WO2000067672A2

WO2000067672A2 - Tissue engineering for making tubular implants

Info

Publication number: WO2000067672A2
Application number: PCT/EP2000/004412
Authority: WO
Inventors: Mark Eastwood
Original assignee: University Of Westminster
Priority date: 1999-05-05
Filing date: 2000-05-04
Publication date: 2000-11-16
Also published as: WO2000067672A3; GB9910377D0; AU5394300A

Abstract

A method for making a tubular structure for surgical implantation into a donor host, characterised in that it comprises: taking a sample of appropriate tissue from the donor host; extracting substantially all cells therefrom; growing the cells in tissue culture; innoculating a generally planar scaffold with the cultured cels; subjecting the innoculated scaffold to further tissue culture; removing the scaffold from tissue culture once the innoculated cells are established; cutting the neo-tissue to a desired profile for the intended use; joining and sealing the edges of the generally planar neo-tissue to form a tubular structure; and subjecting the tubular structure to physiological load conditions until the neo-tissue has substantially similar histological appearance and mechanical properties to the naturally-occurring counterpart is disclosed.

Description

"Tissue Engineering"

This invention relates to tissue engineering; more particularly, it relates to the creation of replacement body tissue and organs or parts thereof for surgical implantation back into a donor host.

Tissue engineering is a new science that combines the application of mechanical engineering with biology. The basic concept is to grow, say, an organ for subsequent implantation under sterile conditions in a laboratory. The implanted organ, which is grown using the patient's own cells on a scaffold or substrate, differs from conventional drug and transplantation therapy as the engineered organ becomes integrated in the patient's body without an immunological response, i.e. rejection. Not only would the implant be self-repairing, but also there would be growth potential. Commonly, commercial tissue culture is based on neonatal foreskin cells and so, being non-specific, foreign body reactions may be anticipated. Tissue culture techniques have enabled cells to be grown outside of the human body for many years, but it has only been in the past few years that the prospect of growing fully functional three-dimensional tissue engineered constructs has become a reality. Major advancements have been made in this science by the interdisciplinary research being carried out by engineers and biologists.

Significant advances in surgical technique have enabled many life saving procedures to be developed, but this has, in many cases, highlighted a serious shortage of graft or donor tissue. This scale of this problem highlights the potential for tissue engineering and the

requirement for tubular engineered organs in particular. For some years, the means has existed to create laminar sheets of a number of body organ tissues, (see, for example, Bell, E. , et al, (1979), Proc. Nat. Acad. Sci. 76, 1274- 1278; Bellows, C , et al, J. Cell Sci. 58: 125-138; Bellows, C , et al (1982), J Ultrastruct Res 78: 178-192; Bouvard, N. , et al, (1992), Biochem Cell Biol 70: 34-42; Wang, C.K. , et al, J. Invest Dermatol 2000 Apr. ; 114(4): 674-680; Yang, E.K. , et al,

Artif Organs 2000 Jan; 24(1): 7-17; Duplan-Perrat, F. , et al, J Invest Dermatol 2000 Feb; 114(2): 365-370; Kim, B.M. , et al, Br J Plast Surg. 1999 Oct; 52(7): 573-578; and Michel, M. , et al, In Vitro Cell Dev Biol Ani 1999 Jun; 35(6): 318-326), which may be advantageously employed for various uses. Common uses are the growth of skin for burns and other accident victims requiring skin grafts, (see, for example, Bowering,

C.K. , J Cutan Med Surg. 1998 Dec; 3 Suppl l:Sl-29-32. Review; Eaglstein, W.H. , J Dermatol. 1998 Dec; 25(12):803-804. Review; Mansbridge, J., et al, Tissue Eng. 1998 Winter; 4(4): 403-414; Jiang, W.G. , and Harding, K.G., Int J Mol Med. 1998 Aug; 2(2): 203-210; Grey, J.E. , et al, J Wound Care. 1998 Jul; 7(7): 324-325; Edmonds, M.E. , et al, Diabet Med. 1997 Dec; 14(12): 1010-1011; Νaughton, G. , et al, Artif Organs. 1997

Νov; 21(11): 1203-1210; Kirsner, R.S. , J Dermatol 1998 Dec; 25(12): 805-811; Medina, J. , et al, Toxicol Appl Pharmacol. 2000 Apr 1; 164(1): 38-45; Flores, F. , et al, Ann Intern Med. 2000 Mar 7; 132(5): 417-418; Wickware, P. , Nature 2000 Jan 27; 403 (6768): 466; and Falabelta, A.F. , et al, Arch Dermatol. 1999 Oct; 135(10): 1219-1222). Sub dermal tissue may also be grown and employed to fill the cavities of acute wounds,

(see, for example, Wang, J.C. , and To, E.W. , Br J Plast Surg. 2000 Jan; 53(1): 70-72; Winfrey, M.E. , et al, Dimens Crit Care Nurs. 1999 Jan-Feb; 18(1): 14-20. Review; and Boyce, S.T. , et al, J Burn Care Rehabil. 1999 Nov-Dec; 20(6): 453-461), caused by the excision of cancerous and other tumours and certain other growths. Sheets of tissue may be grown as thin layers on a number of surfaces, (see, for example, Brunette, D.M. and Chehroudi, B. , J Biomech Eng. 1999 Feb; 121(1): 49-57. Review; den Braber, E.T. , et al, J Biomed Mater Res. 1998 May; 40(2): 291-300; Rajnicek, A. , and McCaig, C , J Cell Sci. 1997 Dec; 110 (Pt 23): 2915-2924; Rajnicek, A., et al, J Cell Sci. 1997 Dec; 110 (Pt 23): 2905-2913; Damji, A. , et al, Exp Cell Res. 1996 Oct 10; 228(1): 114-124; Qu, J. , et al, Oral Dis. 1996 Mar; 2(1): 102-115; Oakley, C , and Brunette, D.M. , Biochem Cell Biol. 1995 Jul-Aug; 73(7-8): 473-489; and Wojciak- Stothard, B. , et al, Cell Biol Int. 1995 Jun; 19(6): 485-490), or substrates as long as these are immersed in a suitable culture medium and culture conditions are carefully controlled.

Thicker layers of tissue may be grown by introducing, as a growing substrate, a sheet of sponge or gel or bio-degradable material, (see, for example, Andre-Frei, V. , et al, Calcif Tissue Int. 2000 Mar; 66(3): 204-211; Werkmeister, J.A. , and Ramshaw, J.A. , Clin Mater. 1992; 9(3-4): 137-138; Langer, R., Ace Chem Res. 2000 Feb; 33(2): 94-101; Fu, K. , et al, Pharm Res. 2000 Jan; 17(1): 100-106; Shastri, N.P. , et al, Proc Νatl Acad Sci

U S A. 2000 Feb 29; 97(5): 1970-1975; Elisseeff, J. , et al, Plast Reconstr Surg. 1999 Sep; 104(4): 1014-1022; Kim, B.S. , et al, Biomaterials. 2000 Feb; 21(3): 259-265; and Serre, CM. , et al, Biomaterials. 1993; 14(2): 97-106), into the culture medium. The growing cells then attach to this material and grow together, eventually forming a sheet of tissue. One limiting factor on thickness is the production of the substrate sheets.

These sheets may be made from animal derived collagen or biodegradable polymers, such as polyglycolic acid, poly(l)lactic acid. Current techniques permit only the production of flat sheets of limited size and thickness. Thus, although it is possible to reconstruct skin, muscle and certain other organ tissues in flat sheets, it has not been possible to create such organ tissue having inherent compound curves or of a tubular configuration that replicate the mechamcal strength and many other characteristics of a natural counterpart organ.

Current use of fixed porcine or human donated heart valves for replacement of human heart valves eventually leads to immunological rejection, calcification and finally disfunctionality, (see, for example, Christie, G.W. , et al, Semin Thorac Cardiovasc Surg.

1999 Oct; 11(4 Suppl 1): 201-205; Christie, G.W. , and Barratt-Boyes, B.G. , Ann Thorac Surg. 1995 Aug; 60 (2 Suppl): S195-199; Mayne, A.S. , et al, J Thorac Cardiovasc Surg. 1989 Aug; 98(2): 170-180; De Biasi, S., et al, Int J Artif Organs. 1980 Sep; 3(5): 271-

276; Sung, H.W. , et al, Biomaterials. 1999 Oct; 20(19): 1759-1772; Gloeckner, D.C. , et al, ASAIO J. 1999 Jan-Feb; 45(1): 59-63; Sadler, L. , et al, Br J Obstet Gynaecol.

2000 Feb; 107(2): 245-253; Mestres, C.A. , and Pomar, J.L. , J Heart Valve Dis. 2000 Jan; 9(1): 169; Melina, G. , et al, J Heart Valve Dis. 2000 Jan; 9(1): 97-103; Zund, G. , et al, Eur J Cardiothorac Surg. 1997 Mar; 11(3): 493-497; Shinoka, T. , et al, Ann

Thorac Surg. 1995 Dec; 60 (6 Suppl): S513-516; Hoerstrup, S.P. , et al, Ann Thorac Surg. 1998 Nov; 66(5): 1653-1657; Shinoka, T. , et al, Circulation, 1996 Nov 1; 94 (9 Suppl): 11164-168; Shinoka, T. , et al, Circulation 1997 Nov 4; 96 (9 Suppl): 11-102-107; Lehner, G. , et al, Eur J Cardiothorac Surg. 1997 Mar; 11(3): 498-504; Eberl, T. , et al, Ann Thorac Surg. 1992 Mar; 53(3): 487-492; Stock, U.A. , et al, J Thorac Cardiovasc

Surg. 2000 Apr; 119(4): 732-740; Zund, G., et a], Eur J Cardiothorac Surg. 1998 Feb; 13(2): 160-164; Fischlein, T. , and Fasol, R. , J Heart Valve Dis. 1996 Jan; 5(1): 58-65; and Bengtsson, L . , and Haegerstrand, A.N. , J Heart Valve Dis. 1993 May; 2(3): 352-

356). Given the limitations of current techniques as described above, it has not been possible until now to create essentially tubular structures, such as arteries, or arterial valves, such as the aortic heart valve. Neither has it been possible to create lengths of tendon sheath, oesophagus, trachea, bronchus, bronchioles, colon, intestine, bladder and urethra or other such structures.

The present invention provides a method for making a tubular structure for surgical implantation into a donor host, characterised in that it comprises: taking a sample of appropriate tissue from the donor host; extracting substantially all cells therefrom; growing the cells in tissue culture; innoculating a generally planar scaffold with the cultured cells; subjecting the innoculated scaffold to further tissue culture; removing the scaffold from tissue culture once the innoculated cells are established; cutting the neo- tissue to a desired profile for the intended use; joining and sealing the edges of the generally planar neo-tissue to form a tubular structure; and subjecting the tubular structure to physiological load conditions until the neo-tissue has substantially similar histological appearance and mechanical properties to the naturally-occurring counterpart.

Also, the present invention provides a similar method for making a tubular tissue structure which is a blood vessel or a heart valve or a part thereof for surgical implantation into a donor host, characterised in that it comprises: taking a sample of appropriate tissue from the donor host; extracting substantially all cells therefrom, starting with endothelial cells; growing the endothelial cells and the other cells in separate tissue cultures; innoculating a generally planar scaffold with the cultured non-endothelial cells; subjecting the innoculated scaffold to further tissue culture; removing the scaffold from tissue culture once the innoculated cells are established; applying a layer of the cultured endothelial cells to at least one side of the scaffold; subjecting the scaffold to further tissue culture; removing the resulting neo-tissue from tissue culture once the endothelial cells are established on at least one side; cutting the neo-tissue to a desired profile for the intended use; joining and sealing the edges of the generally planar neo-tissue to form a tubular structure having endothelial cells at least on the inside; and subjecting the tubular structure to physiological load conditions until the neo-tissue has substantially similar histological appearance and mechanical properties to the naturally-occurring counterpart.

In a preferred embodiment, the latter tubular structure has internal features resulting from the form of the cut profile. Alternatively, suitably-shaped endothelial cell-coated appendages may be added to an endothelial cell-coated side of the neo-tissue prior to formation of the tubular structure. The result is equivalent whether, say, the leaflets in the case of a heart valve are part of the cut profile or are cut separately and are joined and sealed thereto.

Although the present invention is widely applicable, the starting point for the method will often be a sample of vascular material, such as saphenous vein or artery, in order to produce a blood vessel or a heart valve. The tissue sample to be taken is dependent upon the desired tubular structure.

When forming a tubular structure of the desired size and shape, the cut edges of the neo- tissue are preferably joined and sealed using at least one suture and/or fibrin sealant. Generally, a pulse duplicator and/or a mock circulation system may be used to simulate physiological load conditions in the last stage of the present method when applied to the production of a blood vessel or a heart valve. Otherwise, a TCM (tubular culture model) apparatus, for example, may be used to provide appropriate conditioning.

Having indicated the scope of the present invention, it will now be further illustrated and exemplified.

To create the desired tubular structures, a composite sheet of tissue is first grown on a

biodegradable matrix by the generally conventional methods outlined below. In general terms, the sheet of tissue is cut to a suitable size and then folded into a tube. The edges

are then temporarily joined using sutures, surgical staples or any one of the other methods known for joining tissue edges. The resultant structure is then immersed in a subsequent

culture medium for maturing. This maturing, when the cells in the structure are induced to align themselves in set directions and firmly to seal the joint in the tube, is conducted

under the influence of an applied mechanical force through the maturing construct while under culture conditions.

A normal healthy aortic valve has been fully sectioned using standard histological

techniques in section sizes of 20 microns (μm). In addition to this, mechamcal test data has been collected relating to the strength and stiffness of normal healthy aortic valves.

From this complete analysis, there has been determined the percentage constituents of the

structural proteins that consist the connective tissue of a normal healthy aortic heart valve.

The present method will produce a tissue engineered construct that has the same structural properties and constituents of, for example, collagen, elastin, and smooth muscle as the naturally occurring counterpart.

There is now described, with particular reference to the accompanying drawing, Figure 1, one preferred way of accomplishing the method described above.

Appropriate tissues, e.g. arterial, trachea or colon, from the intended recipient of the completed tissue engineered construct are taken by conventional means to use as the cell source. For example, cells for an arterial tissue engineered construct may be obtained from a small section of the saphenous vein. This will contain the full range of cells required for the final tissue engineered construct. Cells are grown from these tissues as either explants (cell migration) or from a collagenase digestion (complete mixed cell population).

The method for the collagenase digestion for the removal of endothelial cells is commonly as follows: a small section of the saphenous vein from the donor/recipient of the tissue engineered heart valve, for example, is taken as the primary cell source. This section of saphenous vein is cut into small cubes, (typically a 1-3 mm cube). These small cubes of saphenous vein are then incubated at 37 °C for 15-30 minutes in a collagenase solution. During this period of incubation, the endothelial cells become separated from the supporting connective tissue. The collagenase solution containing the endothelial cells is then removed from the culture and placed in a 30 ml sterile universal container and centrifuged at 400 G for 5 minutes to separate the cells from the collagenase solution. The endothelial cells are then re-suspended in 5 ml of complete Ml 99 medium (medium containing 1 % penicillin and streptomycin, 15% fetal bovine serum, 1 % 1-glutamine and 1 % endothelial cell growth factor), removed from the universal container and placed into a 75 cm² tissue culmre flask to which a further 15 ml of complete Ml 99 medium is added. The flask is then placed into an incubator at 37 °C.

When the endothelial cells have formed a confluent mono-layer on the base of the tissue culmre flask (after about 5 days in culmre), the cells are removed by the action of enzymatic trypsin. In this procedure, the medium is poured off, then the cell layer is washed with sterile phosphate buffered solution to remove any traces of Ml 99. 5 ml of 10% trypsin is then added to the culture flask and the flask is returned to the incubator for a further 15 min.. At the end of this period, the attachment plaques and secreted connective tissue that has been produced by the cells has been enzymatically removed and so the cells are free in the suspension. The addition of 20 ml of complete medium (as previously described) renders the trypsin inactive. This solution of cells and complete medium is then removed from the tissue culmre flask and placed into a sterile 30 ml universal container for centrifugation at 400 G to separate the cells from the medium. The old medium is poured off and the cells re-suspended in 15 ml of complete medium. This cell/medium suspension is then placed into a tissue culture flask of 225 cm² surface area and placed in an incubator at 37 °C until a confluent mono-layer of cells has again formed. The medium is changed every 2 days to remove the metabolites and reduce the risk of infection. This process is repeated until a sufficient number of cells have been created in the cell bank.

A mixture of interstitial cells, fibroblastic cells, myo-fibroblastic cells and smooth muscle cells are obtained by the same method of collagenase digestion, except the culture period with the collagenase is extended to 45 minutes.

Such tissue culture options are generally conventional.

Another method of extracting interstitial cells, fibroblastic cells, myo-fibroblastic cells and smooth muscle cells is by the explant migration method, (see, for example, Burt, A.M. ,

and McGrouther, D.A. , Animal Cell Biotechnology Academic Press, New York, pp 150- 168).

Cells are grown to confluence, passage and expanded into larger numbers. Cell types derived from these tissue sources include smooth muscle cells, fibroblasts, endothelial cells, urothelial cells, myofibroblasts, interstitial cells, microvascular endothelial cells and

epithelial cells, for example.

When required for use in the tissue engineered construct, the cells are trypsinised from the culture flasks by the method previously described. The cell/medium suspension is

then centrifuged at 400 G for 5 min, then re-suspended in 5 ml of complete medium to increase cell density. The cell suspension is then innoculated/placed onto a collagen

scaffold sheet to form a cell density of 5,000,000 cells/cm³, (as determined by the reverse engineering of the normal human aortic valve), then placed back into an incubator to

allow the cells to become attached into the scaffold, and reorganise the matrix. This process takes about 21 days depending upon the type of cell and the thickness of the

collagenous scaffold. The medium is changed every 2 days to reduce the risk of infection and to remove the metabolites.

For applications that require an endothelial cell layer (e.g. blood vessels, arteries and heart valves), a layer of endothelial cells (prepared as previously described) is placed onto the surface of the collagenous scaffold, at a density of 500,000 cells/cm². After about 5 days in culmre, a monolayer has formed on the surface of what will form the tissue engineered construct. The material is then further cultured under normal culture conditions to allow complete integration of the various components/substrates/cell types, a process taking typically 14-21 days.

Once the basis for the construct has been culmred, e.g. a generally flat sheet of collagenous material complete with the required cell types in the correct proportions, the next stage of making the tissue-engineered construct may begin. In the case of the heart valve, the sheet of neo-tissue is cut into a pre-determined shape (as illustrated in Figure 1) that when folded and connected together with sutures and sealed with fibrin sealant will form the shape of the heart valve, including the leaflets. The final position for the leaflets after suturing is shown by the dotted lines in Figure 1. Alternatively, separate leaflets may be added to the basic profile. For tubular organs without internal features the cell-impregnated collagenous sheet is formed into a basic mbular shape, again sutured and sealed with a fibrin sealant.

To obtain the correct mechanical and physiological properties, further culture of the tissue engineered construct is required that mimics the physiological conditions that the counterpart organ would be subjected to in vivo. In the case of a tissue engineered blood vessel or heart valve, pressure gradients would be applied to the culture medium that flows through the organ in culmre mimicking the systolic and diastolic pressure. A preferred way of achieving this varying pressure gradient is by the use of a heart bypass blood circulation machine or similar mock circulation apparatus. This has the effect of placing the cells under physiological loads associated with the normal function. Other mbular organs may be subjected to regimes of applied mechamcal tension and shear to mimic those found during in vivo use. This precisely controlled mechanical load is applied by using a machine such as the tensioning-Culture Force Monitor and/or a TCM apparatus, (see, for example, Mudera, V.C. , et al, Cell Motil Cytoskeleton 2000 Jan; 45(1): 1-9; Porter, R.A. , et al, Wound Repair Regen. 1998 Mar-Apr; 6(2): 157-166;

Eastwood, M. , et al, Proc Inst Mech Eng [H] . 1998; 212(2): 85-92. Review; Eastwood, M. , et al, Cell Motil Cytoskeleton. 1998; 40(1): 13-21; Brown, R.A. , et al, J Cell Physiol. 1998 Jun; 175(3): 323-332; Brown, R.A. , et al, J Cell Physiol. 1996 Dec; 169(3): 439-447; Eastwood, M. , et al J Cell Physiol. 1996 Jan; 166(1): 33-42; and Eastwood, M. , et al, Biochim Biophys Acta. 1994 Nov 11; 1201(2): 186-192). The effect of this final culture phase that incorporates precisely controlled mechanical/physical stimulation is to orientate the resident cells into the correct position and alignment and to stimulate the cells into synthesising collagen, elastin, fibronectin, proteoglycans and other structural proteins in addition to growth factors, cytokines and chemokines. This process typically takes up to a further 21 days of culture in an appropriate culmre medium

(Ml 99 as previously described) under normal culmre conditions. At the end of this phase, the original collagen scaffold has usually been absorbed by the resident cells and a fully functional matrix has been created. This matrix usually has substantially the same physiological strength as the namrally produced counterpart, also it has similar histological appearance i.e. smooth muscle, collagen and elastin in the microstructure with a distribution of cells in the matrix similar to that found in the natural counterpart.

Once the tissue engineered construct has been completed, it may be implanted into the patient.

In the production of the tissue engineered construct, a collagenous scaffold may be replaced with a biodegradable polymer, such as poly-lactic acid/poly-glycolic acid on a "Dacron" substrate or other suitable material. The basic tissue culmre steps are generally conventional and may be varied within the competence of those skilled in the art. The main features of the present method are the formation of the mbular structure, with or without internal features, and the subsequent conditioning thereof.

Claims

Claims:

1. A method for making a tubular structure for surgical implantation into a donor host, characterised in that it comprises: taking a sample of appropriate tissue from the donor host; extracting substantially all cells therefrom; growing the cells in tissue culmre; innoculating a generally planar scaffold with the culmred cells; subjecting the innoculated scaffold to further tissue culture; removing the scaffold from tissue culture once the innoculated cells are established; cutting the neo- tissue to a desired profile for the intended use; joining and sealing the edges of the generally planar neo-tissue to form a mbular structure; and subjecting the mbular structure to physiological load conditions until the neo-tissue has substantially similar histological appearance and mechamcal properties to the naturally- occurring counterpart.

2. A method for making a tubular tissue structure which is a blood vessel or a heart valve or a part thereof for surgical implantation into a donor host, characterised in that it comprises: taking a sample of appropriate tissue from the donor host; extracting substantially all cells therefrom, starting with endothelial cells; growing the endothelial cells and the other cells in separate tissue cultures; innoculating a generally planar scaffold with the cultured non-endothelial cells; subjecting the innoculated scaffold to further tissue culmre; removing the scaffold from tissue culmre once the innoculated cells are established; applying a layer of the culmred endothelial cells to at least one side of the scaffold; subjecting the scaffold to further tissue culture; removing the resulting neo-tissue from tissue culmre once the endothelial cells are established on at least one side; cutting the neo-tissue to a desired profile for the intended use; joining and sealing the edges of the generally planar neo-tissue to form a tubular structure having endothelial cells at least on the inside; and subjecting the tubular structure to physiological load conditions until the neo-tissue has substantially similar histological appearance and mechanical properties to the naturally-occurring counterpart.

3. A method as claimed in claim 2 wherein the tubular structure has internal features resulting from the form of the cut profile.

4. A method as claimed in claim 2 or claim 3 wherein the tubular structure has internal features resulting from the addition of endothelial cell-coated appendages to an endothelial cell-coated side of the neo-tissue prior to formation of the tubular structure.

5. A method as claimed in any of claims 2 to 4 wherein a sample of vascular material, such as saphenous vein or artery, is taken.

6. A method as claimed in any of claims 1 to 5 wherein a TCM apparatus is used to provide physiological load conditions.

7. A method as claimed in any of claims 2 to 6 wherein a pulse duplicator and/or a mock circulation system is used to provide physiological load conditions. A method as claimed in any of claims 1 to 7 wherein at least one suture and/or fibrin sealant is used to join and seal the edges when forming the tubular structure.