA-dependent PTH effects (7) . To decrease the potency of PTH to activate PKA, however, truncation of the first six rather than the first two amino acids is required. In conclusion, amino acids 1-6 are required for the full interaction of the PTH molecule with a domain of its receptor, which is coupled to activation of adenylate cyclase and, secondarily, of PKA. Interestingly, deletion of the first six amino acids in PTH results in functional antagonism. From classical target cells of PTH, a PTH receptor was cloned (5) . It is named the PTH/PTHrP receptor because it can bind the NH2-terminal portions of both PTH and PTHrP. This receptor is a classical G protein-linked receptor with seven transmembrane domains. The PTH/PTHrP receptor, like many other structurally related receptors, couples to more than one intracellular signaling pathway. By binding to the PTH/PTHrP receptor, PTH may activate either adenylate cyclase, and subsequently PKA, or the phospholipase C (PLC) /protein kinase C (PKC) pathway. There are two major differences for the PLC/PKC-dependent pathway compared to the well-known functional domain responsible for the cAMP-dependent effects of PTH. First, several target cells of PTH, which respond to PTH by the activation of adenylate cyclase, do not respond with activation of the PKC-dependent pathway, e.g., tubule cells derived from the distal part of the nephron. Second, there are two different functional domains within the PTH molecule that may activate PKC in its target cells. One of these functional domains is located between amino acids 28 and 34 (11) . The smallest PTH peptide found to activate PKC covers amino acids 28-32. Site-directed mutagenesis showed that replacement of glutamine by alanine in position 29 destroys its function. Another PKC-activating domain was identified on some bone cells and is located in the NH2-terminal portion. When this is involved, PKC activation can be diminished by deletion of the first two amino acids. These observations suggest that this other PKC-activating domain is located near or is identical with the adenylate cyclase- activating domain. In none of the studies, however, were both PKC-activating domains found to be active on the same target cells. In summary, all classical target cells of PTH respond to PTH by an activation of adenylate cyclase and, subsequently, of PKA and PKC. The corresponding functional domains of PTH are located at the NH2-terminal portion (PKA, PKC) or cover amino acids 28-32 (PKC) . On some bone cell preparations, however, activation of PKC was found only for those covering the NH2-terminal portion.

PTH: activities on classical target cells

On bone, PTH has a dualistic effect. On one hand, PTH exerts anabolic effects on bone cells, e.g., it stimulates the proliferation of osteoblasts or chondrocytes. On the other hand, PTH has catabolic effects on bone as well. It increases bone resorption by acting on osteoclasts. This might happen directly or indirectly, namely, via the release of cytokines from osteoblasts, which belong to the tumor necrosis factor (TNF) family. PTH activates adenylate cyclase on chondrocytes. On most but not all chondrocyte preparations, PTH activates PKC as well. As a consequence of PKC activation, chondrocytes proliferate and their expression of the vitamin D3 receptor increases. On chondrocytes isolated from the resting zone, PTH activates alkaline phosphatase in a PKC-dependent manner, but on chondrocytes isolated from the growth zone, PTH activates alkaline phosphatase in a cAMP-dependent manner. In both cases, PTH(3-34) is devoid of intrinsic activity. This suggests primarily that in target cells of PTH both second messenger pathways, i.e., PKA and PKC activation, may couple to the same biochemical end point. Secondarily, it suggests that responsiveness to both PKC-activating domains described in the literature may be represented in chondrocytes, although probably at different states of maturation. The osteoblasts represent the most thoroughly analyzed cell type of bone responding to PTH. In this cell type, PTH (1-34), often considered as a full biological agonist, activates adenylate cyclase and PKC. PKC activation was demonstrated for PTH (28-48), indicating an involvement of the mid-regional PKC-activating domain. One of the central questions in this field during the last decade has been whether PTH is a mitogen for osteoblasts. The results obtained using cell cultures are still controversial. In some preparations, PTH leads to proliferation in a cAMP-dependent manner. In other preparations, this response requires an activation of PKC. When both pathways are activated together, accumulation of cAMP can be attenuated by a PKC-dependent effect. Therefore, in cells without a PKC responsiveness to PTH, the cAMP-dependent proliferation effect is more pronounced. In exceptional cases, PKC activation by PTH inhibits the proliferation of osteoblasts independently from effects on adenylate cyclase activity. The main question is, therefore, Which cell culture model represents the in vivo situation? In vivo, PTH(l-34) or PTH(28-48) increases bone mass (9) . The results suggest that both PKCdependent and cAMP-dependent anabolic effects of PTH occur in vivo. In contrast to the antagonistic effects of PKC activation on the cAMP-dependent cell proliferation under PTH, the PTH- dependent induction of c-fos and of the vitamin D3 receptor and the activation of the sodium-proton exchanger (NHE) type 3 are synergistically activated by both second messenger pathways. PKA- and PKC-signaling pathways are found to be antagonistic, again in respect to the activation of NHE-I and L-type calcium channels on classical target cells. The third target cell of PTH in the bone is the osteoclast, which is involved in bone resorption and therefore associated with the catabolic effects of PTH. Osteoclasts also respond to PTH by an activation of PKA- and PKC-dependent pathways (6) . The NH2- terminal truncation of PTH, PTH(3-34), does not abolish the activation of PKC by PTH in osteoclasts. The PKC-activating domain of PTH acting on osteoclasts seems to be located at position 28-34. The main effect of PTH on osteoclasts is an increase in the release of protons. This acidifies the surrounding of the cells and supports bone resorption. This effect by PTH is synergistically activated by both intracellular- signaling pathways. It is still not decided whether these direct effects of PTH on osteoclasts are responsible for the PTH-dependent activation of bone resorption. It has also been suggested that PTH acts mainly on osteoblasts, which then release cytokines acting on osteoclasts. Kidney cells, namely, primary cultures of proximal tubule cells, respond to PTH by activation of either the adenylate cyclase pathway or thePKC pathway. The functional domains are the NH2-terminally located functional domain and the 28-34 domain. As an exception, opossum kidney cells, which represent a commonly used cell culture model for studying renal effects of PTH on kidney cells, respond to PTH by an activation of PKC through the NH2-terminally located functional domain. Primary cultures of rat kidney cells derived from different parts of the kidney have also been used. It was found that proximal tubule cells respond to PTH with an activation of both intracellular signal transduction pathways, whereas cells derived from the distal tubule respond only with activation of adenylate cyclase. Like osteoblasts, kidney cells show stronger activation of the adenylate cyclase pathway in cells with downregulated PKC. In summary, PTH acts on all main cell types of bone and kidney. There, it activates the adenylate cyclase pathway via the classical NH2-terminally located functional domain and the PKC-dependent pathways via a midregionally located functional domain. It should be noted that in nearly all cell types thus far investigated PTH activates both second messenger pathways.

PTH: nonclassical target cells

Nonclassical target cells of PTH differ from the classical target cells described above in respect to the major second messenger pathways. They respond to PTH by an activation of PKC but not of adenylate cyclase. Examples are keratinocytes, pancreatic cells, murine T lymphocytes, and cardiomyocytes . The latter cells lose their adenylate cyclase responsiveness to PTH during maturation. Neonatal cardiomyocytes respond to PTH with an accumulation of cAMP, indicating adenylate cyclase activation. Adult cardiomyocytes, however, do not respond in the same way. They selectively respond to PTH by activation of the PLC/PKC pathway. The functional domain of PTH responsible for this effect is located within the 28-34 portion and seems to be identical to the functional domain identified with chondrocytes as target cells. The influence of PTH on the cardiovascular system is not limited to cardiomyocytes (see Ref . 12) . A vasodilatory action of PTH is caused by an activation of adenylate cyclase of smooth muscle cells, which is an exception for the as yet characterized nonclassical target cells, because PTH exclusively activates adenylate cyclase in this vascular cell type. The responsible functional domain for this PTH effect on smooth muscle cells is the classical NH2-terminal portion of the molecule. A third cell population within the cardiovascular system that responds to PTH are pacemaker cells. Initially, it was thought from in vivo studies that the chronotropic effect of PTH is an indirect one, caused by its vasodilatory action. A direct activation of ionic channels on pacemaker cells, however, has meanwhile been demonstrated, indicating that PTH exerts direct chronotropic effects as well (4). PTH has several cardiovascular effects. It is a hypotensive drug because of the relaxation of smooth muscle cells. In intact circulation, a subsequent activation of the baroreflex may indirectly elicit a positive chronotropic response. The chronotropic effect in vivo is further enhanced by a direct action of PTH on pacemaker cells. There are no indications that PTH exerts direct inotropic effects on adult cardiac muscles. Using the PKCactivating domain, PTH also acts as a hypertrophic agonist on cardiomyocytes . The latter observation seems to have significant clinical relevance, because dialysis patients with extremely high serum PTH levels often develop left ventricular hypertrophy. This is reversible upon parathyroidectomy, suggesting that the growth-promoting effect of PTH, identified on cell culture models, has indeed a pathophysiological significance in vivo. In summary, nonclassical target cells show an unusual coupling of the activated PTH receptor to intracellular signaling.

PTHrP: structure and signal transductionpathways In principle, all known target cells for PTH are also target cells for PTHrP because of the structural similarity in their NH2-terminal parts. Six of the first seven amino acids are identical between the two peptide hormones. Therefore, it is not surprising that PTHrP can mimic nearly all functions of PTH mediated by the NH2-terminally located adenylate cyclase-activating domain. The binding domain of PTH and PTHrP, identified between amino acids 18 and 34, does not show a great similarity in the primary structure, but the secondary structure is guite comparable. This seems to enable both peptide hormones to bind to the same receptor, namely, the PTH/PTHrP receptor. In addition, a second PTH receptor has been identified on classical target cells that binds PTH but not PTHrP, which has been named the type 2 receptor (1). Most natural target cells analyzed thus far express the classical PTH/PTHrP receptor. In exceptional cases they also express the type 2 receptor. The PTHrP molecule contains further active parts with no homology to PTH. PTHrP from the rat consists of 141 amino acids and is therefore extended at its COOH-terminal part compared to PTH. In the human two additional variants are found: PTHrP(l-139) and PTHrP ( 1-173) . All three forms are synthesized from a common gene and represent different splice variants. Whether these PTHrP forms differ in their physiological effects on target cells has yet to be elucidated. Some functional domains of PTHrP are located in parts of the molecule with either no or only limited structural similarity to PTH. They couple, nevertheless, to the same second messenger pathways as PTH, namely, to PKA- and PKC-dependent intracellular signaling pathways. A first PTHrP-specific functional domain distinct from PTH couples to the PKA-dependent pathway and is located in the COOHterminal direction from the well-known adenylate cyclase-activating domain of PTH. A second domain seems to be located between amino acids 37 and 107 and stimulates placental calcium transport. A third domain has been found in the region between amino acids 107 and 111. It couples to PKC-activated pathways. These functional domains are located in parts of the molecule with no similarity to PTH. This suggests the existence of PTHrP-specific receptors. Such receptors, however, have not yet been identified.

PTHrP: expression and actions

PTHrP is widely expressed in normal and malignant tissues. Initially, it was identified as a factor released from malignant tissues. It may act as a paracrine or autocrine factor. In most tissuesin which PTHrP is expressed, target cells for PTHrP were found adjacent. They often respond to PTHrP by an activation of adenylate cyclase and subsequently PKA. Vascular smooth muscle cells represent an example. On these cells, the structure-function relationship for the adenylate cyclase-activating domain of PTHrP is identical to that of PTH: PTHrP activates adenylate cyclase, and PTHrP (3-34) and PTHrP (7-34) are devoid of intrinsic activity but antagonize the response to PTHrP not truncated at the NH2- terminal part. This indicates that the first two amino acids are responsible for the interaction between PTHrP and the receptor to activate adenylate cyclase. PTHrP also activates adenylate cyclase in opossum kidney cells, chondrocytes, osteoblasts, and neurons and thereby mimics the activity of the whole molecule in this aspect. In some other cell types, the cAMP-dependent effects of PTHrP could also be mimicked by NH2- terminal truncated PTHrP. Examples are human kidney cells, opossum kidney cells, osteoblasts, and cardiomyocytes. In these latter cell types the functional domain on PTHrP activating adenylate cyclase seems thus to be different from the NH2- terminally located domain of PTH. It is in line with this that PTHrP but not PTH activates adenylate cyclase on cardiomyocytes and osteoblasts from the cell line ROS 17/2.8. In some exceptional target cells of PTHrP, e.g., pancreatic islets or carcinoma cells, it does not activate adenylate cyclase. There are numerous reports showing cAMP-independent PTHrP effects. An accumulation of intracellular calcium was observed in squamous carcinoma and pancreatic islet cells in the presence of PTHrP. This effect of PTHrP can be blocked by PTHrP (7-34) but occurs in the absence of PKA activation. On Walker 256 carcinoma cells, PTHrP acted as amitogen in a cAMP-independent manner. This mitogenic effect of PTHrP requires PKC activation, suggesting that NH2-terminal peptides of PTHrP activate the PLC/PKC pathway. A candidate location of the PKC-activating domain is the peptide sequence 28-34, which represents the PKC- activating domain of PTH. The primary sequence between PTH and PTHrP in this part is different, but the secondary structure is similar. On osteoblasts, PTHrP (28-34) indeed activates PKC (3) . In contrast to PTH, PTHrP does not activate PKC via this domain on cardiomyocytes. With the use of site-directed mutagenesis, it was shown that the difference of the amino acid in position 29, asparagine in the case of PTHrP and proline in the case of PTH, explains the differentresponses (13). In addition, another PKC- activating domain, clearly distinct from that of PTH, was identified between amino acids 107 and 111. Initially, a COOH-terminal portion of PTHrP, PTHrP(107- 139), was characterized as an inhibitor of osteoclastic bone resorption and named osteostatin. The functional domain responsible for this effect covers amino acids 107-111 and is linked to PKC activation. Because this part of PTHrP has no homology to PTH, this effect is unique for PTHrP. A specific receptor to which this part of the peptide hormone binds must be postulated but has yet to be identified. With one exception, an adenylate cyclase activation was not found for this fragment. PKC activation caused by PTHrP (107-111) has been shown on other classical target cells of PTH or PTHrP, e.g., osteoblasts, and nonclassical target cells such as cardiomyocytes . It is an open question whether the identification of active functional domains on PTH and PTHrP molecules is now complete. There are a few reports in the literature indicating the existence of more domains than those identified to date. For example, a partial agonistic activity of PTH (18-48) has been reported on adenylate cyclase activity of periosteal cell culture systems of chick embryos, and PTH (52-84) increases the expression of collagen in proliferating chondrocytes. The effects of PTH and PTHrP are mediated by bioactive fragments produced in vivo with different half-times. This indicates that the identification of different functional domains may have physiological significance.

PTH versus PTHrP in the cardiovascular system

Although various target cells for PTH or PTHrP have been described within the cardiovascular system, the physiological role of PTH and PTHrP in cardiovascular biology has yet to be identified. Excess of PTH or PTHrP, however, is often accompanied by multiple forms of cardiovascular diseases, e.g., left ventricular hypertrophy, atherosclerosis, and hypertension. The cardiovascular system contains three different target cells for PTH and PTHrP: smooth muscle cells, cardiomyocytes, and pacemaker cells. Two cell types express and release PTHrP in a onconstitutive manner, namely, smooth muscle cells and endothelial cells. On the adult cardiomyocyte, PTHrP activates PKA, thereby improving cardiac contractility. This effect can be antagonized by PTH. PTH can activate the PKCdependent pathway leading to myocardial hypertrophy. This response can be antagonized by PTHrP. In adult cardiomyocytes, PTHrP may also activate PKC, but via a functional domain not found on PTH. In smooth muscle cells, PTH and PTHrP synergistically activate the PKA pathway. This leads to vasorelaxation. A local overexpression of PTHrP in vessels has been observed under pathophysiological conditions, as in atherosclerosis and conditions characterized by high blood pressure. The regulation of PTHrP expression in cardiovascular cells, however, is only partly characterized. In smooth muscle cells, PTHrP expression increases under angiotensin II, which is a potent vasoconstrictor. These findings suggest that the expression of the vasorelaxant PTHrP is upregulated in response to a pathophysiological elevation of vasotonus. The regulation of PTHrP expression and release by endothelial cells has not yet been characterized. In the heart, microvascular endothelial cells express PTHrP but, unlike adjacent cardiomyocytes, they themselves do not respond to this hormone. In summary, PTHrP represents a paracrine factor of the cardiovascular system whose role in physiology and pathophysiology is still largely unknown. Concluding remarks

In conclusion, PTH and PTHrP show a great deal of variability in regard to target cells and intracellular signaling. Although PTHrP was initially characterized by its PTH-like activities, it must be considered as a peptide hormone on its own, with several newly discovered effects distinct from those exerted by PTH. The widespread expression of PTHrP in normal tissues and the extensive expression in malignant tissues indicate an important role of this peptide hormone. Local release of PTHrP occurs in several diseases, but the physiological and pathophysiological roles of PTHrP still remain to be clarified.

Experiment 1 :

In the past decade, tissue engineering has emerged as an alternative approach to implant design and tissue regeneration. Design methodologies adapted from current tissue engineering efforts can be applied to regenerate the osteochondral interface.

An in vitro graft system was developed for the regeneration of the osteochondral interface. The native osteochondral interface spans from nonmineralized cartilage to bone, thus one of the biomimetic design parameters for the multiphased osteochondral graft is the calcium phosphate (CA-P) content of the scaffold. The components of this graft system include (1) a hybrid scaffold of hydrogel and polymer- ceramic composite (PLAGA-BG), (2) novel co-culture of osteoblasts and chondrocytes, and (3) a multi-phased scaffold design comprised of three regions intended for the formation of three distinct tissue types: cartilage, interface, and bone. In the current design, the Ca-P content is related to the percent of BG in the PLAGA-BG composite. From the material selection standpoint, one phase of the hydrogel-polymer ceramic scaffold is based on a thermal setting hydrogel which has been shown to develop a functional cartilage-like matrix in vitro. The second phase of the scaffold consists of a composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) . PLAGA-BG is biodegradable, osteointegrative, and able to support osteoblast growth and phenotypic expression. The middle phase, which interfaces the first and second, has a lower Ca-P content than the second phase, being of a mixture of the hydrogel and the PLAGA-BG composite.

The scaffolds utilized in this set of experiments are composed of PLAGA-BG microspheres fabricated using the methods of Lu et al. Briefly, PLAGA 85:15 granules were dissolved in methylene chloride, and 45S5 bioactive glass particles (BG) were added to the polymer solution (0, 25, and 50 weight%BG) . The mixture was then poured into a 1% polyvinyl alcohol solution (sigma Chemicals, St. Louis) to form the microspheres. The microspheres were then washed, dried, and sifted into desired size ranges. The 3-D scaffold construct (7.5 x 18.5 mm) was formed by sintering the microspheres (300-350 μm) at 70°C for over 6 hours.

Bovine articular chondrocytes were harvested aseptically from the carpometacarpal joints of 3 to 4-month old calves by enzymatic digestion. The chondrocytes were plated and grown in fully supplemented Dulbecco' s Modified Eagle Medium (DMEM, with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acids). The chondrocytes were maintained at 37⁰C, 5% CO₂ under humidified conditions.

The composites were sterilized by ethanol immersion and UV radiation. The scaffolds were seeded at 2.0 x 10⁵ cells/sample in 48-well plates. Samples (n=5) were maintained at 37°C for 1, 7, 14, and 21 days. Cell proliferation, alkaline phosphatase (ALP) activity, glycosaminoglycan (GAG) , and mineralization were examined in time. The osteochondral construct consists of three regions, gel- only, gel/microsphere interface, and a microsphere-only region. Isolated bovine chondrocytes were suspended in 2% agarose (Sigma, MO) at 60 x 10⁶ cells/ml. The PLAGA-BG scaffold was integrated with the chondrocyte-embedded agarose hydrogel using a custom mold. Chondrocytes were embedded in the gel-only region and osteoblasts were seeded on the microsphere-only region. All constructs were cultured in fully supplemented DMEM with 50μg/ml of ascorbic acid. The cultures were maintained at 5% CO₂ and 37⁰C, and were examined at 2, 10, and 20 days.

Cell viability was assayed by a live/dead staining assay (Molecular Probe, OR) , where the samples were halved and imaged with a confocal microscope (Olympus, NY) . Proliferation was measured using a fluorescence DNA assay, and ALP activity was determined by a colorimetric enzyme assay. Cell morphology and gel-scaffold integration were examined at 15kV using environmental scanning electron microscope (ESEM, FEI, OR) . For histology, samples were fixed in neutral formalin, embedded in PMMA and sectioned with a microtome. All sections were stained with hematoxylin and eosin, Picrosirius red for collagen, Alizarin Red S for mineralization, and Alcian Blue for GAG deposition.

Chondrocytes maintained viability and proliferated on all substrates tested during the culture period (Figure 18A). As shown in Figure 18B, ALP activity of chondrocytes increased when grown on PLAGA-BG scaffolds, while a basal- level of activity was observed on scaffolds without BG. Chondrocyte ALP activity peaked between days 3 and 7, and these cells elaborated a GAG-rich matrix on the PLAGA-BG composite scaffolds.

The agarose gel layer penetrated into the pores of the PLAGA-BG scaffolds and construct integrity was maintained over time, as seen in Figure 19. Chondrocytes and osteoblasts remained viable in both halves of the construct for the duration of the culturing period.

Chondrocytes remained spherical in both the agarose-only region (G) and the interface (I) region. Chondrocytes (Ch) migrated out of the agarose hydrogel and they attached onto the microspheres in the interface region. These observations were confirmed as these migrating cells did not stain positively for the cell tracking dye used for the osteoblasts. Interestingly, chondrocyte migration was limited to the interface and no chondrocytes were observed in the microsphere region.

Collagen production was evident in both the gel (G) and microsphere (M) regions (Figure 20B) . As shown in Figure 2OA, positive Alcian Blue staining was observed at the interface (I) and within the gel (G), indicative of the deposition of a GAG-rich matrix within these regions by chondrocytes. A mineralized matrix was found within the microsphere region as well as the interface (Figures 2OC, 6 left, 6 right) . Energy dispersive x-ray analysis (EDAX) and microcomputerized tomography (micro-CT) scans revealed that the interfacial region is comprised of a mixture of GAG and amorphous calcium phosphate (Figure 21). This set of experiments focused on the development of a novel osteochondral graft for cartilage repair. Specifically, the PLAGA-BG composite and hydrogel scaffold consisted of a gel-only region for chondrogenesis, a microsphere-only region for osteogenesis, and a combined region of gel and microspheres for the development of an osteochondral interface.

In Experiment 1, the potential of the microsphere composite phase to support chondrocyte growth and differentiation was examined, as they are co-cultured with osteoblasts on the osteochondral scaffold. Cell viability and proliferation were maintained on the scaffolds during culture. In addition, the chondrocytes produced a GAG-rich matrix, suggesting that their chondrogenic potential was maintained in the presence of Ca-P. It is interesting to note that the PLAGA-BG composite promoted the ALP activity of chondrocytes in culture. ALP is an important enzyme involved in cell-mediated mineralization, and its heightened activity during the first week of culture suggest that chondrocytes may participate in the production of a mineralized matrix at the interface.

The osteochondral graft in Experiment 2 supported the simultaneous growth of chondrocytes and osteoblasts, while maintaining an integrated and continuous structure over time. The agarose hydrogel phase of the graft promoted the formation of the GAG-rich matrix. Chondrocytes embedded in agarose have been shown to maintain their phenotypeand develop a functional extracellular matrix in free-swelling culture. More importantly, the osteochondral graft was capable of simultaneously supporting the growth of distinct matrix zones - a GAG-rich chondrocyte region, an interfacial matrix rich in GAG, collagen, and a mineralized collagen matrix produced by osteoblasts. The pre-designed regional difference in BG content across the hybrid scaffold coupled with osteoblast-chondrocyte interactions may have mediated the development of controlled heterogenity on these scaffolds. Previously, such distinct zonal differentiations have only been observed on osteochondral grafts formed in vivo. A reliable in vitro osteochondral model will permit in-depth evaluation of the cell-mediated and scaffold-related parameters governing the formation of multiple tissue zones on a tissue engineered scaffold. Chondrocyte migration into the interface region suggests that these cells may play an important role in the development of a functional interface.

Experiment 2 :

This set of experiments characterizes the growth and maturation of chondrocytes on composite scaffolds (PLAGA- BG) with varying composition ratios of poly-lactide-co- glycolide (PLAGA) and 45S5 bioactive glass (BG) .

For the sample preparation, a water-oil-water emulsion was used (Fig. 22) .

Chondrocytes were harvested asceptically from the bovine carpametacarpal joints (~1 week old) . The cartilage was digested for 2h with protease, 4h with collagenase and resuspended in fully supplemented Dulbecco' s Modified Eagle Medium (DMEM + 10%serum + 1% antibiotics + 1% non-essential amino acids, 50μg/ml ascorbic acid). Composites seeded with cells (64,000 cells/samples) were maintained in a 37°C incubator (5% CO₂) .

At day 1, 3, 7, 14, 21 and 28 days, the samples were harvested and analyzed for cell proliferation (n=5) , ALP activity (n=5), GAG deposition (n=5) and histology.

Chondrocytes were viable and proliferated on all substrates tested. A significantly higher number of cells attached to the 25% composite, and higher number of chondrocytes were found on the 25% samples after 28 days of culture (p<0.05) (Fig. 23) .

From days 1-7, cell number was lower on the 25% substrates (p>0.05), likely due to surface reactions occurring at the PLAGA-BG composite surface. Media pH measured significantly higher alkalinity at days 1 and 3 for 25% BG composites (p<0.05) (Fig. 24).

ALP activity was higher on the 25% PLAGA-BG samples (p<0.05) (Fig. 25). ALP activity peaked at day 7 for the 25% samples, as compared to day 21 for the 0% group (Fig. 25) .

Chondrocytes continued to elaborate on GAG matrix, and GAG content increased with time and peaked on day 21 (Fig. 26).

Chondrocytes penetrated and grew within the pores of the microsphere scaffolds. Mineralization nodules were found on chondrocytes grown on PLAGA-BG composites (Fig. 27).

The second set of experiments further show that PLAGA-BG composite supports chondrocyte proliferation and matrix deposition during the culturing period. The BG surface reactions which lead to the formation of a surface Ca-P layer had a significant effect on the chondrocytes.

PLAGA-BG composites have been shown to be osteoconductive . PLAGA-BG composite with 25% BG caused an increase in ALP activity in articular chondrocytes compared to the control which is consistent with the previous findings with 100% BG. The BG induced mineralization seen here may mimic endochondral bone formation and may be used to facilitate the formation of tidemark in tissue engineered osteochondral grafts.

Experiment 3 :

BACKGROUND

It is well established that molecular transport from the articular surface to the cartilage proper is mediated through dynamic loading⁴⁶'⁴⁷. Physiological loading is also believed to prevent tidemark advancement⁴⁸; O'Connor reported that unloading of the rat hind leg led to thickening of the calcified cartilage layer⁴⁹. Based on these observations and taking into consideration the stratified organization of articular cartilage, it is proposed that cellular communication within cartilage layers plays a regulatory role in cartilage mineralization. Specifically, it is hypothesized that chondrocytes from the surface layer regulate the mineralization potential of chondrocytes residing in the deep zone. To test this hypothesis, the interaction of chondrocytes isolated from the three different zones of articular cartilage using a direct co- culture model was evaluated. The effects of co-culture on chondrocyte mineralization was determined, and conditioned media studies was conducted to evaluate the existence of paracrine effects. Thyroid hormone has previously been shown to induce hypertrophy in both growth plate and aging articular chondrocytes³²'^38"41'⁵⁰. In this study, triiodothyronine (T3), a form of thyroid hormone, was used to stimulate articular chondrocyte hypertrophy and mineralization⁵⁰. Since the growth plate remains open before reaching skeletal maturity, exposure to systemic factors such as T3 in post-natal articular cartilage is not unexpected. Moreover, addition of T3 will simulate the condition following injury to the osteochondral interface, after which the deep zone cartilage may be exposed to systemic factors via invasion of the subchondral bone vasculature⁵¹.

Additionally, the mechanism underlying any potential interactions between zonal sub-populations of articular chondrocytes was investigated. During development, the rate at which the growth plate cartilage mineralizes is highly regulated. It has been shown to be controlled via the parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) negative feedback loop^52"56. During endochondral ossification, the hypertrophic and pre- hypertrophic chondrocytes secrete Ihh which promotes hypertrophy in adjacent chondrocytes^57"59 and induces PTHrP production in immature peri-articular chondrocytes⁵²'⁵⁵'⁶⁰'⁶¹. Whether the PTHrP-Ihh control loop is significant in the regulation of articular chondrocyte mineralization during post-natal development is not known. In situ hybridization of rodent articular cartilage revealed that PTHrP expression is present in chondrocytes residing near the surface of articular cartilage at 20 weeks of age⁶². Based on these observations, it was hypothesized that, similar to growth plate chondrocytes, mineralization by deep zone chondrocytes is regulated by PTHrP, and chondrocytes at the surface and deep zones of articular cartilage may also communicate via the PTHrP-Ihh negative feedback loop. To test this hypothesis, experiments were designed to determine the role of PTHrP in regulating Ihh expression and mineralization potential of deep zone chondrocytes. Specifically, exogenous PTHrP was added to deep zone chondrocytes stimulated by T3, and the resultant effects on cell ALP activity and gene expression were investigated. It was expected that while stimulation of deep zone chondrocytes with T3 will increase Ihh expression, this effect will be countered by the addition of PTHrP. Findings from this study were anticipated to provide insight into the mechanisms governing articular cartilage mineralization, and to have a significant impact on the formulation of future treatment strategies for osteoarthritis as well as functional cartilage repair.

MATERIALS AND METHODS

Cells and Cell Culture

Primary bovine articular chondrocytes were used in this study. The cells were isolated from the knee joints of neonatal calves (~1 week old) obtained from an abattoir (Fresh Farm's Beef, Inc., VT). Due to differences in cartilage thickness between animals and isolation sites, a standardized protocol was developed based on published reports³'⁵'⁶. Specifically, the 10% (by height of the full thickness articular cartilage) closest to the articular surface was considered to be the surface zone, the middle 60% by height was deemed the middle zone, and the 30% closest to the subchondral bone was designated as the deep zone. Zonal populations of chondrocytes were then obtained by enzymatic digestions of tissue derived from each region following the methods of Hidaka et al.^β. Briefly, the tissue was minced and digested overnight in 0.1% w/v collagenase (Sigma, St. Louis, MO) in Dulbecco' s modified Eagle's medium (DMEM, Cellgro-Mediatech, Herndon, VA) supplemented with 1% fetal bovine serum (FBS, Atlanta Biologicals, Atlanta, GA) and 2% antibiotics (10,000 U/mL penicillin, 10 mg/mL streptomycin, Cellgro-Mediatech) . The cells were seeded at high density (4 x 10⁵ cells/cm²) and maintained in fully-supplemented DMEM (10% FBS, 1% nonessential amino acids, 1% antibiotics) . Three sub- populations of chondrocytes were isolated from the tibiofemoral joint: the Surface Zone Chondrocytes (SZC), Middle Zone Chondrocyte (MZC) , and Deep Zone Chondrocytes (DZC) . A full thickness chondrocyte (FC) culture was established from digestion of a mixture of all isolated cartilage tissue pieces, without distinguishing any one cartilage zone from another.

T3 Stimulation of Chondrocytes

Primary chondrocytes were seeded at high density (4 x 10⁵ cells/cm²) , and the cultures were maintained in fully- supplemented media for two to three days after seeding. Cultures were then stimulated with T3 (Sigma, St. Louis, MO) following methods described by Rosenthal et al.⁵⁰ Briefly, all groups were pre-treated for 24 hours with serum-free DMEM supplemented with 1% antibiotic and 0.35 mg/mL bovine serum albumin (Sigma). At day 0, all cultures were stimulated with T3 (0, 10, 100 nM) for 96 hours. After which, all cultures were maintained in mineralization media without T3, which consisted of fully-supplemented DMEM plus 50 μg/mL ascorbic acid (Sigma) and 3 mM β-glycerophosphate (β-GP, Sigma), for up to two weeks. Chondrocyte ALP activity and mineral deposition were assessed at 0, 2, 4, 7, 10, and 14 days.

Co-Culture Models of Zonal Populations of Chondrocytes

To evaluate cellular interactions between chondrocyte populations, direct 2-D co-cultures of DZC with either SZC and/or MZC were established. Specifically, co-cultures of SZC+DZC and MZC+DZC were initiated by seeding at high density (4 x 10⁵ cells/cm²) with a 1:1 mixture of each cell type. The co-culture ratio of' SZC and DZC was chosen to approximate that of the in vivo cell ratio as previous reports have shown that the cell density in the surface zone cartilage is approximately 2 to 3 times higher than that of deep zone cartilage⁷'⁶³. Base on this isolation technique, it is expected the total cell number of SZC and DZC are approximately equal. Individual cultures of SZC, MZC, and DZC, as well as full thickness chondrocytes (FC) served as controls. All chondrocyte cultures were then stimulated with T3 as described above. The effects of co-culture on cell proliferation, ALP activity and mineral deposition were assessed at 2, 4, 7, 10, and 14 days.

Mechanism of Cellular Interactions : Paracrine Effects

To determine whether zonal chondrocyte interactions were mediated through paracrine effects, a conditioned media study was performed. Fresh conditioned media derived from SZC, DZC, and SZC+DZC co-cultures without T3 was collected at day 2 and again at day 4. The media was centrifuged at 150Og for 10 minutes and a 1:1 mix with fresh media with a final T3 concentration of 0 or 10 nM was added to DZC cultures at day 2 and day 4. The mineralization potential of DZC cultures maintained in these conditioned media was examined at day 4.

Mechanism of Cellular Interactions : Role of Parathyroid Hormone-Related Peptide

To evaluate the role of PTHrP in mediating the interaction between zonal chondrocyte populations, PTHrP expression was measured for all groups. In addition, to directly assess its effect on DZC mineralization, exogenous PTHrP was added to DZC cultures stimulated with T3. Specifically, 50 nM of PTHrP (1-40, Sigma) was added along with T3 (0, 10, and 100 nM) to confluent DZC cultures. Cultures of DZC stimulated by T3 but untreated with PTHrP served as controls. The effects of PTHrP on the mineralization potential of DZC were analyzed at 0, 2 and 4 days.

The role of PTHrP-mediated interaction between zonal chondrocytes population was evaluated by blocking the actions of PTHrP using a PTHrP antagonist, PTH (7-34, Bachem, San Carlos, CA)⁶⁴. Exogenous PTH (7-34) at 0, 0.1, 10, and 1000 nM was added to SZC+DZC cultures along with T3 (0, 5OnM) under serum free conditions. The effects of the antagonist on ALP activity of SZC+DZC cultures were analyzed at day 4.

3-D Co-Culture Model To ascertain that the observed responses were not limited to the 2-D model, a 3-D co-culture model was used to verify DZC and SZC interactions. Specifically, DZC were first encapsulated in 2% agarose hydrogel at a concentration of 2^χlO⁷ cell/mL and then co-cultured with a monolayer of surface zone chondrocytes in a culture well (Fig. 9A) . An acellular hydrogel disc separated the DZC-laden disc from the monolayer of surface zone chondrocytes. The effect of co-culture on ALP activity of DZC was evaluated at 1, 3, 7 and 14 days.

End-Point Analysis : Cell Proliferation

Cell proliferation (n=6) was determined by measuring total DNA per sample using the PicoGreen^® assay (Molecular Probes, Eugene, OR) according to the manufacturer's suggested protocol. Briefly, the samples were first rinsed with Phosphate Buffered Saline (PBS, Sigma) and the cells were lysed in 300 μL of 0.1% Triton X solution (Sigma). An aliquot of the sample (20 μL) was then added to 180 μL of the PicoGreen® working solution. Fluorescence was measured with a microplate reader (Tecan, Research Triangle Park, NC) with excitation and emission wavelengths of 485 and 535 nm, respectively. Total cell number in the sample was determined by converting the amount of DNA per sample to cell number using the conversion factor of 7.7 pg DNA/cell⁶⁵.

Table 1. List of oligonucleotides primers used

Endpoint Analysis : Gene Expression

Gene expression (n=5) was measured using reverse transcription followed by real-time polymerase chain reaction (PCR). The oligonucleotides were custom designed and the sequences are summarized in Table 1. Total RNA was isolated using the TRIzol^® reagent (Invitrogen, Carlsbad, CA) extraction method. The isolated RNA was reverse- transcribed into cDNA via the Superscript™ III First-Strand Synthesis System (Invitrogen) following the manufacturer's suggested protocol. The cDNA product was amplified and quantified through real-time PCR using iQ SYBR Green Supermix (BioRad, Hercules, CA) . The expression level of relevant genes indicative of chondrocyte maturation such as ALP, type X collagen, matrix metalloproteinases-13 (MMP 13) , as well as the expression of Ihh and PTHrP were measured and normalized to the expression of the housekeeping gene β-actin. All reactions were run for 40 cycles using the iCycler iQ Real-Time PCR Detection System (BioRad) . Normalized expression levels reported were calculated based on difference between threshold cycles, namely, the difference in threshold cycle values between the gene of interest and the housekeeping gene β-actin.

End-Point Analysis : Mineralization Potential

Cell mineralization potential was determined by examining ALP activity and mineral deposition. Quantitative ALP activity (n=6) was measured using an enzymatic assay based on the hydrolysis of p-nitrophenyl phosphate (pNP-PO₄) to p- nitrophenol (pNP)⁶⁶. The samples were lysed in 0.1% Triton X solution, then added to pNP-PO₄ solution (Sigma) and allowed to react for 30 minutes at 37⁰C. The reaction was terminated with 0.1 N NaOH (Sigma).

A quantitative Alizarin Red-S (Sigma) assay was used to measure mineralization, according to the method described by Puchtler et al.⁶¹. The samples (n=5) were first rinsed with PBS and then fixed in 70% ethanol for 60 minutes at 4⁰C to preserve the mineral. The samples were incubated with the dye solution (40 mM) for 10 minutes, before rinsing with deionized water and PBS. A 10% (w/v) cetylpyridinium chloride solution (CPC, Sigma) was added in order to reconstitute the Alizarin Red-S dye. The amount of Alizarin Red-S, which reflects calcium deposition, was determined at the absorbance wavelength of 570 nm.

End-Point Analysis : Histological Analysis

Histological analysis was performed to visualize extracellular matrix deposition and ALP activity. All samples were washed and fixed for 10 minutes in neutral formalin. Distribution of ALP activity was visualized using Fast Blue RR Salt and AS-MX Phosphate (Sigma) . After fixation, the samples were incubated with 300 μL of dye solution at room temperature for 30 minutes, and then rinsed with deionized water prior to viewing under light microscopy.

For mineral distribution, the samples were stained with 2% Alizarin Red-S for 1 hour and then rinsed with deionized water. In addition to ALP activity and mineralization, the deposition of glycosaminoglycans (GAG) was assessed histologically. After fixation, the samples were stained overnight with 1% Alcian Blue solution (Sigma) and imaged after PBS wash. Two samples (n=2) from each group were used for each histological stain.

Statistical Analysis

Results are presented in the form of mean ± standard deviation, with n equal to the number of samples analyzed. A two-way analysis of variance (ANOVA) was performed to determine the effects of T3 concentration and co-culturing conditions on total cell number, ALP activity and mineral deposition. Similarly, a two-way ANOVA was used to determine effects of combined T3 and PTHrP treatment on cell response. The Tukey-Kramer post-hoc test was used for all pair-wise comparisons, and significance was attained at p<0.05. All statistical analyses were performed using the JMP software (SAS, Cary, NC) .

RESULTS

Effect of T3 on Zonal Popula tions of Chondrocytes As the cultures were seeded at high density and maintained under serum-free conditions, total cell number remained relatively constant for the first four days of culture. Cell number increased after mineralization media was added at day 4 and peaked after one week for all groups examined, however no significant difference was found between groups. The ALP activity of single cultures of SZC and MZC did not respond to T3 stimulation and remained at basal levels. In contrast, ALP activity of DZC increased significantly following T3 stimulation (Fig. IA, p<0.05). Histological staining (Fig. 2B) confirmed these results, with the T3- stimulated DZC cultures exhibiting stronger staining intensity when compared to the DZC control.

Effect of Co-Culture on Chondrocyte Mineralization Potential

When DZC was co-cultured with SZC, the stimulatory effect of T3 on ALP activity was inhibited; with no significant increase in ALP measured in the SZC+DZC group over time (Fig. IB) . A similar response was found in the full thickness cultures (FC, Fig. 1C). As shown in Figure 2, unlike the DZC+MZC group, the co-culture of DZC with SZC completely inhibited the T3-induced increase in ALP activity at day 4. Histological staining (Fig. 2B) confirmed these quantitative findings.

Calcium deposition increased in the chondrocyte cultures after the addition of mineralization media at day 4 in all groups over time (Fig. 3). Stimulation of DZC with 100 nM T3 resulted in a significant increase in calcium deposition at day 7 compared to the control DZC that did not receive T3 (p<0.05). This increase in calcium deposition was not present after day 7, likely due to the termination of T3 stimulation at day 4. When DZC was co-cultured with SZC, no significant increase was found in calcium deposition regardless of T3 stimulation at any time. Similarly, the SZC-only, MZC-only and FC groups accumulated minimal mineral and T3 did not promote calcium deposition in these groups over time. These quantitative results were confirmed by Alizarin Red S staining (Fig. 3B), where DZC stimulated with 100 nM T3 exhibited higher staining intensity compared to DZC control at day 7.

Expression of marker genes characteristic of hypertrophic chondrocytes, including ALP, type X collagen and MMP-13, was determined following T3 stimulation. As expected, both the DZC-only and DZC+MZC co-cultured group showed an increase in gene expression for these hypertrophic markers with T3 stimulation (data not shown) . The expression for ALP at day 4 increased significantly for the DZC-only and DZC+MZC groups, while no increase in ALP was detected in the SZC+DZC group.

Mechanism of Cellular Interactions : Paracrine Effects

As shown in Figure 4, when conditioned media from the DZC group was added to DZC cultures (DZC→DZC), an increase in

ALP activity was measured, and this increase was significantly higher at 10 nM T3 when compared to the untreated DZC control (p<0.05). In contrast, the level of ALP activity in DZC cultures treated with SZC-conditioned media (SZC → DZC) was comparable to the un-stimulated controls. A similar response was observed when the DZC group was stimulated with the (co-cultured) -conditioned media (SZC+DZC→DZC) .

Since chondrocyte mineralization potential was suppressed in the SZC+DZC co-cultured group, gene expression for PTHrP, a known regulator of chondrocyte hypertrophy, was determined. As shown in Figure 5, PTHrP gene expression increased significantly when DZC were co-cultured with SZC. In contrast, the co-culture of DZC with MZC did not lead to increased PTHrP gene expression at day 4.

To further assess the role of PTHrP in mediating SZC and DZC interactions, DZC cultures stimulated with T3 were treated with exogenous PTHrP. As shown in Figure 6, when the DZC culture was treated with PTHrP (50 nM) , the previously observed increase in ALP activity following T3 stimulation was inhibited (Fig. 6A). These findings were corroborated by ALP staining results (Fig. 6B). Treatment with PTHrP also modulated the effect of T3 stimulation on the expression of markers of chondrocyte hypertrophy (Ihh, ALP, Type X collagen) in DZC cultures at day 4 (Fig. 7, p<0.05). Addition of PTHrP also inhibited the increase in ALP expression level for DZC cultures stimulated with 10 and 10OnM T3. Moreover, type X collagen expression level was significantly lower after treatment with PTHrP at 10 nM T3, while PTHrP had no significant effect on MMP13 expression level. Since PTHrP regulates chondrocyte maturation through the PTHrP-Zhh negative feedback loop⁵²'⁵⁵'⁶⁸, gene expression for Ihh was also determined in DZC cultures stimulated with T3. A dose-dependent increase in Ihh expression was measured for DZC cultures stimulated by 10 nM and 100 nM of T3. This increase was however inhibited when the DZC cultures were treated with 50 nM of PTHrP (p<0.05, Fig. 7) .

To further test the role of PTHrP in the interaction between SZC and DZC, a known PTHrP antagonist, PTH (7-34) was added to SZC+DZC co-culture concomitant with T3 stimulation. The PTHrP antagonist had no effect on cell proliferation and was able to block the inhibitory effect of SZC+DZC co-culture on DZC mineralization potential (Fig. 8). At 100OnM PTH (7-34), a significant increase in ALP activity was detected in SZC+DZC co-culture stimulated with T3. These findings were corroborated by ALP staining (data not shown) .

3-D Co-Culture Model

As shown in Figure 9, when SZC and DZC were co-cultured in the 3-D model, a significant decrease in DZC ALP activity was measured when compared with the DZC only control at day 7. Measured ALP activity decreased thereafter for all groups, which is characteristic of the role of ALP in chondrocyte mineralization. Since ALP is an early marker for cartilage calcification and acts as an enzyme to facilitate the hydrolyzation of organic phosphate, its activity will decrease after the onset of mineralization^69"

71

DISCUSSION

The objectives of this study were to determine the role of interactions between zonal populations of articular chondrocytes in post-natal regulation of mineralization and to elucidate the mechanisms governing these cellular interactions. To this end, direct co-culture models of cells derived from the three zones of articular cartilage (surface, middle, and deep) were established, and cellular interactions were found, especially those between chondrocytes derived from the surface and deep zones, which are critical for the inhibition of deep zone mineralization. Moreover, the regulation of chondrocyte mineralization is mediated by local paracrine factors such as PTHrP, most likely through the PTHrP-Ihh negative feedback loop reminiscent of endochondral ossification.

Stimulation with thyroid hormone significantly increased ALP activity in the deep zone chondrocytes and also during their co-culture with middle zone chondrocytes. In contrast, little change in ALP activity was found when deep zone chondrocytes co-cultured with surface zone chondrocytes were stimulated with T3. Gene expression for PTHrP was significantly higher in the co-culture of surface and deep zone chondrocytes, suggesting that PTHrP may be a key modulator of the interactions between these two cell populations. Moreover, exogenous PTHrP inhibited increases in deep zone chondrocyte ALP activity, while decreasing both type X collagen and Ihh expression in these cells. Further evidence supporting the role of PTHrP in mediating surface and deep zone chondrocyte interactions was observed in the blocking of PTHrP in the SZC+DZC co-culture group, which effectively reduced the inhibitory effect of SZC on DZC mineralization potential under T3 stimulation. The potential of PTHrP to regulate growth plate chondrocyte hypertrophy via the PTHrP-Ihh negative feedback loop is well established^52"56, as the presence of PTHrP promotes chondrocyte proliferation and suppresses the maturation of pre-hypertrophic chondrocytes, thereby preventing further mineralization. The findings of this study suggest that a similar mechanism may be implicated in the regulation of articular chondrocyte mineralization.

One limitation of the 2-D model is the dilution effect present in co-culture, as only half of the cell type is present when compared to single cultures. Therefore it is more meaningful to interpret our findings in the context of stimulation with T3 within the same experimental group. For example, DZC plus T3 measured over 100% increase in ALP activity when compared to the DZC control (Fig. 2), while T3 stimulation of MZC+DZC resulted in only a 50% increase over the MZC+DZC control. This lower increase in ALP activity for the MZC-DZC group is likely due to a dilution effect. By the same token, a similar dilution effect is expected when SZC is co-cultured with DZC. Surprisingly, no significant increase was found in SZC-DZC stimulated with T3, suggesting that the observed suppression of ALP activity is likely due to SZC-DZC interactions. This conclusion is further strengthened by the results of the 3- D model (Fig. 8), in which any dilution effect is eliminated by directly evaluating the ALP activity of DZC in co-culture. A similar suppression of the ALP activity was found in 2-D and 3-D co-culture, thus confirming that these zonal chondrocyte interactions are physiologically relevant and not artifacts of the 2-D culturing system. The inhibitory effect in 2-D co-culture however, occurred sooner and at higher magnitudes, as direct co-culture circumvents any potential diffusion limitations associated with the static 3-D system. Future studies will focus on utilizing the 3-D model to elucidate the specific roles of cell sub- populations in the regulation of articular chondrocyte mineralization.

During development, PTHrP is secreted by peri-articular chondrocytes of the epiphyseal growth plate⁵²'⁵⁵'⁵⁸'⁷²; recent studies have shown that post-natal PTHrP expression is localized towards the articular surface⁶²'⁷³. It is thus likely that in our co-culture model, the surface zone chondrocytes are responsible for secreting the PTHrP that led to the suppression of deep zone mineralization. However, the PTHrP expression and conditioned media results point to a more complex interaction. Surface zone chondrocyte-only cultures expressed relatively low levels of PTHrP and accordingly, conditioned media from these cells had no effect on deep zone chondrocyte ALP activity. The up-regulation of PTHrP was only measured in co-culture, which suggests that cellular interaction is a pre-requisite for PTHrP secretion; this is consistent with the active PTHrP-Ihh feedback loop present in endochondral bone formation.

The DZC autocrine interactions, which led to an increase in DZC ALP activity after treatment with DZC-conditioned media, may be directed by a similar mechanism as reported for pre- hypertrophic chondrocytes, which secrete Ihh to promote hypertrophy in adjacent chondrocytes independent of PTHrP during endochondral ossification ^57"59_ Based on these results, the conditioned media from surface and deep zone chondrocyte co-culture was expected to inhibit the ALP activity of deep zone chondrocytes. However, no significant decrease in ALP activity was measured following treatment with the co-cultured media. There are several possible explanations. The 2-D co-culture model permits autocrine and paracrine interactions, as well as physical contact between chondrocyte sub-populations. Preliminary results from our 3-D co-culture model suggest that the co-culture interactions are independent of cell-to-cell contact, as a similar suppressive effect on ALP activity was found in the segregated co-culture of surface and deep zone chondrocytes. Therefore, considering the dilution effect of co-culture (e.g. lower seeding density per cell type) coupled with limitations associated with conditioned media studies (e.g. nutrient depletion vs. dilution effects), these results suggest that the amount of PTHrP present in the conditioned media is not sufficient to suppress the ALP activity of the treated cultures. Yoshida et al. reported that a threshold PTHrP concentration is required for the suppression of growth plate chondrocyte hypertrophy⁵⁴. The conditioned media used here originated from day 4 co-cultures which are presently associated with a significant increase in PTHrP expression. Thus, media from a later time point would likely be more potent. Furthermore, as an active negative PTHrP-Ihh feedback loop is necessary for regulating chondrocyte mineralization, conditioned media study is inherently limited in its inability to fully mimic the in situ cellular communication occurring in co-culture.

Articular chondrocytes isolated from the deep, middle and surface zones exhibit distinct morphology and phenotypes⁵'⁶'⁸'²¹'²⁷. It is thus not surprising that communication exists between cells derived from surface and deep zones of cartilage, since cellular interactions are essential for structural organization and maintenance of the controlled matrix heterogeneity inherent in complex tissues and organs. The results of this study provide a functional rationale for the structural organization of articular cartilage into these three distinct zones and describe an important role for PTHrP in surface and deep zone chondrocyte interactions. Molecular transport increases from the articular surface to cartilage proper under dynamic loading⁴⁶'⁴⁷, and unloaded joints exhibit an accelerated tidemark advancement⁴⁹. Thus regulatory- molecules such as PTHrP secreted by the surface zone chondrocytes are most likely transported to deep zone chondrocytes during joint articulation and physiological loading.

These results suggest that in immature animals, the increased mineralization potential of deep zone chondrocytes induced by systemic factors such as thyroid hormone is regulated by surface zone through the PTHrP-IiIh feedback loop. A major difference between adult and immature articular cartilage resides in the characteristics of the calcified cartilage region. Published studies have reported that the calcified cartilage region is avascular, relatively impermeable, and acts as a barrier against subchondral bone in adult animals¹⁶'^74"77, while substantial vasculature that facilitates molecular transport has been measured across the calcified cartilage region in immature animals⁷⁸. The mechanism regulating the maintenance of the osteochondral interface in adult cartilage is not known. Based on the findings of this study, it is proposed that with age, the continued depletion of chondrocytes at the surface zone during articulation attenuates their regulation of deep zone chondrocytes. This decrease in overall secretion of regulatory molecules (e.g. PTHrP and others) permits deep zone chondrocyte mineralization and advancement of the calcification front into the non- mineralized cartilage region. A resident population of progenitor cells have been identified at the articular surface, periosteum as well as in the synovium⁷⁹'⁸⁰, thus these cells may replenish the population of surface zone chondrocytes. The regulation of deep zone chondrocyte mineralization is reactivated once a sufficient number of surface zone chondrocytes has been regenerated. This newly formed calcified matrix can then serve as the neo- osteochondral interface, separating the remaining articular cartilage from the underlying calcified cartilage and bone.

Interpretation of these cellular interaction findings may also be relevant for understanding the pathology of osteoarthritis. It is possible that the loss of surface zone chondrocytes following microtrauma associated with osteoarthritis may lead to tidemark duplication and the apparent advancement of the calcification zone through deep zone chondrocyte-mediated mineralization. Moreover, damage to the subchondral plate in osteoarthritis could re-expose the deep zone chondrocytes to systemic factors such as T3. In this case, if coupled with wear of the cartilage surface or depletion of surface zone chondrocytes, unchecked mineralization by deep zone chondrocytes can occur. High concentration of thyroid hormone has been shown to induce ectopic mineralization in full thickness cultures of adult chondrocytes⁵⁰. However, it has been reported that PTHrP level is elevated in both osteoarthritic and rheumatic joints⁸¹'⁸². It is likely that in osteoarthritis, the PTHrP- Ihh negative feedback look may be disrupted or compensatory mechanisms are responsible for the maintenance of structural organization in articular cartilage. Recent studies exploring osteoblasts and chondrocytes co- culture⁸³'⁸⁴ have suggested that heterotypic cellular communications may also be relevant for regulating articular chondrocyte mineralization.

The findings of this study are also significant in the context of current efforts in functional cartilage tissue regeneration. Engineered cartilage constructs incorporating a biomimetic zonal distribution of articular chondrocytes have been reported^85'87 and our findings provide additional rationale for such sophisticated mimicry. Furthermore, regeneration of the osteochondral interface on cartilage grafts could be critical for long-term graft stability and functional matrix organization post- implantation. For an engineered cartilage graft to be successful in vivo, a stable osteochondral interface is essential for graft-to-bone integration, biomimetic structural organization and in turn, its extended physiological function. Our results provide initial evidence that zonal chondrocyte cellular communication plays an important role in the regulation and maintenance of the calcified cartilage zone in articular cartilage, and this information can be utilized for the regeneration of a stable and functional osteochondral interface on tissue- engineered cartilage grafts.

CONCLUSION

Cellular communication between zonal sub-populations of articular chondrocytes regulates chondrocyte mineralization potential. Specifically, chondrocytes residing at the articular surface actively suppress mineralization by chondrocytes in the deep layer of articular cartilage. These results suggest that this suppression is regulated, at least in part, through PTHrP. The findings of this study represent the first reported investigation of the role of zonal cellular interactions in articular chondrocyte mineralization and provide a novel rationale for the zonal organization of articular cartilage. These findings also present a mechanism for the post-natal regulation of articular cartilage matrix organization and have implications for osteoarthritis as well as functional cartilage repair.

Experiment 4 : 3-D Culture Model for Studying Interactions Between Subpopulations of Chonodrocytes

BACKGROUND

Using the 2-D co-culture model previously discussed, it was found that cellular communication between zonal sub-populations of articular chondrocytes regulated articular cartilage mineralization. Specifically, chondrocytes residing at the articular surface actively suppress mineralization by chondrocytes in the deep layer of articular cartilage. These results also suggest that this suppression is regulated, at least in part, through parathyroid hormone related peptide (PTHrP). Interpretation of these 2-D results are limited due to the dilution effect on cell response associated with 2-D mixed coculture(Lu H.H. and Wang 2007). This experiment focused on delineating the role that each sub-population of articular chondrocyte plays in regulating articular cartilage mineralization in a 3-D co-culture model.

The objective of this experiment was to further examine the role of cellular communication between sub-populations of articular chondrocytes using a 3-D co-culture model (Fig. 5.1). During development, the rate at which the growth plate cartilage mineralizes is highly regulated. It has been shown that it is controlled via the parathyroid hormone-related peptide (PTHrP) and the Indian hedgehog (Ihh) negative feedback loop(Vortkamp 1996; Karp 2000; Yoshida 2001; Kobayashi 2002; Kronenberg 2006). During endochondral ossification, the hypertrophic and pre-hypertrophic chondrocytes secrete Ihh which promotes hypertrophy in adjacent chondrocytes(Stott and Chuong 1997; St-Jacques 1999; Karaplis 2001) and induces PTHrP production in immature periarticular chondrocytes(Vortkamp 1996; Kobayashi 2002; Kartsogiannis 1997; Lee 1995). In addition, various signaling molecules such as proteins belonging to TGF-β superfamily have been shown to play important role in cartilage mineralization during development (Ballock 1993; Mello and Tuan 2006; Bohme 1995). It is possible that similar to growth plate cartilage, mineralization near the osteochondral interface during articular cartilage development is also regulated by these signaling molecules. It was hypothesized that paracrine interactions between chondrocytes residing at the surface and deep zones of articular cartilage regulate the rate of cartilage mineralization.

MATERIALS AND METHODS

Cells and Cell Culture

Primary bovine articular chondrocytes were used in this study. The cells were isolated from the knee joints of neonatal calves (~1 week old) obtained from an abattoir (Fresh Farm's Beef, Inc., VT). Due to differences in cartilage thickness between animals and isolation sites, a standardized protocol was developed based on published reports(Freeman 1979; Wong 1996; Sun and Kandel 1999). Specifically, the 10% (by height of the full thickness articular cartilage) closest to the articular surface was considered to be the surface zone, the middle 60% by height was deemed the middle zone, and the 30% closest to the subchondral bone was designated as the deep zone. Zonal populations of chondrocytes were then obtained by enzymatic digestions of tissue derived from each region following the methods of Hidaka et α/.(Hidaka 2006). Briefly, the tissue was minced and digested overnight in 0.1% w/v collagenase (Sigma) in DMEM (Cellgro-Mediatech, Herndon, VA) supplemented with 1% FBS (Atlanta Biologicals, Atlanta, GA) and 2% antibiotics (10,000 U/mL penicillin, 10 mg/mL streptomycin, Cellgro-Mediatech). Three sub-populations of chondrocytes were isolated from the tibiofemoral joint: the Surface Zone Chondrocytes (SZC), Middle Zone Chondrocyte (MZC), and Deep Zone Chondrocytes (DZC). A full thickness chondrocyte (FC) culture was established from digestion of a mixture of all isolated cartilage tissue pieces, without distinguishing any one cartilage zone from another.

3-D Co-Culture Model of Zonal Populations of Chondrocytes

A schematic of the 3-D co-culture model is shown in Figure 10. Specifically, cell-seeded agarose hydrogel discs were prepared as previously described (Mauck 2000). The deep zone chondrocyte suspension was then mixed with equal parts of 4% low gelling temperature agarose (Type VII, Sigma) to form a mixture with a final cell concentration of 20 million cells/mL in 2% agarose. After gelling, DZC discs (0 5mm x 2.4mm) were cored out (-0.9 million cells per disc) and maintained in fully-supplemented media for two to three days. The DZC agarose discs were then co-cultured with a monolayer of primary surface zone or middle zone chondrocytes in a 24 well plate seeded at concentration 0.8 million cells per well. The co-culture ratio of SZC and DZC was chosen to approximate that of the in vivo cell ratio as previous reports have shown that the cell density in the surface zone cartilage is approximately 2 to 3 times higher than that of deep zone cartilage(Jadin 2005; Eggli 1988). A 1.5% acellular agarose hydrogel spacer (lmm) separated the chondrocyte disc from the chondrocyte monolayer. The effect of co- culture on the response of DZC, SZC and MZC is examined at day 4, 7, 14 and 21 days.

Mechanism of Cellular Interactions

To evaluate the role of PTHrP in mediating the interaction between zonal chondrocyte populations, PTHrP expression was measured for all groups. In addition, to directly assess its effect on DZC mineralization, exogenous PTHrP was added to DZC cultures stimulated with T3. Specifically, 50 nM of PTHrP (1-37, Bachem, San Carlos, CA) was added along with T3 (0, and 50 nM) to confluent DZC cultures. Cultures of DZC stimulated by T3 but untreated with PTHrP served as controls. The effects of PTHrP on the mineralization potential of DZC were analyzed at 0, 2 and 4 days.

The role of PTHrP-mediated interaction between zonal chondrocyte population was further evaluated by blocking the actions of PTHrP using a PTHrP antagonist, PTH (7- 34, Bachem) (Horiuchi 1983). Exogenous PTH (7-34) at 10 μM was added to SZC+DZC co-cultures along with T3 (5OnM) under serum free conditions. The effects of the antagonist on ALP activity of SZC+DZC cultures were analyzed at day 4.

End-Point Analysis: Cell Proliferation

Cell proliferation (n=6) was determined by measuring total DNA per sample using the

PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturer's suggested protocol. Briefly, the samples were first rinsed with Phosphate Buffered Saline (PBS, Sigma) and the cells were lysed in 300 μL of 0.1% Triton X solution (Sigma). An aliquot of the sample (20 μL) was then added to 180 μL of the PicoGreen® working solution. Fluorescence was measured with a microplate reader (Tecan, Research Triangle Park, NC) with excitation and emission wavelengths of 485 and 535 nm, respectively. Total cell number in the sample was determined by converting the amount of DNA per sample to cell number using the conversion factor of 7.7 pg DNA/cell(Kim 1988).

End-Point Analysis: Gene Expression

Gene expression (n=5) was measured using reverse transcription followed by real-time polymerase chain reaction (PCR). The oligonucleotides were custom designed and the

® sequences are summarized in Table 6.1. Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA) extraction method. Each construct was homogenized in 0.75mL of TRIzol reagent and then mixed with 0.15mL of chloroform (Sigma). After centrifugation at 12,00OG for 15 minutes, the upper aqueous phase was then extracted and mixed with equal volume of 70% ethanol. The resulting solution was then purified with RN Aqueous (Applied Biosystems, Foster City, CA) according to the manufacturer's suggested protocol. The isolated RNA was reverse-transcribed into cDNA via the

TM

Superscript III First-Strand Synthesis System (Invitrogen) following the manufacturer's suggested protocol. The cDNA product was amplified and quantified

TM through real-time PCR using SYBR GreenER qPCR SuperMix (Invitrogen). The expression level of relevant genes were measured and normalized to the expression of the housekeeping gene β-actin. All reactions were run for 45 cycles using the iCycler iQ Real-Time PCR Detection System (BioRad). Normalized expression levels reported were calculated based on difference between threshold cycles, namely, the difference in threshold cycle values between the gene of interest and the housekeeping gene β-actin.

Table 2. List of oligonucleotide primers used

End-Point Analysis: Mineralization Potential

Cell mineralization potential was determined by examining ALP activity and mineral deposition. Quantitative ALP activity (n=6) was measured using an enzymatic assay based on the hydrolysis of p-nitrophenyl phosphate (pNP-PO4) to p-nitrophenol (pNP)(Teixeira 1995). The samples were lysed in 0.1% Triton X solution, then added to pNP-PO4 solution (Sigma) and allowed to react for 30 minutes at 37°C. The reaction was terminated with 0.1 N NaOH (Sigma).

End-Point Analysis: Histological Analysis

Histological analysis was performed to visualize extracellular matrix deposition and ALP activity. All samples were washed and fixed for 10 minutes in neutral formalin. Distribution of ALP activity (n=2) was visualized using AS-BI alkaline solution with fast blue BB (Sigma). After fixation, the samples were frozen sectioned at 7μm and mounted on a glass slide. The slide were then incubated with 300 μL of dye solution at room temperature for 30 minutes, and then rinsed with deionized water prior to viewing under light microscopy.

For other histological analyses samples where embedded in paraffin and sectioned. For mineral distribution samples were stained with 2% Alizarin Red-S or Von Kossa. For glycosaminoglycans (GAG) deposition samples were stained with 1% Alcian Blue solution. All histological stains were visualized with light microscope (Axiovert 25, Zeiss).

Statistical Analysis Results are presented in the form of mean ± standard deviation, with n equal to the number of samples analyzed. A two-way analysis of variance (ANOVA) was performed to determine the effects of T3 and co-culturing conditions on total cell number, ALP activity and gene expression. Similarly, a two-way ANOVA was used to determine effects of combined T3 and PTHrP treatment on cell response. The Tukey-Kramer post- hoc test was used for all pair- wise comparisons, and significance was attained at p<0.05. All statistical analyses were performed using the JMP software (SAS, Cary, NC).

RESULTS

Effect of Co-Culture on Chondrocyte Mineralization Potential

The ALP activity of DZC cultures increased significantly following T3 stimulation similar to what was observed in 2D culture. DZC co-cultured with SZC monolayer in the presence of T3 showed a significantly decrease in DZC ALP activity compared to DZC single culture stimulated with T3 (Fig. 6.2). Unlike the 2D results where SZC+DZC co- culture completely suppressed the effect of T3 on DZC mineralization, SZC+DZC co- culture in this current study only partially suppressed the effect of T3 on DZC mineralization potential. Co-culture of DZC with MZC in the presence of T3 had similar ALP activity compared to DZC single culture treated with T3 ALP (Fig. 11).

Confirming ALP activity, T3 stimulation significantly increased ALP gene expression in DZC cultures (Fig. 12). hi addition, T3 stimulation significantly increased Ihh, and PTHrP/PTH receptor expression level in DZC cultures, but had no effect on type X collagen, MMPl 3 and Runx2 expression level (Fig. 13). Co-culture significantly decreased ALP expression in DZC cultures. Moreover, co-culture significantly decreased Ihh, type X collagen and PTHrP/PTH receptor expression level compared to DZC cultured alone. Interestingly co-culture significantly increased MMP 13 expression level compared to DZC alone.

Mechanism of Cellular Interactions: Role of Parathyroid Hormone-Related Peptide

Gene expression of SZC showed a significant increase in PTHrP expression in co- cultures. A slight increase was seen in TGF-β3 expression level, while no differences were seen in other regulating molecules such as TGF-βl and 2 (Fig. 14).

To further assess the role of PTHrP on DZC hypertrophy, DZC discs were treated with exogenous PTHrP (1-36, 5OnM). As shown in figure 15, a significant decrease in ALP activity was observed in DZC cultures treated with PTHrP (Fig. 6.6a, p<0.05). Histological staining (Fig. 15(b)) confirmed these results, with the T3 -stimulated DZC cultures exhibiting stronger staining intensity when compared to the DZC control. In addition, PTHrP treated groups showed the lowest stain intensity. Treatment with PTHrP also modulated chondrocyte hypertrophic markers in DZC cultures. PTHrP significantly lowered ALP, Ihh, type X collagen, Runx2, and PTHrP/PTH receptor gene expression level in DZCs (Fig. 6.3 and Fig. 6.4). PTHrP had no effect on MMP 13 expression level with was consistent with previous 2-D data, hi addition, when PTHrP antagonist was added to SZC+DZC co-culture in the presence of T3, it completely eliminated the suppressive effect of SZC on DZC mineralization potential (Fig. 6.2).

DISCUSSION

The objective of this study was to validate the observed interactions between zonal populations of articular chondrocytes in post-natal regulation of mineralization found in the 2D model, and to further elucidate the mechanisms governing these cellular interactions. To this end, a 3-D co-culture model of cells derived from the three zones of articular cartilage (surface, middle, and deep) was established, and cellular interactions were found, especially those between chondrocytes derived from the surface and deep zones, which are critical for the inhibition of deep zone chondrocyte mineralization. Moreover, the regulation of chondrocyte mineralization is mediated by local paracrine factors such as PTHrP. The mechanisms governing these cellular interactions are most likely similar to those for regulating osteochondral bone formation.

Stimulation with thyroid hormone significantly increased ALP activity in deep zone chondrocytes, while co-culture with the surface zone chondrocytes significantly suppressed their ALP activity (Fig. 6.1). hi addition, co-culture also significantly decreased Ihh, type X collagen and PTHrP/PTH receptor expression levels. Similar to the 2D study, gene expression for PTHrP was significantly higher in the co-cultured surface zone chondrocytes. Moreover, exogenous PTHrP inhibited the increase in deep zone chondrocyte ALP activity, while decreasing both type X collagen, PTHrP/PTH receptor and Ihh expression in these cells. These findings further confirmed our previous 2-D co- culture results that chondrocytes reside on the surface regulate deep zone cartilage mineralization through PTHrP-ZAA feedback loop.

These results also suggest some alternative regulating mechanism(s) that might exist between surface and deep zone chondrocytes other than the PTHrP-ZAA feedback loop. Gene expression of MMP 13 and Runx2 did not correspond between co-culture and PTHrP treatment. Gene expression of MMP 13 was increased significantly in the SZC+DZC co-culture group, while exogenous PTHrP had no effect on MMP 13 expression, hi addition, PTHrP significantly lowered Runx2 expression level while coculture had no effect on Runx2. These data reveals the complexity that exists in surface zone and deep zone chondrocyte interaction and further studies are required to fully elucidate the underlying mechanisms.

CONCLUSION

Cellular communication between zonal sub-populations of articular chondrocytes regulates chondrocyte mineralization potential. Specifically, chondrocytes residing at the articular surface actively suppress mineralization by chondrocytes in the deep layer of articular cartilage. These results suggest that this suppression is regulated, at least in part, through PTHrP. The findings of this study further support the 2-D results and together these are the first reported investigation of the role of zonal cellular interactions in articular chondrocyte mineralization and provide a novel rationale for the zonal organization of articular cartilage. These findings also present a mechanism for the postnatal regulation of articular cartilage matrix organization and have implications for osteoarthritis as well as functional cartilage repair.

Experiment 5: 3-D Co-Culture of Surface and Deep Zone Chondrocytes BACKGROUND

Osteoarthritis (OA) is the predominant form of arthritis, with 21 million Americans suffering from this degeneratie condition. Pathological changes observed in OA include cartilage thining, largely due to ectopic mineralization of the deep cartilage layer. Understanding the mechanisms that control deep zone chondrocyte mineralization will therefore be important for devising treatments for OA. Experiment 1 and 2 reported that co-culture of surface zone chondrocytes (SZC) and deep zone chondrocytes (DZC) suppressed chondrocyte mineralization potentiall, even when the cultures were simulated by triiodothyronine (T3) , a known chondrocyte hypertrophy promoter. Moreover/ this process may be mediated by the parathyroid hormone-related petide (PTHrP) . These novel findings suggest that SZC-DZC interactions regulate chondrocyte calcification, however the response in as well as the effects of co-culture on the two chondrocyte subpopulations are not discernable in the mixed co-culture model. Moreover, the DZC monolayer and direct SZC-DZC physical contact in 2-D co-culture are non- physiologic. Therefore, the objective of this study is to decipher the relative response of SZC and DZC in co- culture, and determine their respective contributions to the reported suppression of chondrocyte mineralization using a 3-D, segregated, physiologically relevant co- culture model. This experiment will also evaluate if PTHrP facilitates zonal chondrocyte communication in 3-D co- culture. It is hypothesized that SZC regulates DZC hypertrophy and mineralization potential and this interaction is mediated by PTHrP. MATERIALS AND METHODS

Cells and Cell Culture: Articular chondrocytes (1-2 week old calves) were isolated via enzymatic digestions. The top 10% of the cartilage was considered to be SZC, the middle 1/3 was considered MZC, and the bottom 1/3 was considered to be DZC. 3-D Co-Culture Model: The interaction of SZC and DZC was examined in a 3-D co-culture where SZC were seeded at 7xlO⁵ cells/well and DZC were embedded in agarose discs (05x2.4mm, 9xlO⁵ cells/disc). An acellular agarose disc (h=lmm) was used to separate the DZC and SZC in co-culture (Fig. 16) . Control groups include SZC monolayer and DZC disc alone. All cultures were grown in ITS-supplemented DMEM for 1-day before T3 stimulation at OnM or 5OnM. Mechanism of SZC-DZC Interactions : To examine the role of PTHrP in regulating DZC chondrocyte mineralization potential, PTHrP antagonist (PTH, 7-34) was added in order to block PTHrP signaling in co-culture. Additionally, DZC cultures (with or without T3) were treated with exogenous PTHrP (5OnM) . End Point Analysis : Samples were collected on day 4, with total DNA (n=β) determined by Picogreen assay and alkaline phosphatase (ALP, n=6) activity evaluated by both enzymatic assay and histology. Gene expression (n=5) for the PTHrP receptor and hypertrophic markers (ALP, Ihh, Type X collagen) was determined by Real Time PCR.

RESULTS

SZC, DZC Response in Co-Culture - As expected, DZC stimulated by T3 exhibited a significant increase in ALP activity (Fig 16A) with T3. In contrast, when DZC was co- cultured with SZC, the T3-induced increase in its ALP activity was significantly reduced (Fig. 16A) . T3 stimulation promoted higher expression of hypertrophic markers such as ALP, Ihh, and PTHrP/PTH receptor (Fig. 17A, p<0.05), and their expression was significantly decreased in co-culture. SZC measured minimal ALP activity and in co-culture, SZC showed a significantly higher level of PTHrP gene expression compare to SZC control (Fig. 161B) . .Role of PTHrP - When PTHrP antagonist (PTH, 7-34) was added to the SZC+DZC co-culture group, the suppressive effect of SZC on DZC ALP activity was eliminated (Fig 17A) . Moreover, when DZC discs were treated with exogenous PTHrP, a significant decrease in ALP activity was found (Fig. 17B, C) . Co-culture significantly decreased ALP, Ihh, type X collagen and PTHrP/PTH receptor expression levels when compared to DZC disc alone (Fig. 17A) . Interestingly, a similar suppression of these chondrocyte hypertrophic markers was evident in DZC treated with exogenous PTHrP (Fig. 17A) .

DISCUSSION

Due to the specific organization of articular cartilage, chondrocytes isolated from deep zone, middle zone and surface zone exhibit distinct morphology and phenotypes. Our results extends the findings of Jiang et al. to a physiologically relevant 3-D model and demonstrate that cellular communication within different layers of articular cartilage may play a role in regulating cartilage mineralization at the osteochondral interface. The findings of this study suggest that interactions between cells residing in the surface and deep zones are important for the inhibition of deep zone chondrocyte mineralization. Moreover, the regulation of DZC mineralization is mediated by paracrine factors such as PTHrP. The signaling mechanisms governing these cellular interactions are not yet known. It is likely that the PTHrP-IMi feedback loop which is critical in controlling calcification at the growth plate during development may also play a role in post-natal regulations of articular cartilage mineralization. By elucidating the mechanisms of articular chondrocyte mineralization, potential methods to prevent etopic cartilage mineralization in OA can be developed. Moreover, our findings are relevant for osteochondral tissue engineering, as regulating zonal chondrocyte interactions in graft design may promote the formation of a stable osteochondral interface on these grafts.

Experiment 6 : PTHrP Release Study

BACKGROUND

The goal of this study is to determine the release profile of parathyroid hormone related to protein (PTHrP) from alginate beads.

MATERIALS AND METHODS

Two groups (experimental and control) of Alginate beads will be examined for PTHrP release concentration over time. Bead size and wet weight will also be determined.

The experimental group contains 2% medium viscosity alginate beads with 200 nM PTHrP. The control group contains only 2% medium viscosity alginate beads.

Alginate beads are fabricated by: 1) dropping alginate mixture with or without PTHrP into Cacl₂ using syringe through 26G needle, 2) scooping 4 beads into each well of 24-well plate, 3) designating 10 wells the control group, and 10 wells the experimental group, 4) adding ImL media to each well, the media comprising 1% P/S, 0.1% AmpB, 0.1% gentamycin, and DMEM.

For each group, the media is changed daily for 6 wells (n=6) while the other for the other four wells the media is left unchanged. (n=4)

Sample is collected at day 0,1,2,3,4,5,6,7, for the 6 wells where the media is changed daily. Sample is collected at day 7 for the 4 wells where the media is left unchanged. At each time point, the samples are collected by 1) collecting 500 μl media from samples and discard the rest of the media, 2 replacing the old media with ImI fresh media, and 3) freeze media samples until analysis. Each sample will be analyzed for PTHrP release concentration, as determined by Enzyme-Linked Immunosorbent Assay (ELISA) . Bead size and wet weight will also be determined for each sample .

EXPECTED OUTCOMES

PTHrP release from alginate beads will increase over time and the amount of release will be dependent on frequency of media change, i.e., changed daily imitates normal cell culture condition.

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Claims

What is claimed is:

1. A method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit alkali phosphtase (ALP) activity in the subject's deep zone chondrocytes.

2. The method of claim 1, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

3. The method of claim 1, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

4. The method of claim 1, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

5. The method of claim 1, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers .

6. The method of claim 1, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

7. The method of claim 1, wherein the PTHrP is administered via autologous chondrocyte implantation.

8. The method of claim 1, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

9. The method of claim 1, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

10. The method of claim 1, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

11. The method of claim 1, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

12. The method of claim 1, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

13. The method of claim 1, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

14. The method of claim 1, wherein the PTHrP administered is no more than 100 nm in size in size.

15. The method of claim 1, wherein the functional domain of the PTHrP is comprised in PTHrP.

16. The method of claim 1, wherein the functional domain of PTHrP comprises peptides 1-36.

17. The method of claim 1, wherein the subject is a mamma1.

18. The method of claim 1, wherein the subject is human.

19. A method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of type X collagen gene in the subject's deep zone chondrocytes.

20. The method of claim 19, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

21. The method of claim 19, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

22. The method of claim 19, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

23. The method of claim 19, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

24. The method of claim 19, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

25. The method of claim 19, wherein the PTHrP is administered via autologous chondrocyte implantation.

26. The method of claim 19, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

27. The method of claim 19, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

28. The method of claim 19, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

29. The method of claim 19, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

30. The method of claim 19, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

31. The method of claim 19, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

32. The method of claim 19, wherein the PTHrP administered is no more than 100 nm in size in size.

33. The method of claim 19, wherein the functional domain of the PTHrP is comprised in PTHrP.

34. The method of claim 19, wherein the functional domain of PTHrP comprises peptides 1-36.

35. The method of claim 19, wherein the subject is a mammal .

36. The method of claim 19, wherein the subject is human.

37. A method for inhibiting articular cartilage mineralization in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the Indian Hedgehog (Ihh) gene in the subject's deep zone chondrocytes, and thereby inhibit mineralization into calcium phosphate.

38. The method of claim 37, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

39. The method of claim 37, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

Ill

40. The method of claim 37, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

41. The method of claim 37, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

42. The method of claim 37, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

43. The method of claim 37, wherein the PTHrP is administered via autologous chondrocyte implantation.

44. The method of claim 37, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

45. The method of claim 37, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

46. The method of claim 37, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

47. The method of claim 37, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

48. The method of claim 37, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

49. The method of claim 37, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

50. The method of claim 37, wherein the PTHrP administered is no more than 100 nm in size in size.

51. The method of claim 37, wherein the functional domain of the PTHrP is comprised in PTHrP.

52. The method of claim 37, wherein the functional domain of PTHrP comprises peptides 1-36.

53. The method of claim 37, wherein the subject is a mammal .

54. The method of claim 37, wherein the subject is human.

55. A method for preventing osteoarthritis in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP).

56. The method of claim 55, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

57. The method of claim 55, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

58. The method of claim 55, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

59. The method of claim 55, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

60. The method of claim 55, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

61. The method of claim 55, wherein the PTHrP is administered via autologous chondrocyte implantation.

62. The method of claim 55, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

63. The method of claim 55, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

64. The method of claim 55, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

65. The method of claim 55, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

66. The method of claim 55, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

67. The method of claim 55, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

68. The method of claim 55, wherein the PTHrP administered is no more than 100 nm in size in size.

69. The method of claim 55, wherein the functional domain of the PTHrP is comprised in PTHrP.

70. The method of claim 55, wherein the functional domain of PTHrP comprises peptides 1-36.

71. The method of claim 55, wherein the subject is a mammal .

72. The method of claim 55, wherein the subject is human.

73. A method for treating osteoarthritis in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP), thereby preventing osteoarthritis in the subject.

74. The method of claim 73, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

75. The method of claim 73, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

76. The method of claim 73, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

77. The method of claim 73, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

78. The method of claim 73, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

79. The method of claim 73, wherein the PTHrP is administered via autologous chondrocyte implantation.

80. The method of claim 73, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

81. The method of claim 73, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

82. The method of claim 73, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

83. The method of claim 73, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

84. The method of claim 73, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

85. The method of claim 73, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

86. The method of claim 73, wherein the PTHrP administered is no more than 100 nm in size in size.

87. The method of claim 73, wherein the functional domain of the PTHrP is comprised in PTHrP.

88. The method of claim 73, wherein the functional domain of PTHrP comprises peptides 1-36.

89. The method of claim 73, wherein the subject is a mammal .

90. The method of claim 73, wherein the subject is human.

91. A method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit alkaline phosphatase (ALP) activity in the subject's chondrocytes .

92. The method of claim 91, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

93. The method of claim 91, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

94. The method of claim 91, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

95. The method of claim 91, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

96. The method of claim 91, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

97. The method of claim 91, wherein the PTHrP is administered via autologous chondrocyte implantation.

98. The method of claim 91, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

99. The method of claim 91, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

100. The method of claim 91, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

101. The method of claim 91, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

102. The method of claim 91, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

103. The method of claim 91, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

104. The method of claim 91, wherein the PTHrP administered is no more than 100 nm in size in size.

105. The method of claim 91, wherein the functional domain of the PTHrP is comprised in PTHrP.

106. The method of claim 91, wherein the functional domain of PTHrP comprises peptides 1-36.

107. The method of claim 91, wherein the subject is a mammal .

108. The method of claim 91, wherein the subject is human.

109. A method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the type X collagen gene in the subject's chondrocytes.

110. The method of claim 109, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

111. The method of claim 109, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

112. The method of claim 109, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

113. The method of claim 109, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers .

114. The method of claim 109, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

115. The method of claim 109, wherein the PTHrP is administered via autologous chondrocyte implantation.

116. The method of claim 109, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

117. The method of claim 109, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

118. The method of claim 109, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

119. The method of claim 109, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

120. The method of claim 109, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

121. The method of claim 109, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

122. The method of claim 109, wherein the PTHrP administered is no more than 100 nm in size in size.

123. The method of claim 109, wherein the functional domain of the PTHrP is comprised in PTHrP.

124. The method of claim 109, wherein the functional domain of PTHrP comprises peptides 1-36.

125. The method of claim 109, wherein the subject is a mammal .

126. The method of claim 109, wherein the subject is human.

127. A method for promoting articular cartilage repair in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP) effective to inhibit expression of the Ihh gene in the subject's chondrocytes and thereby inhibit mineralization into calcium phosphate.

128. The method of claim 127, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

129. The method of claim 127, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

130. The method of claim 127, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

131. The method of claim 127, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

132. The method of claim 127, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

133. The method of claim 127, wherein the PTHrP is administered via autologous chondrocyte implantation.

134. The method of claim 127, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

135. The method of claim 127, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

136. The method of claim 127, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

137. The method of claim 127, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

138. The method of claim 127, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

139. The method of claim 127, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

140. The method of claim 127, wherein the PTHrP administered is no more than 100 nm in size in size.

141. The method of claim 127, wherein the functional domain of the PTHrP is comprised in PTHrP.

142. The method of claim 127, wherein the functional domain of PTHrP comprises peptides 1-36.

143. The method of claim 127, wherein the subject is a mammal .

144. The method of claim 127, wherein the subject is human.

145. A method for diminishing damaging effects of physical activity on a joint in a subject comprising administering to a deep zone of articular cartilage of the subject an amount of a functional domain of parathyroid hormone-related peptide (PTHrP).

146. The method of claim 145, wherein said articular cartilage includes said deep zone and a surface zone, and said PTHrP is administered to said deep zone.

147. The method of claim 145, wherein the PTHrP administered has a sustained presence in said deep zone of said articular cartilage of said subject.

148. The method of claim 145, wherein the PTHrP is administered as a component of a composition which comprises a pharmaceutically acceptable carrier.

149. The method of claim 145, wherein the PTHrP is administered via materials selected from the group comprising microspheres, alginate beads, hydrogels, and degradable polymers.

150. The method of claim 145, wherein the PTHrP is administered via local injection into said deep zone of said articular cartilage of said subject.

151. The method of claim 145, wherein the PTHrP is administered via autologous chondrocyte implantation.

152. The method of claim 145, wherein the PTHrP is administered via a scaffold apparatus of single or multiple phases, wherein at least one of the phases corresponding to cartilage is impregnated with PTHrP.

153. The method of claim 145, wherein the PTHrP is administered by transporting said PTHrP from a surface zone of said articular cartilage to said deep zone of said articular cartilage via mechanical loading.

154. The method of claim 145, wherein the PTHrP is administered to limit chondrocyte mineralization in said deep zone of said articular cartilage of said subject .

155. The method of claim 145, wherein the PTHrP is administered to mediate interactions between said deep zone and a surface zone of said articular cartilage of said subject.

156. The method of claim 145, wherein an amount of the PTHrP administered into said deep zone of said articular cartilage is in a range of 0.1-150 nM.

157. The method of claim 145, wherein the PTHrP is administered into said deep zone of said articular cartilage of said subject in a plurality of doses over a period of time.

158. The method of claim 145, wherein the PTHrP administered is no more than 100 nm in size in size.

159. The method of claim 145, wherein the functional domain of the PTHrP is comprised in PTHrP.

160. The method of claim 145, wherein the functional domain of PTHrP comprises peptides 1-36.

161. The method of claim 145, wherein the subject is a mammal .

162. The method of claim 145, wherein the subject is human.

163. A scaffold apparatus for inhibiting articular cartilage mineralization in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject .

164. The apparatus of claim 163, wherein at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes .

165. The apparatus of claim 163, wherein one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impreganted with PTHrP.

166. The method of claim 163, wherein the subject is a mammal .

167. The method of claim 163, wherein the subject is human.

168. A scaffold apparatus for preventing osteoarthritis in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

169. The apparatus of claim 168, wherein at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes .

170. The apparatus of claim 168, wherein one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impregnated with PTHrP.

171. The method of claim 168, wherein the subject is a mammal .

172. The method of claim 168, wherein the subject is human.

173. A scaffold apparatus for treating osteoarthritis in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

174. The apparatus of claim 173, wherein at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes .

175. The apparatus of claim 173, wherein one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impregnated with PTHrP.

176. The method of claim 173, wherein the subject is a mammal .

177. The method of claim 173, wherein the subject is human.

178. A scaffold apparatus for promoting articular cartilage repair in a subject, wherein said apparatus comprise multiple phases corresponding to respective zones of the articular cartilage of the subject.

179. The apparatus of claim 178, wherein at least one of said multiple phases is seeded with chondrocytes selected from the group comprising surface zone chondrocytes, middle zone chondrocytes and deep zone chondrocytes .

180. The apparatus of claim 178, wherein one of the multiple phases of the scaffold apparatus is seeded with deep zone chondrocytes, and the phase seeded with deep zone chondrocytes is further impregnated with PTHrP.

181. The method of claim 178, wherein the subject is a mammal .

182. The method of claim 178, wherein the subject is human.