WO2013136089A1 - Polymer-glass composite material - Google Patents

Polymer-glass composite material Download PDF

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Publication number
WO2013136089A1
WO2013136089A1 PCT/GB2013/050663 GB2013050663W WO2013136089A1 WO 2013136089 A1 WO2013136089 A1 WO 2013136089A1 GB 2013050663 W GB2013050663 W GB 2013050663W WO 2013136089 A1 WO2013136089 A1 WO 2013136089A1
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WIPO (PCT)
Prior art keywords
glass
composite material
porous polymer
glass composite
polymer
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PCT/GB2013/050663
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French (fr)
Inventor
Mark Bradley
Gouher Rabani
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The University Court Of The University Of Edinburgh
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Publication of WO2013136089A1 publication Critical patent/WO2013136089A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56Porous materials, e.g. foams or sponges
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817Cartilage-forming cells, e.g. pre-chondrocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3821Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/02Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/06Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/24Materials or treatment for tissue regeneration for joint reconstruction

Definitions

  • the invention relates to a composite material for use as a tissue scaffold, comprising a biodegradable polymer component and a soluble glass component.
  • the material is particularly useful as a scaffold to promote cartilage and bone growth, for example when implanted into a subject.
  • biodegradable polymeric materials to function as scaffolds to promote tissue repair, for example as an alternative to a bone or tissue graft.
  • Biocompatible polymeric materials may be selected both for their resistance to contamination with microorganisms (to minimize post operative infection), physical characteristics (e.g. similarity to the tissue which is to replace the implant) and the rate of biodegradation, in comparison to the rate of cell growth and attachment in vivo.
  • performance across the full range of properties of known materials is still not comparable to grafted tissue. Therefore, the use of synthetic scaffolds made from biodegradable polymers is still generally limited to situations where autograft or allograft procedures are not possible.
  • a porous polymer-glass composite material comprising;
  • a porous polymer- glass composite material for use in a method of treatment by surgery comprising; at least 80wt% of a biodegradable organic polymeric material; and
  • porous polymer-glass composite material comprises around, or less than, 20wt% of the water soluble glass.
  • the polymer material and the water soluble glass may constitute less than 100wt% of the composite material and the porous polymer- glass composite material may comprise one or more other materials, in addition to the polymer material and the water soluble glass (for example one or more biocompatible excipients or filler materials).
  • the porous polymer-glass composite material is for use as a tissue scaffold, in a method of surgery.
  • the porous polymer-glass composite is particularly useful for promoting bone growth or repair, and or for promoting cartilage growth or repair. It has been found that the stiffness of the porous polymer-glass composite material is improved, in relation to a porous polymeric material alone, when the water soluble glass is present in an amount up to around 20wt%, and that the stiffness of the composite materials having greater amounts of glass is reduced. It has also been found that porous polymer-glass composite materials having larger amounts of water soluble glass do not deform elastically at low strains.
  • porous polymer-glass composite materials comprising less than around 20wt% (and preferably around 5wt%) of water soluble glass deform elastically, or near elastically, at low strains (i.e. from zero strain to at least around 10-15% strain, and typically between 0%-30% strain).
  • the material deforms elastically up to and in some cases beyond 50% strain.
  • the porous polymer-glass composite material comprises less than around 10wt% of water soluble glass.
  • the porous polymer-glass composite material comprises around 5wt% of water soluble glass.
  • the optimum amount of water soluble glass is 5wt%; i.e. the highest compressive Young's Modulus is observed for porous polymer-glass composite material comprising around 5wt% of water soluble glass.
  • the water soluble glass is not a silicate glass.
  • the porous polymer-glass composite material of the present invention preferably makes use of a biocompatible water soluble glass material which, over a period of time, dissolves completely in vivo and is cleared completely from a patient's system, and which is silicon free (or substantially silicon free).
  • the water soluble glass is a water soluble phosphate glass.
  • the phosphate glass comprises zinc or strontium.
  • Zinc is a solution rate modifier and it has been found that phosphate glass comprising an amount of zinc advantageously reduces the dissolution rate of the water soluble phosphate glass.
  • zinc is present in an amount of zinc oxide less than 10 mole% of the total oxides.
  • Zinc may be present in an amount of zinc oxide between 1 -6 mole% of the total oxides, and most preferably in an amount of zinc oxide less than 5 mole% of the total oxides.
  • Strontium when present may be present in the same amount as defined for zinc above.
  • the phosphate glass material may be a A b (M l 2 0) x (M"0) y (P 2 0 5 ) z material, wherein; M 1 is a metal from group I of the periodic table;
  • M" is a divalent metal, preferably from group II of the periodic table
  • A is one or more further materials, typically oxide materials, and b > 0
  • M" is divalent metal from other than group 2 of the periodic table and may for example be zinc.
  • the phosphate glass material may comprise an amount of one or more further materials A, which are typically metal or metalloid oxide materials, and thus b>0.
  • the phosphate glass may further comprise boron, silver and/or copper, and most preferably zinc. Therefore it may be that x+y+z ⁇ 1 .
  • the phosphate glass is a sodium-calcium-phosphate glass, most preferably further comprising zinc.
  • the water soluble glass may be in the form of a powder, as flakes or as fibres.
  • the water soluble glass is in the form of flakes or fibres, or any other particulate form having a comparable particle dimension, so as to provide a comparable ratio of surface area to mass of glass.
  • Flakes or fibres of glass material may be mixed with the biodegradable polymer material to provide a composite material having a uniform composition, and it has been found that flakes or fibres of glass advantageously have a slower dissolution rate than glass in the form of a powder. Accordingly, cell proliferation into the composite material, which is known to be promoted by the presence of the glass material, occurs over a longer period of time and cytotoxicity resulting from high rates of dissolution in vivo, is also reduced.
  • the particles of water soluble glass have an average dimension of between 5 ⁇ -50 ⁇ .
  • the water soluble glass may comprise glass filaments, the filaments having an average diameter of 5 ⁇ -50 ⁇ , or the water soluble glass may comprise glass flakes having an average thickness of 5 ⁇ -50 ⁇ .
  • the particles may have an average smallest dimension of between 1 0 ⁇ -30 ⁇ , and more preferably between 1 5 ⁇ -25 ⁇ .
  • the dissolution rate of the water soluble glass is preferably less than 0.05 mg.cm “2 .hr “1 and more preferably less than 0.01 mg.cm “2 .hr “1 . In a preferred embodiment, the dissolution rate of the water soluble glass is around 0.005 mg.cm “2 .hr “1 .
  • dissolution rate of a material we mean the mass of the material dissolved per hour in pure water at standard temperature and pressure. Dissolution rate may be expressed per unit of surface area and per unit mass of the material, and so be a property of composition of the material.
  • the composition of the soluble glass for example the amount and type of solution rate modifiers (for example zinc) to provide a material having a predetermined dissolution rate (per mg of material, per cm 2 of material surface). It is also known to vary particle dimensions (e.g. filament diameter) of soluble glass, to vary the surface area per gram of material, so as to vary the rate at which the material dissolves.
  • solution rate modifiers for example zinc
  • particle dimensions e.g. filament diameter
  • the porous polymer-glass composite material may comprise any suitable type of biodegradable polymeric material, and may comprise a single polymer or copolymer, or a mixture of one or more polymers or copolymers.
  • the porous polymer- glass composite material comprises biodegradable polyester material.
  • the porous polymer-glass composite material comprises polycapro lactone (PCL) and/or polylactic acid (PLA), or more preferably poly-L-lactic acid (PLLA).
  • the polymeric material comprises a mixture of PCL and PLLA, preferably in a ratio of between around 70/30 and 90/1 0, or more preferably in a ratio of or around 80/20, of PCL/PLLA by mass.
  • the polymeric material comprises polyglycolic acid, polyglycolactide or one or more polysaccharides.
  • the polymer-glass composite material comprises an open interconnected pore network, so as to facilitate cell penetration, for example in use of the composite material as a tissue scaffold.
  • the average pore size is in the range of 1 00 ⁇ -700 ⁇ , and more preferably or the range of from 250 ⁇ -500 ⁇ .
  • the pore size distribution of the pores is narrow.
  • the sizes of 90%, or more preferably 95% or more, of pores lie in the range from 1 00 ⁇ -700 ⁇ , or more preferably 1 00 ⁇ -700 ⁇ , or 250 ⁇ -500 ⁇ .
  • a porous polymer-glass composite material having an appropriate microporosity, and interconnectivity of pores enables rapid vascularisation and ingrowth to occur in vivo.
  • pore interconnectivity facilitates resorption of the composite material over the 6- 1 8 month tissue growth/regrowth period, in use of the material as a tissue scaffold.
  • Porous polymer-glass composite materials may be prepared by a variety of methods, including mixing of glass into a polymer solution, or mixing of glass into a monomeric solution, and performing polymerisation in situ. The solvent may then be rapidly evaporated (e.g. at elevated temperature and then at elevated temperature under vacuum), so as to form pores in the polymeric material.
  • very limited pore size control is possible by this method.
  • the porous polymer-glass composite material is obtained by a method of adding the glass to a polymer solution (of a first solvent) further comprising a soluble porogen, i.e. a particulate material having a high dissolution rate, in comparison to either the polymeric material or the glass, in a second solvent.
  • a soluble porogen i.e. a particulate material having a high dissolution rate
  • the porogen is water soluble, and may for example be a salt, e.g. sodium chloride, PEG, or a sugar, or any other suitable material having a much higher solubility rate in water than the glass or the polymeric material.
  • the first solvent may then be removed (e.g. by drying, under vacuum, or freeze-drying) and the polymer-glass composite material washed in the second solvent, to dissolve the porogen.
  • the average size and size distribution of the pores which are left when the porogen has been removed reflect the particle size distribution of the porogen, and the porosity of the polymer-glass composite material reflects the amount of porogen used. Therefore, preparation of the polymer-glass composite material by a method using porogen enables both the pore size distribution and the porosity of the composite to be controlled with greater accuracy than has been previously possible.
  • An alternative method is to employ thermally induced phase separation (as described in La Carrubba et al International Journal of Material Forming, 2008, vol 1 ., No. 1 , Supp/1 p 619-622) to induce porosity and this may be used with and without salt leaching in combination with the glass.
  • the composite material typically has a porosity of 60 - 85%.
  • the porous polymer-glass composite material is for use as a tissue scaffold.
  • the porous polymer-glass composite material is for use as a bone or cartilage scaffold.
  • cells for example chondrocytes or osteoblasts
  • the water soluble glass dissolves, and the polymeric material biodegrades and is replaced by cells.
  • the materials of the present invention can be adapted to biodegrade and dissolve over a time period compatible with rate of growth or regrowth of a particular tissue type. For example, for bone or cartilage tissue, growth or regrowth typically occurs over a 6-18 month period.
  • the porous polymer-glass composite material is for use as a bone scaffold, to promote bone growth or regrowth and/or repair, and is entirely resorbed in vivo over a 10-12 month period.
  • the porous polymer-glass composite material may further comprise cells within pores of the porous polymer-glass composite material.
  • the porous polymer-glass composite material may be seeded with cells, such as human or animal cells.
  • the polymer-glass composite material may be seeded with cells in vitro and then implanted into a subject, to thereby increase the rate of tissue regrowth.
  • the cells are osteoblasts (bone cells) or chondrocytes (cartilage cells).
  • the cells are mesenchymal stem cells such as adipose derived mesenchymal stem cells.
  • a method of preparing a porous polymer-glass composite material comprising the steps of:
  • porogen material has a low dissolution rate in the first solvent (in comparison to the polymeric material), or is more preferably substantially insoluble in the first solvent.
  • the polymeric material has a low dissolution rate (in comparison to the porogen material), or is more preferably substantially insoluble, in the second solvent.
  • the glass is a soluble glass and has a low, or very low, dissolution rate in the first solvent, or is substantially insoluble in the first solvent.
  • the soluble glass material is soluble in the second solvent, and the dissolution rate of the particulate porogen material in the second solvent is greater than the dissolution rate of the glass in the second solvent.
  • the dissolution rate of the porogen in the second solvent is much higher than the dissolution rate of the soluble glass in the second solvent by, for example, one or more orders of magnitude; e.g. two, or three or more orders of magnitude higher, or more preferably five to ten orders of magnitude higher.
  • the dissolution rate of the glass is less than 0.05 mg.cm “2 .hr “1 (e.g. around 0.005mg.cm “2 .hr “1 ) and the dissolution rate of the porogen material (for example sodium chloride) is in the range from 5-10mg.cm “2 .hr “1 (and in some embodiments greater than 10 mg.cm “2 .hr “1 ), in the second solvent.
  • the porogen material for example sodium chloride
  • the glass is a water soluble glass (for example a water soluble phosphate glass) and the second solvent is water.
  • the particulate porogen material may be any suitable water soluble particulate material having a much higher dissolution rate in water than the water soluble glass (for example a salt, such as sodium chloride, or a sugar).
  • the invention also extends to a structure composed of porous polymer-glass composite material (of the first or second aspect, or obtained or obtainable by the method of the third aspect), having a smallest dimension of greater than 20mm, or greater than 40mm.
  • the invention provides for larger porous polymer-glass composite structures than has previously been possible and the composite material may therefore be used in a wider range of surgical procedures.
  • the glass may be in the form or a powder, granules or flakes, or may be in the form of fibres.
  • the step of removing substantially all of the porogen material by washing the polymer-glass composite material in the second solvent produces a porous polymer-glass composite material having an interconnected open pore network.
  • the polymeric material may comprise a single polymer, or may comprise one or more polymers or co-polymers.
  • the polymeric material may comprise biodegradable polymeric material (such as one or a mixture of polyesters).
  • the first solvent may be a volatile organic solvent, such as an alcohol, ether or chlorinated solvent.
  • the first solvent may be chloroform.
  • Glasses, such as phosphate glasses are typically insoluble in organic solvents (and organic solvents in which selected polymeric material is soluble may be selected, in which glasses are substantially or completely insoluble).
  • the mixture of a solution of a polymeric material in a first solvent, a glass material, and a particulate porogen material is an admixture, and thus the components of the mixture may be added in any sequence.
  • the step of mixing typically comprises vortexing, or any method of mixing suitable to produce a mixture of uniform composition (i.e. in which the porogen material and glass are intimately mixed into, and distributed or dispersed within, the solution of polymeric material).
  • the first solvent may be removed by any suitable method, including by placing the mixture under vacuum, or placing the mixture at elevated temperature.
  • the method does not comprise placing the mixture under vacuum at elevated temperature.
  • the first solvent is removed by freeze-drying, that is to say freezing the mixture using dry ice, or liquid nitrogen or any other suitable cryogenic material, and placing the mixture under vacuum whilst frozen. It has been found that rapid removal of the first solvent, particularly if the mixture has not set or solidified, results in the formation of large pores and has a detrimental effect on the structural properties of the porous polymer-glass composite material which is produced. Freeze drying, or otherwise removing the first solvent without causing sudden vaporisation of solvent in the bulk of the material, prevents the formation of large pores and a wider pore size distribution.
  • the method preferably comprises removing substantially all of the first solvent by freeze drying the mixture of; a solution of polymeric material in the first solvent; the glass material, and; the particulate porogen material.
  • the method may be a method of preparing a porous polymer-glass composite material of the first aspect.
  • the method may further comprise seeding the porous polymer-glass composite material with cells, for example osteoblasts, chondrocytes or mesenchymal stem cells such as adipose derived mesenchymal stem cells.
  • cells for example osteoblasts, chondrocytes or mesenchymal stem cells such as adipose derived mesenchymal stem cells.
  • the method may further comprise differentiating mesenchymal stem cells, for example by use of an osteogenic medium (or a chondrogenic medium).
  • the invention also extends to a porous polymer-glass composite material obtained or obtainable by the method of the third aspect.
  • a method of treatment by surgery comprising implanting an implant into a subject, the implant comprising (or consisting of) a porous polymer glass composite material according to the first, second or third aspects.
  • the cells may for example be osteoblasts, chondrocytes or mesenchymal stem cells such as adipose derived mesenchymal stem cells.
  • the method comprises seeding the porous polymer-composite material with mesenchymal stem cells, differentiating the mesenchymal stem cells, for example by use of an osteogenic medium (or a chondrogenic medium), and implanting an implant comprising or consisting of the said composite material.
  • first feature is referred to herein as comprising (or comprises) a second feature
  • first feature may consist of, or consist essentially of, the second feature, or may include the second feature in addition to other features.
  • Figure 1 shows porous polymer-glass composite material prepared using solvent evaporation method (1 ) and (b) shows porous polymer-glass composite material prepared using porogen leaching method (2).
  • Figure 2 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0%, 5%, 20% and 30% of CG151 powder, in PCL/PLLA (80/20) polymeric material, as strain is reduced from 60% to 0%.
  • Figure 3 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank”), 5%, 10% and 30% of CG151 powder, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to destruction.
  • Figure 4 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank”), 5%, 20% and 30% of CG101 powder, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to 85%.
  • Figure 5 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank”), 5%, 10%, 20% and 30% of CG107 fibre, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to 85%.
  • Figure 7 shows results of an MTT assay of MG-63 cell proliferation on porous polymer glass composite materials after 24 hours, 48 hours and 1 week, normalised to results obtained from a polystyrene control sample.
  • Comparative results are also shown for 100% fibre glass samples ("CP.140” and "CP.107”) and a sample comprising CG107 fibres ground into a powder (“C.P.107p”).
  • Figure 8 shows the number of ADSCs on scaffold sample after 1 , 7, 14 and 21 days. Osteogenic media was added after 1 day. Results were obtained using a DNA assay, on a hydroxyaptite sample ("HA"), a blank sample of a porous PCL/PLLA (80/20) material ("BL”) and a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material ("5%”). The blank and polymeric materials were made using the porogen leaching method (2).
  • Figure 9 shows representative images of live cells (green) and dead cells (red), 24 hours post seeding of two sample scaffold materials seeded with ADSCs and treated with osteogenic media. The magnification scale bar represents 200 ⁇ .
  • the image labelled "BL” is a blank sample of a porous PCL/PLLA (80/20) material and the image labelled 5% is a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material.
  • the materials were made using the freeze drying and porogen leaching method (2).
  • Figure 10 shows representative images of RUNX2 staining of ADSC seeded scaffold materials treated with osteogenic media.
  • the magnification scale bar represents 100 ⁇ .
  • Images labelled "HA”, "BL” and “5%” are of materials similarly labelled in relation to Figure 8. Comparative images of ADSCs grown on culture plastic and treated with osteogenic media (image labelled "OS”) and ADSCs grown on culture plastic and not treated with osteogenic media (image labelled "NOR”), Images are also shown of MG63 seeded culture plastic samples, labelled MG63+ and MG63- ("+” and "- " designating samples with and without immunocytochemical staining).
  • Figure 1 1 shows representative images of Col1 a1 staining of ADSC seeded scaffold materials.
  • the scale bar represents 100 ⁇ . Samples are labelled as for Figure 10.
  • Figure 12 shows representative images of Osteocalcin staining of ADSC seeded scaffold materials. The scale bar represents 100 ⁇ . Samples are labelled as for Figure 10.
  • Figure 13 shows gene expression of Runx2, measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples were treated with osteogenic media after 1 day. Results are shown for a hydroxyaptite sample ("HA"), a blank sample of a porous PCL/PLLA (80/20) material (“BL”) and a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material (“5%”). Results are also shown for control samples of ADSCs grown on culture plastic and treated with osteogenic media (labelled "OS”) and ADSCs grown on culture plastic and not treated with osteogenic media (labelled "NO”)
  • Figure 14 shows gene expression of collagen type 1 (Col1 a1 ), measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples are labelled as for Figure 13.
  • Figure 15 shows gene expression of osteocalcin, measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples are labelled as for Figure 13.
  • Figure 16 shows alkaline phosphatase staining (dark blue) of ADSC seeded samples (column marked "+cells") 13 days after exposure to osteogenic media. Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
  • Figure 17 shows expanded views of ADSC seeded samples of Figure 16. Images are filtered to show alkaline phosphate staining (top row), DAPI staining (middle row) and both alkaline phosphate and DAPI staining (bottom row.) The scale image bar represents 200 ⁇ .
  • Figure 18 shows alizarin red staining of ADSC seeded samples (column marked "+cells”) after 14 days (13 days after exposure to osteogenic media). Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
  • Figure 19 shows Von Kossa staining (black) of ADSC seeded samples (column marked “+cells”) after 14 days (13 days after exposure to osteogenic media). Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
  • Figure 20 shows Von Kossa staining of ADSC seeded samples after 14 days (13 days after exposure to osteogenic media).
  • the magnification scale bar represents 200 ⁇ . Images are labelled as above.
  • Figure 21 shows DSC data for a porous glass polymer material containing PCL/PLLA (80/20) and 5% CG107, and prepared using porogen leaching method (2), before sterilisation.
  • Figure 22 shows DSC data for the porous glass polymer material containing PCL/PLLA (80/20) and 5% CG107, and prepared using porogen leaching method (2), after sterilisation with gamma radiation.
  • Figure 23 shows images of stained, sterilized samples 8 hours after seeding with ADSCs (A) without re-wetting (B) after re-wetting with PBS and (C) after re-wetting with denatured ethanol and PBS.
  • Figure 24 shows an attachment assay of ADSC cells comparing gamma sterilised dry sample, gamma sterilised PBS conditioned sample and IMS sterilised and PBS conditioned sample.
  • Figures 25(a)-(d) shows photographs of sheep 2 taken during in vivo testing.
  • Figure 26 shows representative microscopical pictures of implanted sites after 4 weeks, of (a) Left femur of sheep 1 , site 2 (b) right humerus of sheep 1 , site 5 (c) left femur of sheep 2, site 2 (d) left humerus of sheep 2, site 4.
  • Sodium Chloride was obtained from Fisher Scientific and sieved using a 500 micron and a 240 micron sieve.
  • a range of phosphate glass materials were obtained from Giltech Limited. Samples were provided as powders or fibres. Properties and approximate dissolution rates are set out in Table 1 .
  • CorGlaes samples are tertiary sodium calcium phosphate glasses.
  • CG140 and CG107 additionally comprise amounts of zinc, which is present as a solution rate modifier.
  • CorGlaes is a trade mark of Giltech Limited.
  • CG101 and CG151 were used as received in the powder form.
  • CG107 and CG140 fibre were flocculated and the fibres cut in to lengths of 0.8-1 .2cm.
  • fibres were also ground to form a powder (and these samples are designated CG140p and CG107p).
  • Porous polymer-glass composite materials were prepared by each of three methods.
  • An attachment and proliferation assay was carried out using MG-63 (human osteosarcoma) as a prototype of human bone cells.
  • a standard MTT assay was used to quantify the number of cell attachment and proliferation, as follows:
  • Samples were sterilised by autoclave, placed in a 6-well plate and washed extensively with sterile deionised water, sterile phosphate buffered saline solution (PBS) and Dulbecco's modified Eagle's medium (DMEM).
  • Washed MG-63 (Human osteosarcoma) cells (ca. 500,000 cells per well) were seeded in DMEM (500 ⁇ _) for 8 hours. The samples were then immersed in DMEM and incubated for a further 24 hours. The porous polymer-glass composite samples were then transferred to another 6-well plate.
  • the samples were then incubated for 24, 48 hours and 7 days (and media was changed every second day). The media was removed and the samples were washed with PBS to remove unattached cells, transferred to a new 6-well plate and then treated with trypsin/EDTA (2.5%, 200 ⁇ _) for 30 minutes to detach cells which were attached to the composite samples.
  • DMEM (1 mL) was added and the solution was drawn up into, and expelled from, a pipette, in order to distribute the detatched cells evenly.
  • the cells were replated on a 6 well plate 100 ⁇ _ ⁇ / ⁇ and incubated for 12 hours. Cells were then washed twice with PBS. All media was then replaced with a mixture of 50 ⁇ _ of fresh media (phenol red free) and 50 ⁇ _ of MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) in phosphate buffered saline (“PBS”) (1 mg/ml_) and the cells incubated for 4 hours at 37°C.
  • PBS phosphate buffered saline
  • ADSCs Human adipose derived mesenchymal stem cells
  • ADSCs were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% foetal calf serum (FCS), L-glutamine (2mM), penicillin (100 lll/mL) and streptomycin (100 ⁇ g/mL) (obtained from Sigma-Aldrich Ltd., Poole, UK), termed growth media.
  • DMEM Dulbecco's Modified Eagle medium
  • FCS foetal calf serum
  • L-glutamine (2mM) penicillin
  • streptomycin 100 ⁇ g/mL
  • HA hydroxyapatite material
  • BL polycaprolactone and poly-L-lactic acid
  • CP.107 porous polymer-glass composite material made by the freeze drying and porogen leaching method described above, comprising 5% CG107 in an 80:20 mixture of polycaprolactone and poly-L-lactic acid
  • ADSCs were also cultured on tissue culture plastic, with and without treatment by osteogenic media, in six of the remaining wells, in the same manner. ADSC seeded culture plastic samples are labelled OS (with osteogenic media) and NOR (without osteogenic media).
  • MG63 cells were also cultured on tissue culture plastic, without osteogenic media, in six of the remaining cells in the same manner. MG63 seeded culture plastic samples are labelled MG63+ and MG63- ("+" and "-" designating samples with and without immunocytochemical staining). Note that both MG63+ and MG63- labelled samples were DAPI stained, as discussed below. DNA extraction and precipitation
  • Samples were removed from culture wells, and placed in separate, sterile 1 .5ml_ Eppendorf tubes and 500 ⁇ of phenol added to each well. Each well was scraped with a pipette tip and then the phenol transferred to a sterile, 1 .5 ml Eppendorf tube. 100 ⁇ of sterile Dnase free water was added and each tube was then vortexed for 30 seconds. The tubes were then centrifuged at 2,000 rpm for 5 minutes and the aqueous phase transferred to a sterile 1 .5ml Eppendorf tube.
  • the precipitated DNA was collected by centrifugation of each sample at 10,000 rpm for 45 minutes.
  • the ethanol was removed and the precipitate (in the form of a pellet) was washed with 70%(v/v) ethanol and again centrifuged at 10,000 rpm for 15 minutes.
  • the ethanol was removed and the pellets allowed to air dry for 5 minutes.
  • the dried DNA was re-suspended in 10 ⁇ of sterile DNase free water and stored at -20 °C. Fixation
  • each well were washed three times with PBS and fixed in 100% methanol at -20 °C for 10 minutes, then washed in PBS again three times prior to staining.
  • Alkaline phosphatase activity was assessed by histochemical staining of samples prepared in a 24 well plate as discussed above. Alkaline phosphatase histochemical staining was performed using a commercial analytical test kit (Leukocyte Alkaline Phosphatase Kit, Sigma-Aldrich Ltd., Poole, UK) following manufacturer instructions. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected.
  • ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate were fixed as discussed above, washed thoroughly with distilled water and incubated in 2% Alizarin red solution (pH 4.3) for 2 minutes, and then washed three times in distilled water. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected.
  • Mineralised extra cellular matrix identification - phosphate (Von Kossa histochemistry)
  • ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate were fixed as discussed above, washed thoroughly with distilled water and incubated in 5% silver nitrate solution before being exposed to a 60-watt light bulb at a distance of approximately 5 cm for 1 hour. Following exposure, cells were washed in distilled water and incubated in 5% sodium thiosulphate solution for 5 minutes. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected.
  • RNA extraction and qRT-PCR Total RNA was isolated from human ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate, as discussed above. On days 1 , 7, 14 and 21 after seeding RNA was extracted using a commercial Trizol reagent (Invitrogen, Paisley, UK) and following maunfacturers instructions. (Trizol is a trade mark of Molecular Research Center, Inc.)
  • RNA samples were transcribed to cDNA using Superscript III First strand Synthesis SuperMix for RT-PCR (Invitrogen). (Superscript is a trade mark of Life Technologies Coproration).
  • Runx2 Staining ADSCs seeded on samples, as described above, were blocked with blocking buffer (10% goat serum, 1 % BSA and 0.1 % Triton X-100) for 1 hour at room temperature and then incubated overnight at 4 e C with Anti-human flunx-P/CBFAI antibody (obtained from R&D Systems, Abingdon, UK) at 1 :200 dilution in antibody diluent (a modified PBS formulation buffer with protein carrier and preservatives).
  • blocking buffer 10% goat serum, 1 % BSA and 0.1 % Triton X-100
  • Col1 a1 Staining ADSCs seeded on samples, as described above, were blocked with Dako protein block (obtained from Dako Limited) for 1 hour at room temperature and then incubated overnight at 4 e C with mouse monoclonal anti-collagen type I antibody (Sigma-Aldrich) at 1 :200 dilution in antibody diluent.
  • Dako protein block obtained from Dako Limited
  • mouse monoclonal anti-collagen type I antibody Sigma-Aldrich
  • Osteocalcin Staining ADSC seeded on samples, as described above, were blocked with blocking buffer for 45 min at room temperature and incubated overnight at 4 e C in anti-human osteocalcin antibody (obtained from R&D Systems, Abingdon, UK) at 1 :200 dilution in PBS.
  • DAPI Staining All samples were also stained with DAPI solution (4',6-diamidino-2- phenylindole solution), which binds strongly to all cells and is a blue fluorescent dye.
  • Samples of porous polymer-glass composite material prepared using the freeze-drying porogen leaching method discussed above, was used for in-vivo studies.
  • the samples comprised 5% CG107 in a polymeric material consisting of an 80:20 mixture of polycaprolactone:poly-L-lactic acid.
  • Each sheep was weighed and then injected with a thiopental-pentobarbital mixture (thiopental was obtained from Merial Animal Health Limited and sodium pentobarbital was obtained from Ceva Animal Health Ltd.) and atropine sulphate. The sheep were then placed under 0 2 -isoflurane inhalant aesthetic (0.5-4% mixture of AErrane, obtained from Baxter Healthcare Ltd. - "AErrane" is a trade mark of Baxter International Inc.) for continued general anaesthesia during the surgical procedure. Each sheep was also given a pre-operative analgesic treatment of buprenorphine and flunixin. As a prophylactic measure, pre-operative antibiotics (penicillin procaine and penicillin benzathin) were also intramuscularly administered.
  • pre-operative antibiotics penicillin procaine and penicillin benzathin
  • Selected surgical areas were clipped free of wool and the skin scrubbed with povidone iodine, wiped with 70% isopropyl alcohol, painted with povidone iodine solution and draped.
  • the surgical procedure was performed using standard aseptic techniques. Femoral defects were created as follows: a cutaneous incision was made on each latero-distal femoral condyle. The muscles were separated using blunt dissection in order to access the femur. The periostum was removed from the femoral epiphysis and metaphysis to expose the implant sites. Drilling was conducted (perpendicular to the bone surface) to create a cylindrical hole with a final diameter of 5-6mm and a depth of 10mm.
  • Humeral defects were created as follows: a skin incision over the shoulder joint was made from the acromion to the middle of the proximal third of the humerus on each lateral humeral major tubercle. Subcutaneous tissues and deep fascia was dissected and a proximal-distal incision of the deltoid muscle was made. The infraspinalis muscle was retracted caudally by blunt dissection. A drill guide was placed centrally in the groove of the humeral major tubercle. Drilling was conducted perpendicular to the bone surface to create a hole with a final diameter of 5mm and a depth of 10mm. Histopatholoqical analysis
  • One central cross section of each site was obtained by the microcutting and grinding technique described by Donath K, Brunner G., "A method for the study of undecalcified bone and that with attached soft tissues.”; J. Oral Pathol, 1 1 , 318-326, 1982.
  • Each section was be dehydrated in alcohol solutions of increasing concentration, cleared in xylene and embedded in polymethylmetacrylate (PMMA). The sections were then stained with Goldner Trichrome staining, for qualitative and semi-quantitative analysis. Quantitative and semi-quantitative Histological evaluation analysis
  • the performance and the local tissue effects evaluation is based on the qualitative and semi-quantitative histological analysis, according to the criteria set out in Tables 3 and 4.
  • porogen leaching method (2) yields a porous polymer- glass composite material with a narrow pore size distribution and does not produce any large voids ( Figure 1 b). Pore sizes ranged from 250-500 ⁇ and the pores were interconnected. The pore network thus facilitated tissue ingrowth and vascularisation, as discussed below.
  • composite materials may be prepared using other water soluble porogens, including other soluble salts and sugars.
  • the amount and size of crystals can be varied to alter pore size and distribution.
  • porous polymer-glass composite material In order to be suitable for use as a tissue scaffold, in particular for the promotion of bone growth or repair, the porous polymer-glass composite material must have sufficient structural integrity to be easily handled in theatre.
  • FIG. 2 shows stress-strain plots for samples comprising 0%, 5%, 20% and 30% CG151 , as strain is reduced from 60% to zero. All glass containing samples showed some degree of plastic deformation (i.e. the plots do not pass through (0,0)) and the degree of deformation increases with percentage glass content. Furthermore, the stiffness of the material comprising 5% CG151 was greater than other glass containing samples, and the sample without glass (i.e. the gradient of the stress-strain plots of figure 2 is greatest for the 5% CG151 sample).
  • Figure 3 shows stress-strain data measured on further samples comprising 0% ("blank"), 5%, 10% and 30% of CG151 powder, during destructive testing as strain was increased from 0% to 85%.
  • the material with 5% CG151 again showed greater stiffness than either the blank material (comprising 0% glass) or materials with a larger percentage of glass.
  • Table 5 shows a least squares linear fit of the data shown in Figure 3.
  • the parameter "a” provides an approximate measure of stiffness.
  • Table 6 shows a least squares linear fit of the data shown in Figure 4.
  • MG63 Attachment & Proliferation Assay In order to assess potential performance as a scaffold support for bone grown or repair/regrowth, the viability and proliferation of human bone cells within porous polymer-glass composite materials was assessed using the MTT assay described above. Porous samples were prepared according to the freeze drying and porogen leaching method (3), comprising PCL/PLLA (80/20) polymeric material and varying amounts of the glass materials set out in table 1 .
  • Figure 6 shows results of an MTT assay of MG-63 cell proliferation on materials comprising CG101 powder and CG151 powder (in the amounts of 0% ("blank"), 10%, 15%, 20% and 30%). Results are normalised against results obtained from a control sample of polystyrene. Assays were conducted after 24 hours, 48 hours and one week.
  • FIG. 7 shows similar results of MTT assays conducted on samples comprising CG140 fibres and CG107 (in both powered and fibre form). Assays were conducted on samples comprising 0% glass ("blank") and 20%, 30% and 100% CG140, and on samples comprising 5%, 20%, 30% and 100% CG107. The blank material containing no glass was seen to support both attachment and proliferation, in line with earlier reports.
  • Adispose derived mesenchymal stem cells are multipotent cells, capable of self renewal, and possess the potential to differentiate along several lineages, including differentiation into adipocytes, chondrocytes and osteoblasts (the main cell types in fat, cartilage and bone, respectively). The ease of expansion and osteogenic potential of these cells make them ideally suited to study the osteogenic properties of bone graft replacements in vitro.
  • Runx2 is a transcription factor required for osteogenic differentiation and its presence is indicative of osteoblast cells or of cells becoming, committed to the osteoblast lineage.
  • Figure 10 shows RUNX2 staining on cells attached to scaffolds or grown on tissue culture plastic.
  • Figure 1 1 shows col1 a1 staining on cells attached to scaffolds or grown on tissue culture plastic.
  • Col1 a1 (also known as collagen type 1 ) is the most abundant protein in bone (approx 30% of bone is protein, of this 90% is type 1 collagen). Mature collagen fibres can be clearly are seen on the seeded sample containing 5% CG107 and on the seeded sample without glass ("BL"). The amount of collagen present is similar to the positive control sample ("OS"). Collagen type I staining was also detected on the seeded hydroxyapatite ("HA”), however visual inspection confirmed that this was intracellular and not the mature form seen for the other samples.
  • OS positive control sample
  • HA hydroxyapatite
  • porous polymer-glass composite material comprising PCL/PLLA (80/20) polymer material containing 5% CG107, and porous PCL/PLLA (80/20) polymer without glass, both support osteoblast production of collage type 1 to a greater extent than hydroxyapatite.
  • Figure 12 shows osteocalcin staining on cells attached to scaffolds or grown on tissue culture plastic.
  • Osteocalcin is one of the most bone specific proteins, found in bone prior to mineralization. The strongest staining again was seen on seeded porous composite materials comprising PCL/PLLA (80/20) and containing 5% CG107. As can been seen in Figure 12, the staining for this sample was greater than that observed for the blank porous polymer sample ("BL"), the hydroxyapatite sample, and the control samples ("OS” and "NOR"). These results therefore also confirm that osteoblasts are present on the "5%” material and, in addition, indicate that the cells have produced producing an organic matrix upon the scaffold.
  • BL blank porous polymer sample
  • OS hydroxyapatite sample
  • NOR control samples
  • Alkaline phosphatase is an enzyme expressed by osteoblasts prior to mineralisation.
  • Leukocyte Alkaline Phosphatase Kit in the manner discussed above, alkaline phosphatase present in each of the samples was stained dark blue.
  • alkaline phosphatase was observed to be present on all seeded sample, after exposure to osteogenic media.
  • the porous PCL/PLLA 80/20 polymer material containing 5% CG107 showed strongest staining.
  • Calcium phosphate crystals appear once osteoblasts produce a mature matrix capable of supporting mineralisation, and may be stained using alizarin red dye.
  • Figure 18 shows alizarin red staining on samples on day 14.
  • the porous PCL/PLLA (80/20) polymer material containing 5% CG107 showed even staining over whole the whole sample scaffold, whereas the blank sample ("BL") of the same polymer material without glass showed uneven staining.
  • the hydroxyapatite sample showed deep staining throughout, due to its composition. The same result was observed for the sample which was not seeded. Von Kossa staining
  • FIGS 19 and 20 show dark staining in the centre of the porous composites material comprising PCL/PLLA (80/20) polymer material and 5% CG107. Similar staining was seen on the blank porous polymer sample (no glass), but the staining was not as strong as 5%.
  • porous polymer- glass composite material In order to be suitable for use as a tissue scaffold, it is vital that the porous polymer- glass composite material can be subjected to sterilization using standard industrial procedures and still retain the required physical properties. For this reason a sample of porous material containing PCL/PLLA (80/20) and 5% CG107 (and prepared using porogen leaching method (2)) was sterilised using gamma irradiation and the samples tested using differential scanning calorimetry.
  • Body weight data are presented in Table 9.
  • the body weight of sheep 1 slightly decreased (1 kg) between implantation and termination. This loss of body weight was low and not associated to clinical abnormality and was therefore considered as not clinically significant.
  • the non-implanted test article did not withstand the technical preparation and appeared fully dissolved in the embedding media.
  • the test article was dissolved but left footprints evoking an open porous material.
  • the pores of the test article were moderately invaded by a markedly vascularized fibromesenchymal tissue.
  • the fibromesenchymal tissue was infiltrated by macrophages and multinucleated giant cells of moderate grade, along with a slight grade of lymphocytes. Slight signs of material degradation, mediated by the phagocytizing cells were observed.
  • the level of local inflammation was likely influenced by the material degradation process. Direct bone-material contact could not be established as the test article material could not be observed. Bone ingrowth (defined here as newly formed bone of moderate grade) was frequently observed within the peripheral portion of the test article.
  • Figure 26 shows representative microscopal pictures of the implanted sites after 4 weeks. The regions of the pictures are labelled as follows:
  • porogen leaching method provides porous polymer-glass composite materials with an optimum porosity (narrower pore size distribution, elimination or large voids) and optimal handling properties (improved mechanical stiffness).
  • the materials should preferably comprise a soluble glass with a solution rate of less than 0.01 mg.cm " 2 .hr "1 . Solution rates at or below these values are such that the glasses resorb over a period of 6-18 months, which is ideal for tissue growth and regrowth. It has been shown that glasses with fast solution rates are not suitable for promotion of cell growth particularly bone cells.
  • CG107 is a particularly suitable soluble glass material, for use in tissue scaffolds.
  • porous polymer glass composite materials of the present invention are able to support osteogenic differentiation of multipotent cells to a greater extent than porous polymer materials without glass, or hydroxyapatite materials.
  • the materials of the present invention also promote greater attachment and proliferation of cells than porous polymer materials without glass, or hydroxyapatite materials.

Abstract

Disclosed is a porous polymer-glass composite material comprising at least 80wt% of a biodegradable polymeric material; and water soluble glass. The polymer-glass composite material may be prepared by porogen leaching, which provides for control over the size and size distributions of the pores. The mechanical properties and biocompatibility of the polymer-glass composite material enables its use as a tissue scaffold, to promote tissue regrowth.

Description

Polymer-Glass Composite Material
Field of the Invention
The invention relates to a composite material for use as a tissue scaffold, comprising a biodegradable polymer component and a soluble glass component. The material is particularly useful as a scaffold to promote cartilage and bone growth, for example when implanted into a subject.
Background to the Invention
It is known to implant biodegradable polymeric materials to function as scaffolds to promote tissue repair, for example as an alternative to a bone or tissue graft.
Biocompatible polymeric materials may be selected both for their resistance to contamination with microorganisms (to minimize post operative infection), physical characteristics (e.g. similarity to the tissue which is to replace the implant) and the rate of biodegradation, in comparison to the rate of cell growth and attachment in vivo.
A large range of biocompatible and biodegradable polymeric materials, including co- polymers and polymer mixtures, are known for use with medical implants. However performance across the full range of properties of known materials is still not comparable to grafted tissue. Therefore, the use of synthetic scaffolds made from biodegradable polymers is still generally limited to situations where autograft or allograft procedures are not possible.
It has also been proposed to use glass materials which are soluble in vivo to promote tissue growth, either alone or mixed with polymeric materials (US 7,531 ,005). However, while the physical properties of soluble glass materials can be modified by the addition of polymer to form composite materials, composite materials have not yet been developed having both the improved physical properties and which have the required properties in vivo which would be required for the composites to be adequate alternatives to tissue grafts. It has been found that cell adhesion, penetration and rate of cell growth through materials of this type is extremely sensitive to the physical structure and composition of these materials. For example, it has been found that use of soluble glass materials alone, or of materials with a high percentage of soluble glass materials, can increase local acidity and cause cell death. Consequently, neither soluble glass materials, nor glass/polymer composite materials have yet been developed which have been shown to be suitable for use as scaffolds suitable for medical implantation.
Accordingly, it is an object of the present invention to address the foregoing disadvantages, and to provide composites suitable for use as an alternative to autografted or allografted tissues.
Summary of the Invention According to a first aspect of the invention, there is provided a porous polymer-glass composite material comprising;
at least 80% by weight (i.e. 80wt%) of a biodegradable organic polymeric material; and a water soluble glass. According to a second aspect of the invention, there is provided a porous polymer- glass composite material for use in a method of treatment by surgery, comprising; at least 80wt% of a biodegradable organic polymeric material; and
a water soluble glass. Thus the porous polymer-glass composite material comprises around, or less than, 20wt% of the water soluble glass. The polymer material and the water soluble glass may constitute less than 100wt% of the composite material and the porous polymer- glass composite material may comprise one or more other materials, in addition to the polymer material and the water soluble glass (for example one or more biocompatible excipients or filler materials).
Preferably the porous polymer-glass composite material is for use as a tissue scaffold, in a method of surgery. The porous polymer-glass composite is particularly useful for promoting bone growth or repair, and or for promoting cartilage growth or repair. It has been found that the stiffness of the porous polymer-glass composite material is improved, in relation to a porous polymeric material alone, when the water soluble glass is present in an amount up to around 20wt%, and that the stiffness of the composite materials having greater amounts of glass is reduced. It has also been found that porous polymer-glass composite materials having larger amounts of water soluble glass do not deform elastically at low strains. In contrast, porous polymer-glass composite materials comprising less than around 20wt% (and preferably around 5wt%) of water soluble glass deform elastically, or near elastically, at low strains (i.e. from zero strain to at least around 10-15% strain, and typically between 0%-30% strain). In preferred embodiments, for example in composite materials comprising around 5wt% glass, the material deforms elastically up to and in some cases beyond 50% strain.
Preferably the porous polymer-glass composite material comprises less than around 10wt% of water soluble glass.
Preferably the porous polymer-glass composite material comprises around 5wt% of water soluble glass. In a preferred embodiment, the optimum amount of water soluble glass is 5wt%; i.e. the highest compressive Young's Modulus is observed for porous polymer-glass composite material comprising around 5wt% of water soluble glass.
Preferably the water soluble glass is not a silicate glass. The use of silicon-containing materials in vivo has been associated with deleterious long term health effects and therefore the porous polymer-glass composite material of the present invention preferably makes use of a biocompatible water soluble glass material which, over a period of time, dissolves completely in vivo and is cleared completely from a patient's system, and which is silicon free (or substantially silicon free). Preferably the water soluble glass is a water soluble phosphate glass.
Preferably the phosphate glass comprises zinc or strontium. Zinc is a solution rate modifier and it has been found that phosphate glass comprising an amount of zinc advantageously reduces the dissolution rate of the water soluble phosphate glass. Preferably, zinc is present in an amount of zinc oxide less than 10 mole% of the total oxides. Zinc may be present in an amount of zinc oxide between 1 -6 mole% of the total oxides, and most preferably in an amount of zinc oxide less than 5 mole% of the total oxides. Strontium, when present may be present in the same amount as defined for zinc above.
The phosphate glass material may be a Ab(Ml 20)x(M"0)y(P205)z material, wherein; M1 is a metal from group I of the periodic table;
M" is a divalent metal, preferably from group II of the periodic table;
A is one or more further materials, typically oxide materials, and b > 0
and wherein (x+y+z+b) = 1 . In some embodiments, M" is divalent metal from other than group 2 of the periodic table and may for example be zinc. In some embodiments b=0, and x+y+z= 1 . The phosphate glass material may comprise an amount of one or more further materials A, which are typically metal or metalloid oxide materials, and thus b>0. For example the phosphate glass may further comprise boron, silver and/or copper, and most preferably zinc. Therefore it may be that x+y+z< 1 .
Typically, z is less than around 0.85 and more typically between around 0.3 and 0.6, and x+y is typically between around 0.4 and 0.6. In a preferred embodiment, the phosphate glass is a sodium-calcium-phosphate glass, most preferably further comprising zinc.
The water soluble glass may be in the form of a powder, as flakes or as fibres. Preferably the water soluble glass is in the form of flakes or fibres, or any other particulate form having a comparable particle dimension, so as to provide a comparable ratio of surface area to mass of glass. Flakes or fibres of glass material may be mixed with the biodegradable polymer material to provide a composite material having a uniform composition, and it has been found that flakes or fibres of glass advantageously have a slower dissolution rate than glass in the form of a powder. Accordingly, cell proliferation into the composite material, which is known to be promoted by the presence of the glass material, occurs over a longer period of time and cytotoxicity resulting from high rates of dissolution in vivo, is also reduced.
In some embodiments, the particles of water soluble glass have an average dimension of between 5μηι-50μΓΤΐ. For example, the water soluble glass may comprise glass filaments, the filaments having an average diameter of 5μηι-50μΓΤΐ, or the water soluble glass may comprise glass flakes having an average thickness of 5μηι-50μΓΤΐ. The particles may have an average smallest dimension of between 1 0μηι-30μΓΤΐ , and more preferably between 1 5μηι-25μΓΤΐ .
The dissolution rate of the water soluble glass is preferably less than 0.05 mg.cm"2.hr"1 and more preferably less than 0.01 mg.cm"2.hr"1. In a preferred embodiment, the dissolution rate of the water soluble glass is around 0.005 mg.cm"2.hr"1. By "dissolution rate" of a material we mean the mass of the material dissolved per hour in pure water at standard temperature and pressure. Dissolution rate may be expressed per unit of surface area and per unit mass of the material, and so be a property of composition of the material. It is well known to those skilled in the art to vary the composition of the soluble glass, for example the amount and type of solution rate modifiers (for example zinc) to provide a material having a predetermined dissolution rate (per mg of material, per cm2 of material surface). It is also known to vary particle dimensions (e.g. filament diameter) of soluble glass, to vary the surface area per gram of material, so as to vary the rate at which the material dissolves.
The porous polymer-glass composite material may comprise any suitable type of biodegradable polymeric material, and may comprise a single polymer or copolymer, or a mixture of one or more polymers or copolymers. Preferably, the porous polymer- glass composite material comprises biodegradable polyester material. In some embodiments the porous polymer-glass composite material comprises polycapro lactone (PCL) and/or polylactic acid (PLA), or more preferably poly-L-lactic acid (PLLA). In a preferred embodiment, the polymeric material comprises a mixture of PCL and PLLA, preferably in a ratio of between around 70/30 and 90/1 0, or more preferably in a ratio of or around 80/20, of PCL/PLLA by mass.
In some embodiments, the polymeric material comprises polyglycolic acid, polyglycolactide or one or more polysaccharides. Preferably the polymer-glass composite material comprises an open interconnected pore network, so as to facilitate cell penetration, for example in use of the composite material as a tissue scaffold. Preferably the average pore size is in the range of 1 00μηι-700μΓΤΐ, and more preferably or the range of from 250μηι-500μΓΤΐ.
Preferably the pore size distribution of the pores is narrow. Preferably the sizes of 90%, or more preferably 95% or more, of pores lie in the range from 1 00μηι-700μΓΤΐ , or more preferably 1 00μηι-700μηι , or 250μηι-500μηι.
A porous polymer-glass composite material having an appropriate microporosity, and interconnectivity of pores, enables rapid vascularisation and ingrowth to occur in vivo. In addition, pore interconnectivity facilitates resorption of the composite material over the 6- 1 8 month tissue growth/regrowth period, in use of the material as a tissue scaffold.
It has been found that materials having even a small number of very large pores, for example having sizes of the order of 1 0"3 m (as can be present in materials having a larger pore size distribution) do not have satisfactory mechanical properties. In addition, large pores define concomitantly large voids, and tissue ingrowth to fill the voids can take much longer than for smaller pores, in use of the materials as tissue scaffolds. Very small pores inhibit tissue ingrowth and vascularisation. Porous polymer-glass composite materials may be prepared by a variety of methods, including mixing of glass into a polymer solution, or mixing of glass into a monomeric solution, and performing polymerisation in situ. The solvent may then be rapidly evaporated (e.g. at elevated temperature and then at elevated temperature under vacuum), so as to form pores in the polymeric material. However, only very limited pore size control is possible by this method.
Preferably, the porous polymer-glass composite material is obtained by a method of adding the glass to a polymer solution (of a first solvent) further comprising a soluble porogen, i.e. a particulate material having a high dissolution rate, in comparison to either the polymeric material or the glass, in a second solvent. Preferably the porogen is water soluble, and may for example be a salt, e.g. sodium chloride, PEG, or a sugar, or any other suitable material having a much higher solubility rate in water than the glass or the polymeric material. The first solvent may then be removed (e.g. by drying, under vacuum, or freeze-drying) and the polymer-glass composite material washed in the second solvent, to dissolve the porogen. The average size and size distribution of the pores which are left when the porogen has been removed reflect the particle size distribution of the porogen, and the porosity of the polymer-glass composite material reflects the amount of porogen used. Therefore, preparation of the polymer-glass composite material by a method using porogen enables both the pore size distribution and the porosity of the composite to be controlled with greater accuracy than has been previously possible. An alternative method is to employ thermally induced phase separation (as described in La Carrubba et al International Journal of Material Forming, 2008, vol 1 ., No. 1 , Supp/1 p 619-622) to induce porosity and this may be used with and without salt leaching in combination with the glass.
The composite material typically has a porosity of 60 - 85%.
Preferably, the porous polymer-glass composite material is for use as a tissue scaffold. Preferably, the porous polymer-glass composite material is for use as a bone or cartilage scaffold.
In use, cells (for example chondrocytes or osteoblasts) penetrate into the porous composite material and proliferate on the scaffold. The water soluble glass dissolves, and the polymeric material biodegrades and is replaced by cells. Advantageously, the materials of the present invention can be adapted to biodegrade and dissolve over a time period compatible with rate of growth or regrowth of a particular tissue type. For example, for bone or cartilage tissue, growth or regrowth typically occurs over a 6-18 month period. In a preferred embodiment, the porous polymer-glass composite material is for use as a bone scaffold, to promote bone growth or regrowth and/or repair, and is entirely resorbed in vivo over a 10-12 month period.
The porous polymer-glass composite material may further comprise cells within pores of the porous polymer-glass composite material. For example, the porous polymer- glass composite material may be seeded with cells, such as human or animal cells. Thus, the polymer-glass composite material may be seeded with cells in vitro and then implanted into a subject, to thereby increase the rate of tissue regrowth.
In some embodiments, the cells are osteoblasts (bone cells) or chondrocytes (cartilage cells). In some embodiments, the cells are mesenchymal stem cells such as adipose derived mesenchymal stem cells.
According to a third aspect of the invention, there is provided a method of preparing a porous polymer-glass composite material, comprising the steps of:
(1 ) preparing a mixture of; a solution of a polymeric material in a first solvent; a glass material, and; a particulate porogen material which is soluble in a second solvent;
(2) removing substantially all of the first solvent to produce a polymer-glass composite material, having particles of porogen material dispersed therein; and
(3) removing substantially all of the porogen material by washing the polymer-glass composite material in the second solvent, to produce a porous polymer-glass composite material.
Preparation of porous polymer-glass composite materials by the method of the present invention, wherein porogen material is leached from polymer-glass composite material, enables much greater control over porosity (which is related to the amount of porogen material used) and pore size and size distribution (which is related to the particle size and particle size distribution of the porogen material used) than previously known methods. Preferably the porogen material has a low dissolution rate in the first solvent (in comparison to the polymeric material), or is more preferably substantially insoluble in the first solvent. Preferably the polymeric material has a low dissolution rate (in comparison to the porogen material), or is more preferably substantially insoluble, in the second solvent.
In some embodiments, the glass is a soluble glass and has a low, or very low, dissolution rate in the first solvent, or is substantially insoluble in the first solvent.
In some embodiments, the soluble glass material is soluble in the second solvent, and the dissolution rate of the particulate porogen material in the second solvent is greater than the dissolution rate of the glass in the second solvent. Preferably the dissolution rate of the porogen in the second solvent is much higher than the dissolution rate of the soluble glass in the second solvent by, for example, one or more orders of magnitude; e.g. two, or three or more orders of magnitude higher, or more preferably five to ten orders of magnitude higher.
In some embodiments, the dissolution rate of the glass is less than 0.05 mg.cm"2.hr"1 (e.g. around 0.005mg.cm"2.hr"1) and the dissolution rate of the porogen material (for example sodium chloride) is in the range from 5-10mg.cm"2.hr"1 (and in some embodiments greater than 10 mg.cm"2.hr"1), in the second solvent.
In some embodiments, the glass is a water soluble glass (for example a water soluble phosphate glass) and the second solvent is water. The particulate porogen material may be any suitable water soluble particulate material having a much higher dissolution rate in water than the water soluble glass (for example a salt, such as sodium chloride, or a sugar).
It is postulated that the small amount of dissolution which occurs on the surface of a glass material which is soluble in the second solvent facilitates penetration of the second solvent into the composite material, and thus facilitates dissolution and removal of the porogen material. Thus, known limitations in the use of porogen leaching methods (caused by the time taken to leach the porogen from polymeric material, in turn limiting the size or thickness of a piece of composite material made by this method) is reduced.
The invention also extends to a structure composed of porous polymer-glass composite material (of the first or second aspect, or obtained or obtainable by the method of the third aspect), having a smallest dimension of greater than 20mm, or greater than 40mm. As a consequence, the invention provides for larger porous polymer-glass composite structures than has previously been possible and the composite material may therefore be used in a wider range of surgical procedures.
The glass may be in the form or a powder, granules or flakes, or may be in the form of fibres. Preferably, the step of removing substantially all of the porogen material by washing the polymer-glass composite material in the second solvent, produces a porous polymer-glass composite material having an interconnected open pore network. The polymeric material may comprise a single polymer, or may comprise one or more polymers or co-polymers. The polymeric material may comprise biodegradable polymeric material (such as one or a mixture of polyesters).
The first solvent may be a volatile organic solvent, such as an alcohol, ether or chlorinated solvent. The first solvent may be chloroform. Glasses, such as phosphate glasses are typically insoluble in organic solvents (and organic solvents in which selected polymeric material is soluble may be selected, in which glasses are substantially or completely insoluble). The mixture of a solution of a polymeric material in a first solvent, a glass material, and a particulate porogen material is an admixture, and thus the components of the mixture may be added in any sequence. The step of mixing typically comprises vortexing, or any method of mixing suitable to produce a mixture of uniform composition (i.e. in which the porogen material and glass are intimately mixed into, and distributed or dispersed within, the solution of polymeric material).
The first solvent may be removed by any suitable method, including by placing the mixture under vacuum, or placing the mixture at elevated temperature. Preferably the method does not comprise placing the mixture under vacuum at elevated temperature.
In a preferred embodiment, the first solvent is removed by freeze-drying, that is to say freezing the mixture using dry ice, or liquid nitrogen or any other suitable cryogenic material, and placing the mixture under vacuum whilst frozen. It has been found that rapid removal of the first solvent, particularly if the mixture has not set or solidified, results in the formation of large pores and has a detrimental effect on the structural properties of the porous polymer-glass composite material which is produced. Freeze drying, or otherwise removing the first solvent without causing sudden vaporisation of solvent in the bulk of the material, prevents the formation of large pores and a wider pore size distribution. Thus, the method preferably comprises removing substantially all of the first solvent by freeze drying the mixture of; a solution of polymeric material in the first solvent; the glass material, and; the particulate porogen material.
The method may be a method of preparing a porous polymer-glass composite material of the first aspect.
The method may further comprise seeding the porous polymer-glass composite material with cells, for example osteoblasts, chondrocytes or mesenchymal stem cells such as adipose derived mesenchymal stem cells.
The method may further comprise differentiating mesenchymal stem cells, for example by use of an osteogenic medium (or a chondrogenic medium).
The invention also extends to a porous polymer-glass composite material obtained or obtainable by the method of the third aspect.
Further preferred and optional features of the third aspect correspond to preferred and optional features of the first and second aspects.
According to a fourth aspect of the invention, there is provided a method of treatment by surgery, comprising implanting an implant into a subject, the implant comprising (or consisting of) a porous polymer glass composite material according to the first, second or third aspects.
The method may be a method or repairing damaged tissue, for example bone or cartilage tissue. The method may comprise obtaining cells from a subject, seeding the porous polymer-glass composite material with the said cells, and implanting an implant an implant into a subject, the implant comprising (or consisting of) a porous polymer glass composite material according to the first, second or third aspects, seeded with cells from the subject.
The cells may for example be osteoblasts, chondrocytes or mesenchymal stem cells such as adipose derived mesenchymal stem cells. In some embodiments, the method comprises seeding the porous polymer-composite material with mesenchymal stem cells, differentiating the mesenchymal stem cells, for example by use of an osteogenic medium (or a chondrogenic medium), and implanting an implant comprising or consisting of the said composite material.
Further preferred and optional features correspond to preferred and optional features of the first through third aspects.
Where a first feature is referred to herein as comprising (or comprises) a second feature, we mean that the first feature may consist of, or consist essentially of, the second feature, or may include the second feature in addition to other features.
Description of the Drawings The invention will now be described with reference to the following figures, in which:
Figure 1 (a) shows porous polymer-glass composite material prepared using solvent evaporation method (1 ) and (b) shows porous polymer-glass composite material prepared using porogen leaching method (2).
Figure 2 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0%, 5%, 20% and 30% of CG151 powder, in PCL/PLLA (80/20) polymeric material, as strain is reduced from 60% to 0%.
Figure 3 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank"), 5%, 10% and 30% of CG151 powder, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to destruction.
Figure 4 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank"), 5%, 20% and 30% of CG101 powder, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to 85%. Figure 5 shows a plot of compressive stress against compressive strain of porous polymer-glass composite materials prepared using porogen leaching method (2), comprising 0% ("blank"), 5%, 10%, 20% and 30% of CG107 fibre, in PCL/PLLA (80/20) polymeric material, as strain is increased from 0% to 85%.
Figure 6 shows results of an MTT assay of MG-63 cell proliferation on porous polymer glass composite materials after 24 and 48 hours, normalised to results obtained from a polystyrene control sample. Results are shown for porous polymer-glass composite materials prepared using porogen leaching method (2), and comprising 0% glass ("blank"), and X% by weight of each of CG101 powder (labelled "C.P.101 (X%)") and CG151 powder (labelled "C.P.151 (X%)"), where X% = 10%, 15%, 20% and 30%.
Figure 7 shows results of an MTT assay of MG-63 cell proliferation on porous polymer glass composite materials after 24 hours, 48 hours and 1 week, normalised to results obtained from a polystyrene control sample. Results are shown for porous polymer- glass composite materials prepared using porogen leaching method (2), and comprising 0% glass ("blank"), and X% by weight of each of CG140 fibre (labelled "C.P.140(X%)", where X% = 20% and 30%) and CG107 fibre (labelled "C.P.107(X%)", where X% = 5% and 20%). Comparative results are also shown for 100% fibre glass samples ("CP.140" and "CP.107") and a sample comprising CG107 fibres ground into a powder ("C.P.107p").
Figure 8 shows the number of ADSCs on scaffold sample after 1 , 7, 14 and 21 days. Osteogenic media was added after 1 day. Results were obtained using a DNA assay, on a hydroxyaptite sample ("HA"), a blank sample of a porous PCL/PLLA (80/20) material ("BL") and a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material ("5%"). The blank and polymeric materials were made using the porogen leaching method (2). Figure 9 shows representative images of live cells (green) and dead cells (red), 24 hours post seeding of two sample scaffold materials seeded with ADSCs and treated with osteogenic media. The magnification scale bar represents 200μηι. The image labelled "BL" is a blank sample of a porous PCL/PLLA (80/20) material and the image labelled 5% is a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material. The materials were made using the freeze drying and porogen leaching method (2).
Figure 10 shows representative images of RUNX2 staining of ADSC seeded scaffold materials treated with osteogenic media. The magnification scale bar represents 100μηι. Images labelled "HA", "BL" and "5%" are of materials similarly labelled in relation to Figure 8. Comparative images of ADSCs grown on culture plastic and treated with osteogenic media (image labelled "OS") and ADSCs grown on culture plastic and not treated with osteogenic media (image labelled "NOR"), Images are also shown of MG63 seeded culture plastic samples, labelled MG63+ and MG63- ("+" and "- " designating samples with and without immunocytochemical staining).
Figure 1 1 shows representative images of Col1 a1 staining of ADSC seeded scaffold materials. The scale bar represents 100μηι. Samples are labelled as for Figure 10.
Figure 12 shows representative images of Osteocalcin staining of ADSC seeded scaffold materials. The scale bar represents 100μηι. Samples are labelled as for Figure 10. Figure 13 shows gene expression of Runx2, measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples were treated with osteogenic media after 1 day. Results are shown for a hydroxyaptite sample ("HA"), a blank sample of a porous PCL/PLLA (80/20) material ("BL") and a porous polymer-glass composite material comprising 5% is CG107 in a PCL/PLLA (80/20) polymeric material ("5%"). Results are also shown for control samples of ADSCs grown on culture plastic and treated with osteogenic media (labelled "OS") and ADSCs grown on culture plastic and not treated with osteogenic media (labelled "NO")
Figure 14 shows gene expression of collagen type 1 (Col1 a1 ), measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples are labelled as for Figure 13.
Figure 15 shows gene expression of osteocalcin, measured for ADSC seeded samples after 0, 1 , 7, 14 and 21 days. Samples are labelled as for Figure 13. Figure 16 shows alkaline phosphatase staining (dark blue) of ADSC seeded samples (column marked "+cells") 13 days after exposure to osteogenic media. Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
Figure 17 shows expanded views of ADSC seeded samples of Figure 16. Images are filtered to show alkaline phosphate staining (top row), DAPI staining (middle row) and both alkaline phosphate and DAPI staining (bottom row.) The scale image bar represents 200μηι.
Figure 18 shows alizarin red staining of ADSC seeded samples (column marked "+cells") after 14 days (13 days after exposure to osteogenic media). Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
Figure 19 shows Von Kossa staining (black) of ADSC seeded samples (column marked "+cells") after 14 days (13 days after exposure to osteogenic media). Images are labelled in accordance with Figure 10. Comparative images are also shown for samples which were not seeded ("-cells"). The magnification scale bar represents 5mm.
Figure 20 shows Von Kossa staining of ADSC seeded samples after 14 days (13 days after exposure to osteogenic media). The magnification scale bar represents 200μηι. Images are labelled as above.
Figure 21 shows DSC data for a porous glass polymer material containing PCL/PLLA (80/20) and 5% CG107, and prepared using porogen leaching method (2), before sterilisation.
Figure 22 shows DSC data for the porous glass polymer material containing PCL/PLLA (80/20) and 5% CG107, and prepared using porogen leaching method (2), after sterilisation with gamma radiation. Figure 23 shows images of stained, sterilized samples 8 hours after seeding with ADSCs (A) without re-wetting (B) after re-wetting with PBS and (C) after re-wetting with denatured ethanol and PBS. Figure 24 shows an attachment assay of ADSC cells comparing gamma sterilised dry sample, gamma sterilised PBS conditioned sample and IMS sterilised and PBS conditioned sample.
Figures 25(a)-(d) shows photographs of sheep 2 taken during in vivo testing.
Figure 26 shows representative microscopical pictures of implanted sites after 4 weeks, of (a) Left femur of sheep 1 , site 2 (b) right humerus of sheep 1 , site 5 (c) left femur of sheep 2, site 2 (d) left humerus of sheep 2, site 4. Detailed Description of an Example Embodiment Materials
Medical grade biodegradable polymers polycaprolactone (Mw 100,000 Da, PCL) and Poly-L-lactic acid (PLLA, Mw 152,000 Da, Mn 99,000 Da) were obtained from Boerhinger Ingelheim and Evonik respectivey
Sodium Chloride was obtained from Fisher Scientific and sieved using a 500 micron and a 240 micron sieve.
A range of phosphate glass materials were obtained from Giltech Limited. Samples were provided as powders or fibres. Properties and approximate dissolution rates are set out in Table 1 .
Table 1
Product Code Form Particle size / Approx Dissolution
Fibre diameter Rate (mg.cm-2.hr-1)
CorGlaes Pure 101 Powder 20μηι 0.25
("CG101 ")
CorGlaes Pure 151 Powder 19μηι 0.035
("CG151 ") CorGlaes Pure 140 Fibre 15μηι 0.02
("CG140")
CorGlaes Pure 107 Fibre 15μηι 0.005
("CG107")
All CorGlaes samples are tertiary sodium calcium phosphate glasses. CG140 and CG107 additionally comprise amounts of zinc, which is present as a solution rate modifier. CorGlaes is a trade mark of Giltech Limited.
CG101 and CG151 were used as received in the powder form. CG107 and CG140 fibre were flocculated and the fibres cut in to lengths of 0.8-1 .2cm.
For some experiments, fibres were also ground to form a powder (and these samples are designated CG140p and CG107p).
Preparation of Porous Polymer-Glass Composite Materials
Porous polymer-glass composite materials were prepared by each of three methods.
(1 ) Solvent Evaporation
Glass was added to a solution of polycaprolactone and poly-L-lactic acid in chloroform (4 ml per gram of polymeric material) and the mixture thoroughly vortexed. The sample was placed in an oven at 40 °C for 4 days and then dried under vacuum for a further 3 days at 40 °C, to yield a porous polymer-glass composite material.
(2) Solvent Evaporation and Porogen Leaching
Glass was added to a solution of polycaprolactone and poly-L-lactic acid in chloroform (4 ml per gram of polymeric material) and the mixture thoroughly vortexed. NaCI (in an amount of 4g per gram of polymeric material) was added and the mixture thoroughly vortexed. The sample was then placed in the oven at 40 °C for 4 days. The resulting material was then washed with copious amounts of deionised water for 4 days and then dried in the oven at 40 °C for a further 4 days, to yield a porous polymer-glass composite material. (3) Freeze Drying and Porogen Leaching
Glass was added to a solution of polycaprolactone and poly-L-lactic acid in chloroform (4 ml per gram of polymeric material) and the mixture thoroughly vortexed. NaCI (in an amount of 4g per gram of polymeric material) was added and the mixture thoroughly vortexed. The sample was then frozen in dry ice for 3 minutes and then placed under vacuum for 24 hours, and allowed to thaw. The resulting material was then washed with copious amounts of deionised water for 4 days and then dried in the oven at 40 °C for a further 4 days, to yield a porous polymer-glass composite material. Porous polymeric material samples were also prepared by porogen leaching, by the method (2) set out above, but without the addition of water soluble phosphate glass. Samples comprising an 80/20 mixture of polycaprolactone and poly-L-lactic are hereinafter referred to as "BL". Sterilisation
All samples were sterilised by gamma radiation (25-35kGy) and sealed in a 150mm x 170mm Peel pouch consisting of film and porous paper. Tensile testing
Tensile testing was conducted using an Instron 3367 Tensile/Compression machine. Compression tests were performed at 2mm/s. In vitro Testing
Attachment & Proliferation Assay (MTT assay, MG63J
An attachment and proliferation assay was carried out using MG-63 (human osteosarcoma) as a prototype of human bone cells. A standard MTT assay was used to quantify the number of cell attachment and proliferation, as follows:
Samples were sterilised by autoclave, placed in a 6-well plate and washed extensively with sterile deionised water, sterile phosphate buffered saline solution (PBS) and Dulbecco's modified Eagle's medium (DMEM). Washed MG-63 (Human osteosarcoma) cells (ca. 500,000 cells per well) were seeded in DMEM (500μΙ_) for 8 hours. The samples were then immersed in DMEM and incubated for a further 24 hours. The porous polymer-glass composite samples were then transferred to another 6-well plate.
The samples were then incubated for 24, 48 hours and 7 days (and media was changed every second day). The media was removed and the samples were washed with PBS to remove unattached cells, transferred to a new 6-well plate and then treated with trypsin/EDTA (2.5%, 200μΙ_) for 30 minutes to detach cells which were attached to the composite samples. DMEM (1 mL) was added and the solution was drawn up into, and expelled from, a pipette, in order to distribute the detatched cells evenly.
The cells were replated on a 6 well plate 100μΙ_Λ/νβΙΙ and incubated for 12 hours. Cells were then washed twice with PBS. All media was then replaced with a mixture of 50μΙ_ of fresh media (phenol red free) and 50μΙ_ of MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) in phosphate buffered saline ("PBS") (1 mg/ml_) and the cells incubated for 4 hours at 37°C.
After the incubation the resulting formazan crystals were dissolved by adding 100 μΙ_ of MTT solubilisation solution (10% Triton-X 100 in acidic isopropanol (0.1 M HCI)). The absorbance was measured spectrophotometrically at a wavelength of 570 nm.
The values were compared to the same amount of cells seeded in a well and incubated for 24, 48 hours and 7 days (media changed every second day). The cells seeded in a well only (and not a porous polymer-glass composite scaffold) were also washed with PBS, treated with trypsin/EDTA(2.5%, 200μΙ_) for 10 min and diluted with DMEM (1 mL), and replated (Ι ΟΟμ-Jwell). In vitro testing with adipose derived mesenchymal stem cells
Cell culture Human adipsoe tissue samples were obtained under informed consent from donors undergoing liposuction for cosmetic reasons. Human adipose derived mesenchymal stem cells (ADSCs) were isolated from tissue samples and expanded using a method published previously (Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang Jl, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH.Mol Biol Cell. 2002, 13(12):4279-95.). ADSCs were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% foetal calf serum (FCS), L-glutamine (2mM), penicillin (100 lll/mL) and streptomycin (100μg/mL) (obtained from Sigma-Aldrich Ltd., Poole, UK), termed growth media. Cells were maintained for at 37 °C, 5% C02, until the adherent cells were confluent. Confluent cells were passaged by treatment with TrypLE Select (Invitrogen) for use in experiments or reseeded. All experiments were conducted using cells at passage 4. (TrypLE is a trade mark of Life Technologies Corporation).
Cell Attachment and Proliferation Assays Cell attachment and proliferation assays with adipose derived mesenchymal stem cells were conducted on three materials; a hydroxyapatite material ("HA"), a porous polymeric material comprising an 80:20 mixture of polycaprolactone and poly-L-lactic acid ("BL"), and a porous polymer-glass composite material made by the freeze drying and porogen leaching method described above, comprising 5% CG107 in an 80:20 mixture of polycaprolactone and poly-L-lactic acid ("CP.107").
Three samples of each material were placed into a 24-well tissue culture plate. Each well was seeded with 2x104 ADSC in 100μί of growth media. At 2, 4, 6 and 8 hours after seeding (during the attachment period) and at 1 , 7, 14 and 21 days after seeding (during the proliferation period), the samples were washed three times with PBS.
Induction of Osteogenic Differentiation
Osteogenic differentiation of cells was induced by culture in osteogenic medium ("OS"), comprising 50μΜ ascorbic acid phosphate (obtained from Wako Chemicals GmbH), 10mM β-glycerophosphate and 100 nM dexamethasone. Osteogenic media was instroduced to samples after an attachment period of 1 day.
ADSCs were also cultured on tissue culture plastic, with and without treatment by osteogenic media, in six of the remaining wells, in the same manner. ADSC seeded culture plastic samples are labelled OS (with osteogenic media) and NOR (without osteogenic media).
MG63 cells were also cultured on tissue culture plastic, without osteogenic media, in six of the remaining cells in the same manner. MG63 seeded culture plastic samples are labelled MG63+ and MG63- ("+" and "-" designating samples with and without immunocytochemical staining). Note that both MG63+ and MG63- labelled samples were DAPI stained, as discussed below. DNA extraction and precipitation
Samples were removed from culture wells, and placed in separate, sterile 1 .5ml_ Eppendorf tubes and 500μΙ of phenol added to each well. Each well was scraped with a pipette tip and then the phenol transferred to a sterile, 1 .5 ml Eppendorf tube. 100μΙ of sterile Dnase free water was added and each tube was then vortexed for 30 seconds. The tubes were then centrifuged at 2,000 rpm for 5 minutes and the aqueous phase transferred to a sterile 1 .5ml Eppendorf tube.
In order to precipitate the DNA, 0.1 volumes of 3 M sodium acetate (pH 5.5) and 2 volumes of absolute ethanol was added to the aqueous phase and each sample then incubated overnight at -20 °C.
The precipitated DNA was collected by centrifugation of each sample at 10,000 rpm for 45 minutes. The ethanol was removed and the precipitate (in the form of a pellet) was washed with 70%(v/v) ethanol and again centrifuged at 10,000 rpm for 15 minutes.
The ethanol was removed and the pellets allowed to air dry for 5 minutes. The dried DNA was re-suspended in 10μΙ of sterile DNase free water and stored at -20 °C. Fixation
After 1 , 7, 14 and 21 days of seeding (experimental time-points), each well were washed three times with PBS and fixed in 100% methanol at -20 °C for 10 minutes, then washed in PBS again three times prior to staining.
Alkaline phosphatase activity
Alkaline phosphatase activity (ALP) was assessed by histochemical staining of samples prepared in a 24 well plate as discussed above. Alkaline phosphatase histochemical staining was performed using a commercial analytical test kit (Leukocyte Alkaline Phosphatase Kit, Sigma-Aldrich Ltd., Poole, UK) following manufacturer instructions. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected.
Mineralised extra cellular matrix identification - Calcium (Alizarin red staining)
ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate, were fixed as discussed above, washed thoroughly with distilled water and incubated in 2% Alizarin red solution (pH 4.3) for 2 minutes, and then washed three times in distilled water. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected. Mineralised extra cellular matrix identification - phosphate (Von Kossa histochemistry)
ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate, were fixed as discussed above, washed thoroughly with distilled water and incubated in 5% silver nitrate solution before being exposed to a 60-watt light bulb at a distance of approximately 5 cm for 1 hour. Following exposure, cells were washed in distilled water and incubated in 5% sodium thiosulphate solution for 5 minutes. At each time-point, images were taken of the whole well/sample and images were also taken at each of 10 randomly selected substrate locations. Representative images were then selected.
RNA extraction and qRT-PCR Total RNA was isolated from human ASDCs seeded onto samples or on tissue culture plastic, in a 24 well plate, as discussed above. On days 1 , 7, 14 and 21 after seeding RNA was extracted using a commercial Trizol reagent (Invitrogen, Paisley, UK) and following maunfacturers instructions. (Trizol is a trade mark of Molecular Research Center, Inc.)
Total RNA samples were transcribed to cDNA using Superscript III First strand Synthesis SuperMix for RT-PCR (Invitrogen). (Superscript is a trade mark of Life Technologies Coproration).
For the quantitative RT-PCR, 60ng of template cDNA was added to each PCR reaction. Quantitative PCR was performed using a Qiagen Corbett Rotor-Gene 600. Specific primers were used for the following genes: Actin, RUNX2, Collagen type I, Osteocalcin and Alkaline phosphatase, with qPCR Master Mix (Promega Corporation). Standard cycling conditions were used (initial denaturation step at 95eC for 20 sec, followed by 40 amplification cycles of 3 sec duration at 95eC and then 30 sec at 60eC).
Data analysis was performed using Microsoft Excel. Levels of expression of each gene of interest were normalized to actin gene levels at Day 0.
Immunocvtochemical staining
Runx2 Staining: ADSCs seeded on samples, as described above, were blocked with blocking buffer (10% goat serum, 1 % BSA and 0.1 % Triton X-100) for 1 hour at room temperature and then incubated overnight at 4eC with Anti-human flunx-P/CBFAI antibody (obtained from R&D Systems, Abingdon, UK) at 1 :200 dilution in antibody diluent (a modified PBS formulation buffer with protein carrier and preservatives). Samples were washed three times with PBS (1 mL) on an orbital shaker at 40 rpm for 5 min and then incubated for 1 hour in the dark and at room temperature, with anti-rat secondary antibody conjugated with Alexa Fluor 488 fluorescent dye (obtained from Invitrogen) at 1 :400 dilution in antibody diluent. (Invitrogen is a trade mark of Life Technologies Corporation and Alexa Fluor is a trade mark of Molecular Probes, Inc.). Col1 a1 Staining : ADSCs seeded on samples, as described above, were blocked with Dako protein block (obtained from Dako Limited) for 1 hour at room temperature and then incubated overnight at 4eC with mouse monoclonal anti-collagen type I antibody (Sigma-Aldrich) at 1 :200 dilution in antibody diluent.
Samples were then washed three times with PBS (1 ml_) on an orbital shaker at 40 rpm for 5 min and then incubated for 1 hour at room temperature with an anti-mouse secondary antibody conjugated with Alexa Fluor 488 at 1 :1000 dilution in PBS antibody diluent. The samples were protected from light during incubation.
Osteocalcin Staining: ADSC seeded on samples, as described above, were blocked with blocking buffer for 45 min at room temperature and incubated overnight at 4eC in anti-human osteocalcin antibody (obtained from R&D Systems, Abingdon, UK) at 1 :200 dilution in PBS.
Cells were washed three times with PBS (1 mL) on an orbital shaker at 40 rpm for 5 min and then incubated with anti-mouse secondary conjugated with Alexa Fluor 488 at 1 :1000 dilution in PBS at room temperature for 1 hour in the dark. DAPI Staining : All samples were also stained with DAPI solution (4',6-diamidino-2- phenylindole solution), which binds strongly to all cells and is a blue fluorescent dye.
Image acquisition and analysis All images were collected using a Zeiss Axio Observer Z1 microscope with an LD Plan- Neofluar 20x/0.4 Korr Ph 2 objective lens (manufactured by Carl Zeiss Ltd.). The microscope was coupled to a Zeiss AxioCamMR3 camera. The image acquisition and processing software used was Zeiss Axiovison Rel 4.8 and Axiovison Version 4.7.1 .0, respectively. Visualisation of immunofluorescent staining was conducted using Dako fluorescent imaging medium (obtained from Dako UK Ltd).
RNA extraction and RT-PCR
Total RNA was isolated from human ADSCs cultured on tissue culture plastic (without exposure to osteogenic media ("NOR") and exposed to osteogenic media after 1 day ("OS") and from human ADSCs cultured on hydroxyapatite ("HA"), on porous PCL/PLLA (80/20) prepared by porogen leaching method (2) (blank sample "BL") and on porous polymer-glass composite comprising PCL/PLLA (80/20) and 5% CG107, prepared by porogen leaching method (2) ("5%").
On days 1 , 7, 14 and 21 after seeding RNA was extracted using a commercial Trizol reagent (Invitrogen, Paisley, UK) and following maunfacturers instructions. Total RNA samples were transcribed to cDNA using Superscript III First strand Synthesis SuperMix for RT-PCR (Invitrogen). For the quantitative RT-PCR, 60ng of template cDNA was added to each PCR reaction. Quantitative PCR was performed using a Qiagen Corbett Rotor-Gene 600.
Specific primers were used for the following genes: Actin, RUNX2, Collagen type I, Osteocalcin and Alkaline phosphatase, with qPCR Master Mix (Promega). Standard cycling conditions were used (initial denaturation step at 95eC for 20 sec, followed by 40 amplification cycles of 3 sec duration at 95eC and then 30 sec at 60eC). Data analysis was performed using Microsoft Excel. Levels of expression of each gene of interest were normalized to actin gene levels at Day 0. In Vivo Studies
Samples of porous polymer-glass composite material, prepared using the freeze-drying porogen leaching method discussed above, was used for in-vivo studies. The samples comprised 5% CG107 in a polymeric material consisting of an 80:20 mixture of polycaprolactone:poly-L-lactic acid.
Two sheep were bilaterally implanted in the femur and the humerus with test articles of the above sample material. The sheep were implanted at three sites on each femur (medio-distal condyle) and at one site on each humerus (latero-proximal major tubercle) as described in Table 2, showing the study design. Table 2.
Figure imgf000027_0001
Surgical procedure Prior to the surgical procedure, food was withheld for 24 hours, and water withheld for 12 hours, before implantation.
Each sheep was weighed and then injected with a thiopental-pentobarbital mixture (thiopental was obtained from Merial Animal Health Limited and sodium pentobarbital was obtained from Ceva Animal Health Ltd.) and atropine sulphate. The sheep were then placed under 02-isoflurane inhalant aesthetic (0.5-4% mixture of AErrane, obtained from Baxter Healthcare Ltd. - "AErrane" is a trade mark of Baxter International Inc.) for continued general anaesthesia during the surgical procedure. Each sheep was also given a pre-operative analgesic treatment of buprenorphine and flunixin. As a prophylactic measure, pre-operative antibiotics (penicillin procaine and penicillin benzathin) were also intramuscularly administered.
Selected surgical areas were clipped free of wool and the skin scrubbed with povidone iodine, wiped with 70% isopropyl alcohol, painted with povidone iodine solution and draped.
Monitoring During the surgery, a rectal temperature probe and a rumen tube was placed. Electrocardiogram (ECG) and oxygen saturation was monitored. Implantation procedure
The surgical procedure was performed using standard aseptic techniques. Femoral defects were created as follows: a cutaneous incision was made on each latero-distal femoral condyle. The muscles were separated using blunt dissection in order to access the femur. The periostum was removed from the femoral epiphysis and metaphysis to expose the implant sites. Drilling was conducted (perpendicular to the bone surface) to create a cylindrical hole with a final diameter of 5-6mm and a depth of 10mm.
Humeral defects were created as follows: a skin incision over the shoulder joint was made from the acromion to the middle of the proximal third of the humerus on each lateral humeral major tubercle. Subcutaneous tissues and deep fascia was dissected and a proximal-distal incision of the deltoid muscle was made. The infraspinalis muscle was retracted caudally by blunt dissection. A drill guide was placed centrally in the groove of the humeral major tubercle. Drilling was conducted perpendicular to the bone surface to create a hole with a final diameter of 5mm and a depth of 10mm. Histopatholoqical analysis
Histopatholoqical slide preparation
The 16 humeral and femoral implanted sites, and one unimplanted test article, were structurally characterized. One central cross section of each site was obtained by the microcutting and grinding technique described by Donath K, Brunner G., "A method for the study of undecalcified bone and that with attached soft tissues."; J. Oral Pathol, 1 1 , 318-326, 1982. Each section was be dehydrated in alcohol solutions of increasing concentration, cleared in xylene and embedded in polymethylmetacrylate (PMMA). The sections were then stained with Goldner Trichrome staining, for qualitative and semi-quantitative analysis. Quantitative and semi-quantitative Histological evaluation analysis
Qualitative and semi-quantitative histological evaluation of the local tissue effects and the performance was conducted for each implanted site, based on an adapted ISO 10993-6 standard.
Evaluation and statistical analysis
The performance and the local tissue effects evaluation is based on the qualitative and semi-quantitative histological analysis, according to the criteria set out in Tables 3 and 4.
Table 3: Histological evaluation system - Cell type/response
Figure imgf000029_0001
Table 4: Histological evaluation system - Performance response
Score
Performance
0 1 2 3 4 Groups of 4-
Minimal Broad band Extensive band
7 capillaries
capillary of capillaries of capillaries with
Neovascularization 0 proliferation with with supporting supporting
focal. supporting fibroblastic fibroblastic
1 -3 buds structures structures structures
Fibrocytes/
Moderately
fibroconnective tissue, 0 Narrow band Thick band Extensive band thick band
fibrosis/encapsulation
Moderate Marked
Slight extent Severe extent extent of extent of
Osteolysis 0 of bone of bone
bone bone
resorption resorption resorption resorption
Slight = Moderate.
Marked. »
Osteoblastic cells 0 equivalent to >normal Very marked normal bone
normal bone bone
Moderate Marked
Slight extent Severe extent extent of extent of
Newly formed bone 0 of bone of bone
bone bone
formation formation formation formation
= 0% of
bone to
Osteointegration 1 - 25% 26% - 50% 51 % - 75% 76% - 1 00% implant
contact
~ 0% bone
Osteoconduction ingrowth/ 1 - 25% 20% - 50% 51 % - 76% 76% - 1 00% on growth
0 (primary Slight (initial
Severe (up to
Bone remodeling* woven signs of Moderate Marked
corticalization) bone) remodeling)
Severe (1 00%
Material degradation^ 0 Slight Moderate Marked
degraded)
Implant migration
Severe (100% (peripheral 0 Slight Moderate Marked
disseminated) dissemination/diffusion)
A compared with a non-implanted device
* compared with distant bone density Results
Effect of Method of Preparation on Pore Structure of Porous Polymer-Glass Composite Materials
It has been found that preparation of porous polymer-glass composite materials using porogen leaching methods (2) and (3) yields materials with a narrower pore size distribution than materials prepared by solvent evaporation method (1 ). Solvent evaporation method (1 ) produces materials having an uneven distribution of pore sizes, and including a proportion of extremely large pores up to 1 .0-1 .5cm diameter (Figure 1 a).
In comparison, preparation by porogen leaching method (2), yields a porous polymer- glass composite material with a narrow pore size distribution and does not produce any large voids (Figure 1 b). Pore sizes ranged from 250-500μηι and the pores were interconnected. The pore network thus facilitated tissue ingrowth and vascularisation, as discussed below.
In other embodiments (not shown), composite materials may be prepared using other water soluble porogens, including other soluble salts and sugars. The amount and size of crystals can be varied to alter pore size and distribution.
Stress Testing
In order to be suitable for use as a tissue scaffold, in particular for the promotion of bone growth or repair, the porous polymer-glass composite material must have sufficient structural integrity to be easily handled in theatre.
A detrimental effect on the properties was qualitatively observed for samples comprising more than 5% of phosphate glass material. At higher percentages of glass, the samples became brittle and crumbly, making them difficult to handle.
Compression stress testing was then conducted on a series of porous samples prepared using method (3), and comprising PCL/PLLA (80/20) polymeric material and varying amounts of CG151 powder. Figure 2 shows stress-strain plots for samples comprising 0%, 5%, 20% and 30% CG151 , as strain is reduced from 60% to zero. All glass containing samples showed some degree of plastic deformation (i.e. the plots do not pass through (0,0)) and the degree of deformation increases with percentage glass content. Furthermore, the stiffness of the material comprising 5% CG151 was greater than other glass containing samples, and the sample without glass (i.e. the gradient of the stress-strain plots of figure 2 is greatest for the 5% CG151 sample).
Figure 3 shows stress-strain data measured on further samples comprising 0% ("blank"), 5%, 10% and 30% of CG151 powder, during destructive testing as strain was increased from 0% to 85%. The material with 5% CG151 again showed greater stiffness than either the blank material (comprising 0% glass) or materials with a larger percentage of glass.
Table 5 shows a least squares linear fit of the data shown in Figure 3. The parameter "a" provides an approximate measure of stiffness.
Table 5
Slopes: y
Figure imgf000032_0001
values closer to 1 indicate a closer fit to the experimental data)
Similar results were observed for materials comprising CG101 powder (Figure 4). Table 6 shows a least squares linear fit of the data shown in Figure 4. Table 6
Figure imgf000033_0001
Compressive stress-strain destructive testing was also conducted on a series of samples containing CG107 fibres. Similar results were observed (Figure 5). Table 7 shows a least squares linear fit of the data shown in Figure 5. Stiffness over the whole measured range is comparable for the blank sample and the sample containing 5% CG107 fibre, however the material comprising CG107 clearly demonstrates greater stiffness in the range 0-30% strain and performs more consistently overall. Table 7
Figure imgf000033_0002
In vitro Testing
MG63 Attachment & Proliferation Assay In order to assess potential performance as a scaffold support for bone grown or repair/regrowth, the viability and proliferation of human bone cells within porous polymer-glass composite materials was assessed using the MTT assay described above. Porous samples were prepared according to the freeze drying and porogen leaching method (3), comprising PCL/PLLA (80/20) polymeric material and varying amounts of the glass materials set out in table 1 . Figure 6 shows results of an MTT assay of MG-63 cell proliferation on materials comprising CG101 powder and CG151 powder (in the amounts of 0% ("blank"), 10%, 15%, 20% and 30%). Results are normalised against results obtained from a control sample of polystyrene. Assays were conducted after 24 hours, 48 hours and one week.
The sample labelled C.P.151 (30%), containing 30% by weight of CG151 powder, showed an attachment of cells after 24 hours. However, after 48 hours, no attachment or proliferation was seen (within the error limits of the experiment). Figure 7 shows similar results of MTT assays conducted on samples comprising CG140 fibres and CG107 (in both powered and fibre form). Assays were conducted on samples comprising 0% glass ("blank") and 20%, 30% and 100% CG140, and on samples comprising 5%, 20%, 30% and 100% CG107. The blank material containing no glass was seen to support both attachment and proliferation, in line with earlier reports. CG140 degraded in the media within 7 days, and therefore the proliferation all of the samples containing CG140 was never higher than the blank material, and was less than the blank material after 7 days. Results obtained from assays conducted on materials containing CG107 showed generally greater attachments and proliferation, and by far the best results were obtained for the material containing 5% CG107 fibre ("CP.107(5%)"). Results for material containing 5% CG107 in powder form were poor in comparison. Notably, materials containing higher than 5% CG107 caused the media to become acidic after approximately 24 hours, resulting in cell death. The media was changed on a more regular basis to maintain cell growth for these samples, however these initial results suggest that materials comprising higher than approximately 20% glass may not be suitable for use as tissue scaffolds. In vitro testing with ADSCs
Adispose derived mesenchymal stem cells (ADSC) are multipotent cells, capable of self renewal, and possess the potential to differentiate along several lineages, including differentiation into adipocytes, chondrocytes and osteoblasts (the main cell types in fat, cartilage and bone, respectively). The ease of expansion and osteogenic potential of these cells make them ideally suited to study the osteogenic properties of bone graft replacements in vitro. Attachment, proliferation and differentiation of ADSCs on a scaffold consisting of a porous polymer-glass composite material, comprising PCL/PLLA (80/20) polymer and 5% CG107 fibre, was tested and results compared to tests conducted on hydroxyapatite and a blank sample ("BL") of the porous PCL/PLLA (80/20) polymer without glass.
The number of cells present was measured by the DNA assay described above. Results are shown in Figure 8.
In further seeding experiments, cells were seeded onto blank and 5% CG107 samples and analysed for viability after 24 hours. Samples were washed with propidium iodide solution in order to stain dead cells (red), and with Calcein AM dye solution in order to stain live cells (green). Representative images of each sample are shown in figure 9.
No dead cells were seen on the sample containing CG107 fibre, demonstrating that all the cells attached to the scaffold were alive after 24 hours. Only a single dead cell was observed on the blank sample. This result suggests both scaffolds are non-toxic and that cells attached to these materials are viable.
Immunofluorescent staining
Runx2 is a transcription factor required for osteogenic differentiation and its presence is indicative of osteoblast cells or of cells becoming, committed to the osteoblast lineage.
Figure 10 shows RUNX2 staining on cells attached to scaffolds or grown on tissue culture plastic.
As clearly shown in Figure 10, the strongest nuclear staining was seen on the seeded sample containing 5% CG107 ("5%"), and was at similar levels to the MG63 and ADSC seeded OS positive controls. These results indicate that the 5% material supports early ostegenic differentiation to a greater extent than the material with no glass ("BL") or the hydroxyapatite sample ("HA").
Figure 1 1 shows col1 a1 staining on cells attached to scaffolds or grown on tissue culture plastic.
Col1 a1 (also known as collagen type 1 ) is the most abundant protein in bone (approx 30% of bone is protein, of this 90% is type 1 collagen). Mature collagen fibres can be clearly are seen on the seeded sample containing 5% CG107 and on the seeded sample without glass ("BL"). The amount of collagen present is similar to the positive control sample ("OS"). Collagen type I staining was also detected on the seeded hydroxyapatite ("HA"), however visual inspection confirmed that this was intracellular and not the mature form seen for the other samples. These results suggest that a porous polymer-glass composite material comprising PCL/PLLA (80/20) polymer material containing 5% CG107, and porous PCL/PLLA (80/20) polymer without glass, both support osteoblast production of collage type 1 to a greater extent than hydroxyapatite. Figure 12 shows osteocalcin staining on cells attached to scaffolds or grown on tissue culture plastic.
Osteocalcin is one of the most bone specific proteins, found in bone prior to mineralization. The strongest staining again was seen on seeded porous composite materials comprising PCL/PLLA (80/20) and containing 5% CG107. As can been seen in Figure 12, the staining for this sample was greater than that observed for the blank porous polymer sample ("BL"), the hydroxyapatite sample, and the control samples ("OS" and "NOR"). These results therefore also confirm that osteoblasts are present on the "5%" material and, in addition, indicate that the cells have produced producing an organic matrix upon the scaffold.
Gene Expression
In order to confirm the results of the immunocytochemical staining (Runx2, col1 a1 and osteocalcin, discussed above) gene expression of each of the proteins was analysed. Each of figures 13-15 graphs show that for the "5%" sample, percentage increases gene expression during this period exceeds that observed for the other materials. These results indicate that cells differentiate faster and that more cells become osteoblasts, when ADSCs are seeded on the 5% CG107 sample than on any of the other ADSC seeded samples.
Alkaline phosphatase staining
Alkaline phosphatase is an enzyme expressed by osteoblasts prior to mineralisation. In Using the Leukocyte Alkaline Phosphatase Kit (in the manner discussed above), alkaline phosphatase present in each of the samples was stained dark blue. As exemplified by the images shown in Figures 16 and 17, alkaline phosphatase was observed to be present on all seeded sample, after exposure to osteogenic media. The porous PCL/PLLA 80/20 polymer material containing 5% CG107 showed strongest staining.
Alizarin Red staining
Calcium phosphate crystals appear once osteoblasts produce a mature matrix capable of supporting mineralisation, and may be stained using alizarin red dye.
Figure 18 shows alizarin red staining on samples on day 14. The porous PCL/PLLA (80/20) polymer material containing 5% CG107 showed even staining over whole the whole sample scaffold, whereas the blank sample ("BL") of the same polymer material without glass showed uneven staining.
As expected, the hydroxyapatite sample showed deep staining throughout, due to its composition. The same result was observed for the sample which was not seeded. Von Kossa staining
The Von Kossa stain also detects phosphate which is present when osteoblasts produce a mature matrix capable of mineralisation. Figures 19 and 20 show dark staining in the centre of the porous composites material comprising PCL/PLLA (80/20) polymer material and 5% CG107. Similar staining was seen on the blank porous polymer sample (no glass), but the staining was not as strong as 5%.
As expected, the staining of the hydroxyapatite sample was again very strong.
The results of the Von Kossa staining are therefore consistent with the alizarin red staining results.
Post Sterilisation Testing
In order to be suitable for use as a tissue scaffold, it is vital that the porous polymer- glass composite material can be subjected to sterilization using standard industrial procedures and still retain the required physical properties. For this reason a sample of porous material containing PCL/PLLA (80/20) and 5% CG107 (and prepared using porogen leaching method (2)) was sterilised using gamma irradiation and the samples tested using differential scanning calorimetry.
There was no visual change to the sample after sterilisation and the porosity and average pore size did not change significantly. In addition, DSC thermal analysis confirmed the melting point remained unchanged (Figures 21 and 22).
In vitro testing confirmed the samples are still functional but required to be wetted with buffer solutions (Figures 23 and 24). Samples of the 5% CG107 material which had been sterilised were seeded with ADSCs and incubated over a period of one day, the media was seen to penetrate the conditioned samples seen as pink staining (Figure 23). Cell counts were taken after 2, 4, 6 and 8 hours (Figure 24). Data and images were acquired on gamma sterilized materials seeded without wetting, gamma sterilised materials conditioned with PBS and Denatured Ethanol (IMS, 70%) and conditioned with PBS.
The results show that all samples remained functional, but that re-wetting of the materials with buffer solution after sterilisation was beneficial. It is anticipated that re- wetting would occur naturally in vivo or during surgery. In Vivo Testing- Peri-operative observations
During the pre-operative clinical examinations of the sheep, no abnormalities were observed. Surgery was performed according to the previously described procedure. No major difficulties were encountered during the surgical procedure. It should be noted that the cylindrical shape of the test articles was easily maintained at implantation. No particular peri-operative observations were noted (see table 8). The diameter of the defects was 4 mm in diameter and 9 to 1 1 mm length
Table 8 Peri-operative observations at implantation
Figure imgf000039_0001
Left 4 4.0 10.0 NTR
Humerus
Right 5 4.0 10.0 NTR
NTR: Nothing to report
Follow up
Body weight data
Body weight data are presented in Table 9. The body weight of sheep 1 slightly decreased (1 kg) between implantation and termination. This loss of body weight was low and not associated to clinical abnormality and was therefore considered as not clinically significant.
Table 9 - Body weight data
Figure imgf000040_0001
Clinical observations
Detailed clinical observations are presented in Table 10. One and ten days following implantation, Sheep 1 and sheep 2 respectively showed abnormal feces. This clinical sign was observed for three days. A partial food diet was given for 2 days. No other clinical abnormalities were observed during the whole follow-up period in any sheep
Table 10 - Follow-up
Figure imgf000041_0001
Macroscopical observations at termination
Detailed macroscopical observations at termination are reported in Table 1 1 . No signs of adverse local effects were observed at the level of the implanted sites 1 . Sheep 1 showed hematoma at the level of the subcutaneous tissue suture. This observation was considered to be related to the surgery. The colouration of the bone surrounding the test articles was slightly darker in 4 (two femoral and one humeral) out of 16 sites and slightly yellow in 6 (femoral) out of 16 sites. Four sites presented red blots on the surrounding bone. For the other sites, the coloration was similar to non-implanted bone. A soft tissue was observed on the test article in 4 (three femoral and one humeral) out of 16 sites. All the implanted test articles showed a red coloration.
Table 11 Macroscopical observations at termination
Bone tissues
Site Soft tissues
Color Observations
Slightly yellow
Red coloration with several
of the implant
Femur Left NTR Hard red spots (n=5,
Implant slightly 1 to 5 mm
over level diameter)
Figure imgf000042_0001
Slight red coloration of
1 NTR Hard Similar to bone the implant
Implant hardly visible
Dark red coloration of the implant
2 NTR Hard Similar to bone
Right Implant slightly under level (around 1 mm)
Similar to bone
Red coloration Red spots (7 x
of the implant 2 mm and 3
3 NTR Hard Implant slightly mm diameter)
over level on the cranial
(around 1 mm) side
Slight red coloration of the implant
Left 4 NTR Hard Similar to bone
Soft tissue covering the
Humerus
implant
Similar to bone
Slight red Slightly dark
Right 5 NTR Hard coloration of around the
the implant implanted site
Histopathological analysis
A total number of 16 sections were analyzed. The individual semi-quantitative data and identifications of the photomicrographs are reported in Table 12.
Non-implanted test article
The non-implanted test article did not withstand the technical preparation and appeared fully dissolved in the embedding media.
Implant sites
The test article was dissolved but left footprints evoking an open porous material. The pores of the test article were moderately invaded by a markedly vascularized fibromesenchymal tissue. The fibromesenchymal tissue was infiltrated by macrophages and multinucleated giant cells of moderate grade, along with a slight grade of lymphocytes. Slight signs of material degradation, mediated by the phagocytizing cells were observed. The level of local inflammation was likely influenced by the material degradation process. Direct bone-material contact could not be established as the test article material could not be observed. Bone ingrowth (defined here as newly formed bone of moderate grade) was frequently observed within the peripheral portion of the test article. Surprisingly, in 2 sites (sheep 2, Left 2 and 3) out of 16, there was a deep bone ingrowth (marked grade) within the defect associated with a complete degradation of the test article and resumed local inflammation (no inflammation). No obvious particulate formation or peripheral dissemination of test article was detected. No evidence of cytotoxicity was observed.
Table 12 - Semi-quantitative histopathological analysis
Figure imgf000044_0001
Figure 26 shows representative microscopal pictures of the implanted sites after 4 weeks. The regions of the pictures are labelled as follows:
10 - bone ingrowth 1 1 - marked bone ingrowth
12 - Vascularised fibromesenchymal tissue
14 - bone marrow Summary
It has been shown that the porogen leaching method provides porous polymer-glass composite materials with an optimum porosity (narrower pore size distribution, elimination or large voids) and optimal handling properties (improved mechanical stiffness).
For use as a tissue scaffold for the growth or repair of bone or cartilage, the materials should preferably comprise a soluble glass with a solution rate of less than 0.01 mg.cm" 2.hr"1. Solution rates at or below these values are such that the glasses resorb over a period of 6-18 months, which is ideal for tissue growth and regrowth. It has been shown that glasses with fast solution rates are not suitable for promotion of cell growth particularly bone cells.
It has been shown that CG107 is a particularly suitable soluble glass material, for use in tissue scaffolds.
It has been shown that the acidic environment created by soluble glass materials is cytotoxic, and that materials comprising high proportions of the soluble glasses are not suitable for use as tissue scaffolds.
It has been demonstrated that porous polymer glass composite materials of the present invention are able to support osteogenic differentiation of multipotent cells to a greater extent than porous polymer materials without glass, or hydroxyapatite materials. The materials of the present invention also promote greater attachment and proliferation of cells than porous polymer materials without glass, or hydroxyapatite materials.
Similarly, greater mineralisation and deposition of a more extensive organic matrix was also shown for the porous polymer-glass composite materials of the present invention. The in vivo study showed the test article did not induce any major adverse local tissue effect and showed in average a moderate bone ingrowth after 4 weeks of intraosseous implantation

Claims

A porous polymer-glass composite material comprising;
- at least 80wt% of a biodegradable polymeric material; and
- a water soluble glass.
A porous polymer-glass composite material for use in a method of treatment by surgery, comprising;
- at least 80wt% of a biodegradable polymeric material; and
- a water soluble glass.
A porous polymer-glass composite material according to claim 2, for use as a tissue scaffold.
A porous polymer-glass composite material according to claim 2 or claim 3, for use in a method of bone growth or repair, or for use in a method of cartilage growth or repair.
A porous polymer-glass composite material according to any preceding claim, comprising less than around 5wt% of water soluble glass.
A porous polymer-glass composite material according to any preceding claim, wherein the water soluble glass may is a water soluble phosphate glass.
A porous polymer-glass composite material according to claim 6, wherein the phosphate glass comprises zinc.
A porous polymer-glass composite material according to claim 6 or claim 7, wherein the phosphate glass material has the formula Ab(Ml 20)x(M"0)y(P205)z wherein; M1 is a metal from group I of the periodic table;
- M" is a divalent metal;
- A is one or more further materials, typically oxide materials, and b > 0 and wherein (x+y+z+b) = 1 . A porous polymer-glass composite material according to claim 8, wherein one or more of;
- M" is a metal from group II of the periodic table, or zinc
b > 0 and A comprises one or more further metal or metalloid oxide materials
- the phosphate glass comprises boron, silver, copper and/or zinc
10. A porous polymer-glass composite material according to any preceding claim, wherein the water soluble glass is in the form of flakes or fibres.
1 1 . A porous polymer-glass composite material according to any preceding claim, wherein particles of water soluble glass have an average smallest dimension of between 5μηι-50μΓΤΐ.
A porous polymer-glass composite material according to any preceding claim, wherein the dissolution rate of the water soluble glass is less than 0.05 mg.cm" 2.hr"1
A porous polymer-glass composite material according to any preceding claim, wherein the biodegradable polymeric material comprises polycaprolactone (PCL) and/or polylactic acid (PLA).
14. A porous polymer-glass composite material according to any preceding claim, having an open, interconnected pore network.
15. A porous polymer-glass composite material according to any preceding claim, having an average pore size in the range of 1 00μηι-700μΓΤΐ and/or wherein the sizes of at least 90%, of pores lie in the range from 1 00μηι-700μΓΤΐ.
A porous polymer-glass composite material according to any preceding claim, comprising cells within pores of the porous polymer-glass composite material.
17. A porous polymer-glass composite material according to claim 16, seeded with osteoblasts, chondrocytes or mesenchymal stem cells.
18. A method of preparing a porous polymer-glass composite material, comprising the steps of:
(1 ) preparing a mixture of; a solution of a polymeric material in a first solvent; a glass material, and; a particulate porogen material which is soluble in a second solvent;
(2) removing substantially all of the first solvent to produce a polymer-glass composite material, having particles of porogen material dispersed therein; and
(3) removing substantially all of the porogen material by washing the polymer- glass composite material in the second solvent, to produce a porous polymer- glass composite material.
19. A method according to claim 18, wherein the dissolution rate of the glass is less than 0.05 mg.cm"2.hr"1 and the dissolution rate of the porogen material is in the range from 5-10mg.cm"2.hr"1 in the second solvent.
20. A method according to claim 18 or claim 19, wherein the second solvent is water.
21 . A method according to any one of claims 18 to 20, wherein the first solvent is a volatile organic solvent.
22. A method according to any one of claims 18 to 21 , comprising removing the first solvent by freeze drying.
23. A method according to any one of claims 18 to 22, wherein the glass is in the form of flakes or fibres.
24. A method according to any one of claims 18 to 23, wherein the polymeric material comprises biodegradable polymer material.
25. A method according to any one of claims 18 to 24, comprising seeding the porous polymer-glass composite material with cells, for example osteoblasts, chondrocytes or mesenchymal stem cells.
26. A method according to claim 25, comprising differentiating mesenchymal stem cells.
27. A porous polymer-glass composite material obtained or obtainable by the method of any one of claims 18 to 26.
28. A structure composed of porous polymer-glass composite material according to any one of claims 1 to 17 or 27, having a smallest dimension of greater than 20mm.
29. A method of treatment by surgery, comprising implanting an implant into a subject, the implant comprising a porous polymer glass composite material according to any one of claims 1 to 17 or 27, or a structure according to claim 28.
30. A method according to claim 29, comprising obtaining cells from a subject, seeding the porous polymer-glass composite material with the said cells, and implanting an implant an implant into a subject.
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Citations (3)

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