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Effect of fibrin glue coating on the formation of new cartilage
Effect of Fibrin Glue Coating on the Formation of New Cartilage X. Fei, B.-K. Tan, S.-T. Lee, C.-L. Foo, D.-F. Sun, and S.-E. Aw
A
RTICULAR cartilage has little capacity to regenerate itself. Pain and disability associated with cartilage damage in joints are difficult to treat and this has prompted the search for means of restoring hyaline cartilage on articular surfaces. One promising method entails implanting autologus chondrocytes on denuded joint surfaces.1–3 To obtain sufficient quantities of chondrocytes for this purpose, researchers have employed a variety of substances to promote the growth of chondrocytes in vitro.4 –10 Examples include the use of serum-free media,11 growth factors,12 vitamin A, C, and D,13,14 synovial fluid,15 and calcium.16 Others analyzed the components of extracellular matrix (ECM) such as collagen type II, fibronectin, and laminin and investigated their effects on chondrocyte activity.17,18 Reports indicate that these interact with integrin receptors on cell membranes. Qi and Scully19 showed that type II collagen regulated chondrocyte activity by mediating the response of chondrocytes to transforming growth factor-beta 1. Similarly, Nehrer et al20 reported that the type and pore size of collagen influenced the behavior of canine chondrocytes. Furthermore, it was reported that chondrocytes in suspension produced more ECM than cells seeded on flat surfaces.21,22 Fibrin glue is widely used as a sealant in surgery,23 commonly after dural repair, liver resection, and in burn surgery. Reports suggest that fibrin glue promotes neovascularization and connective tissue regeneration.24 –27 In this study, fibrin glue was used to coat cell-polymer constructs consisting of chondrocytes seeded on polyglactin (PGA) and poly-L-lactic acid (PLLA) polymers. The aim was to investigate the effect of fibrin glue coating on chondrocyte growth and extracellular matrix production in vitro and in vivo.
cartilage was used principally for in vitro studies, whereas rabbit cartilage was used for in vivo experiments.
Polymer Constructs PGA fibers were obtained from unbraided surgical sutures (0-0 Vicryl suture material, Ethicon, Somerville, NJ). Random tangles of PGA fibers were placed in cylindrical moulds and bonded with 2% of PLLA in methylene chloride.30 Scaffolds consisting of a mixture of PGA and PLLA (50% to 50%) (PGLA) (Medisorb Technologies International LP) were created using the solventcasting particulate-leaching technique.29 The constituents of fibrin glue were bovine fibrinogen (Sigma) in aprotinin solution and thrombin (Sigma) in phosphate-buffered solution containing calcium chloride.31 Fibrin glue was cast in cylindrical moulds to produce semisolid disks. To create coated constructs, polymer scaffolds were cast with fibrin glue in similar moulds. All constructs were irradiated with 2.5 ⫻ 106 rads for sterilization. Cultured chondrocytes were injected into the constructs and the composite units were used for the in vitro and in vivo studies.
Laboratory Study
MATERIALS AND METHODS Chondrocytes
Ninety units, 0.3 cm in diameter and 0.5 cm in thickness, were divided into three groups of 30 units each and cultured in glass bottles for 3, 13, and 30 days. Each bottle contained a magnetic stir bar and was maintained in a CO2 incubator. Each group of 30 constructs was subdivided into six subgroups (5 units per subgroup). For in vitro culture, the units were placed in individual pockets on a cotton sheet. This customized carrier was in turn suspended in culture by copper suspension wires. The six subgroups were chondrocyte-PGA with fibrin glue coating (group I), without fibrin glue coating (group II), chondrocyte-PGLA with fibrin glue coating (group III), without coating (group IV), PGA-fibrin glue without cells (group V), and PGLA-fibrin glue without cells (group VI). Each cell-polymer unit contained approximately 1.5 ⫻ 105 chondrocytes. At predetermined time intervals, the cell-polymer constructs were extracted for analysis. They were weighed, frozen, and lyophilized. The units were then digested with 1 mL papain buffer solution (3 mg/mL) at 60°C for 16 to 20 hours. The cell number per sample was estimated by DNA quantification using
Chondrocytes were derived from human cartilage and rabbit pinna. Cartilage harvest was performed under aseptic conditions. The cells were isolated by digestion with type II collagenase as previously described.28,29 These were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY) containing 4500 mg/L glucose and 584 mg/L glutamine, 10% fetal calf serum (FCS; GIBCO), proline (46 g/mL), ascorbic acid (50 g/mL), insulin (5 g/mL), transferrin (125 g/mL), penicillin (100 U/mL), and streptomycin (100 g/mL) (Sigma, St Louis, Mo). Human
From the Departments of Clinical Research (X.F., D.-F.S., S.-E.A.) and Plastic Surgery (B.-K.T., C.-L.F., S.-T.L.), Singapore General Hospital, Singapore. Supported by the Department of Clinical Research, Singapore General Hospital, Singapore. Address reprint requests to Dr Xun Fei, Scientific Officer, Department of Clinical Research, Singapore General Hospital, Outram Rd, Singapore 169608.
0041-1345/00/$–see front matter PII S0041-1345(99)00939-2 210
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 210–217 (2000)
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Fig 1. Histologic appearance of fibrin glue-coated cell-polymer constructs cultivated for 3 days (A), 13 days (B), and 30 days (C). (Toluidine blue staining, magnification ⫻200). Notice the chondrocyte attached to the PGA fiber (small arrow). Cross-section of PGA fiber, which stained positively for ECM, is indicated by the big arrow.
Hoechst 33258 dye.32 Total collagen content was determined based on the hydroxyproline content (determined by reaction with chloramine T and P-dimethylaminobenzaldehyde33). For histologic analysis, the constructs were fixed in 10% formalin, frozen sectioned (5 m thick), and stained with H&E. Toluidine blue was used to stain glycosaminoglycan (GAG). Type I, type II, and type IV collagens were detected by immunohistochemistry.
Animal Study The cell-polymer constructs were transplanted in three rabbits. Each rabbit received four constructs: (1) chondrocyte-PGA, (2) chondrocyte-PGA with fibrin glue coating, (3) chondrocyte-PGLA, and (4) chondrocyte-PGLA with fibrin glue coating. Each unit contained approximately equal number of autologous chondrocytes: rabbit I, 0.8 ⫻ 106 cells each; rabbit II, 5 ⫻ 106 cells each; and rabbit III, 0.3 ⫻ 106 cells each. The animals were euthanized on days 10, 60, and 141, respectively. Prior to transplantation, each construct was encircled with a steel wire for easy identification during retrieval. These cell-polymer constructs were placed subcutaneously (SC) on paravertebral fascia. At harvest, the explants were weighed and measured. Sections were obtained for histologic analysis. Cell survival was evaluated with apoptosis fluorescence immunostaining (Promega, Madison, Wis). Type II collagen was detected with anti-type II collagen immunostaining (green colored) and cell nuclei were labeled with propidium iodide (orange colored). GAG expression was detected with toluidine blue.
Statistical Evaluation The DNA concentration and collagen content were reported as mean ⫾ standard error (M ⫾ SD). Independent t tests were performed to determine statistical significance.
RESULTS Laboratory Study
Histomorphology. At day 3, sections of fibrin glue-coated PGA constructs showed an intact fibrin glue envelope, which appeared bright pink with H&E and bright blue with toluidine blue. Chondrocytes were concentrated at the center of the construct. Dividing cells were found mainly attached to the PGA fibers, whereas nondividing cells were scattered in void spaces. Both spherical and spindle-shaped cells were present. At day 13, the ECM was present and distributed throughout the construct. Uniform staining of the PGA fiber surface was seen, indicating the existence of ECM on the fiber surface. The fiber cores were not stained. On day 30, cross-sections of the fibers were distinctly stained, suggesting that the components of ECM, such as collagen and GAG, had penetrated the core of the fibers. Degraded PGA material with refractive shine was noted (Fig 1). Type 1 and type II collagen can be detected by immunohistochemistry. Sections from uncoated PGA constructs on days 3, 13, and 30 presented with less ECM than in the coated counterparts. The deposition of ECM on the surface of the fibers was observed on day 13 and the penetration of ECM in the cores of the fibers was seen on day 30. The morphology of chondrocytes and the degraded PGA materials were similar to those in the coated units (Fig 2). Physical Properties of Cell-Polymer Constructs
Over 30 days of culture, the wet weight of the coated PGA constructs decreased to 70% of the wet weight on day 3,
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FEI, TAN, LEE ET AL
Fig 2. Histologic appearance of uncoated cell-polymer constructs cultivated for 3 days (A), 13 days (B), and 30 days (C). (Toluidine blue staining, magnification ⫻200). Notice the chondrocyte attached to the PGA fiber (small arrow). Cross-section of PGA fiber, which stained positively for ECM, matrix, is indicated by the big arrow.
whereas the wet weight of uncoated counterparts decreased to 50%. These data suggest that coated constructs retained mass better than uncoated constructs. Biochemical Components
Measured profiles of DNA and collagen content in the fibrin glue-coated and uncoated units are given in Figures 3 and 4. There was a time-dependent increase in DNA content over 4 weeks of cultivation. There was a timedependent increase in DNA content in coated and un-
Fig 3. DNA content in coated and uncoated PGA units (ng/mL). M ⫾ SD; n ⫽ 4, P ⬎ .05.
coated constructs. The collagen content was slightly higher in the uncoated constructs than that in the coated ones. The difference of the amount between the two groups was not significant statistically (P ⬎ .05; Fig 3). The collagen content increased more rapidly in the coated groups than in the uncoated groups. Collagen
Fig 4. Collagen content in coated and uncoated PGA units (g/mL). M ⫾ SD; n ⫽ 4, group t test, P ⬍ .01 on day 30.
FIBRIN GLUE COATING
Fig 5. DNA content in coated and uncoated PGLA units. M ⫾ SD; n ⫽ 4, P ⬍ .05 on day 30.
content in each coated construct on day 30 was 1.00 ⫾ 0.09 g, about 1.37 times that of the uncoated units (0.73 ⫾ 0.05 g). The difference between coated and uncoated groups was statistically significant (P ⬍ .01; Fig 4). In the PGLA samples, the cell numbers increased more rapidly in the coated constructs than in the uncoated ones. The DNA amount on day 30 was 13.0 ⫾ 2.9 ng per construct, approximately twice that of the uncoated ones (6.5 ⫾ 1.3 ng per construct; Fig 5). Similarly, the collagen content was higher in the coated constructs than in the uncoated ones. On day 30, collagen content in the coated constructs was 0.23 ⫾ 0.02 g, about 1.96 times the average collagen content of the uncoated ones (0.12 ⫾ 0.03 g). The difference between the two groups was statistically significant (P ⬍ .01; Fig 6). Animal Study
Cell-polymer constructs buried SC in three rabbits were explanted on days 10, 60, and 141. The four constructs (chondrocytes-PGA, chondrocytes-PGA with fibrin glue coating, chondrocytes-PGLA, and chondrocytes-PGLA with coating) did not demonstrate any obvious difference in volume on day 10. The physical dimensions of the constructs are given in Table 1. On day 60, the size difference was noticeable. The coated PGA construct was thicker and larger (14.1 ⫻ 14.1 ⫻ 6.1 mm), whereas the uncoated was thinner and smaller (7.0 ⫻ 7.0 ⫻ 1.7 mm; Fig 7). The wet weight of the coated PGA construct was approximately 64% of the weight on day 10, whereas its counterpart was only 26%. Similar results were observed for day 141 samples. The coated PGLA construct appeared more solid and was
213
Fig 6. Collagen content in coated and uncoated PGLA units (g/unit). Mean ⫾ SD; n ⫽ 4, group t test, P ⬍ .05 on day 30.
thicker than its counterpart, which appeared membranous (Table 1, Fig 8). The wet weight of the coated PGLA construct was approximately 40% of the weight on day 10, whereas its counterpart was only 16%. Only three units were harvested on day 141. The missing one (PGA with fibrin glue) had extruded due to infection. Histologic sections of coated constructs appeared significantly more compact and homogenous than the uncoated counterparts on day 60. Connective tissue was seen extending centripetally, distributed among clusters of chondrocytes. Chondrocytes at the periphery adjacent to the fibrin glue layer were spherical in shape, closely resembling well-differentiated chondrocytes. However, those located at the core of the unit were spindle shaped. In addition, higher levels of matrix were detected at the periphery, evidenced by deep staining with toluidine blue (Figs 9, 10). Most cells in both groups with or without coating were chondrocytes evidenced by type II collagen labeling. The majority of cells were viable, confirmed by apoptosis immunostaining. Both groups had neovascular ingrowth. The presence of polymer fractions was noted. Sections of the day 141 and coated construct showed grossly the same features, but with less cartilage component and more neovascular ingrowth. In the uncoated construct, the cartilaginous matrix was reduced to a narrow strip. Comparison of PGA and PGLA Cores
The results in this study demonstrated that the PGA cores were better than the PGLA cores in terms of size and chemical components in both fibrin glue-coated and uncoated conditions. For the in vitro study, in the coated
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FEI, TAN, LEE ET AL Table 1. Physical Data of Chondrocyte-Polymer Explants From Rabbit PGA Coated
Rabbits
Duration of Implant (d)
Wet Wt (mg)
1 2 3
10 60 141
1030 666 —
Uncoated
Size (mm)
Wet Wt (mg)
16.1 ⫻ 15.6 ⫻ 5.9 14.1 ⫻ 14.1 ⫻ 6.1 —
790 210 97
PGLA Coated
Size (mm)
Wet Wt (mg)
17.7 ⫻ 16.6 ⫻ 5.4 7.0 ⫻ 7.0 ⫻ 1.7 12.9 ⫻ 10.8 ⫻ 5.1
1053 393 424
constructs with the same initial cell number, the collagen amount in PGA units on day 30 was 3.57 ⫾ 0.26 g/mL, whereas that in the PGLA units was 1.30 ⫾ 0.12 g/mL. In the uncoated constructs, the collagen amount in PGA units was 2.05 ⫾ 0.23 g/mL, whereas that in PGLA counterparts was 0.58 ⫾ 0.05 g/mL. For the in vivo study, in the coated constructs with the same initial size, the size in PGA explant on day 60 was 14.1 ⫻ 14.1 ⫻ 6.1 mm, whereas the size of the PGLA counterpart was 12.6 ⫻ 12.6 ⫻ 4.9 mm. As a further example of an in vivo experiment (Table 2 and Fig 11), three paired cell-polymer constructs without fibrin glue coating, with the same initial cell number and the same size in the operation day, were harvested from two rabbits on day 278. The size of the first pair was 11.1 ⫻ 15.4 ⫻ 2.1 mm (PGA) vs 3.9 ⫻ 3.9 ⫻ 0.4 mm (PGLA). The size of the second pair was 16.6 ⫻ 9.8 ⫻ 1.2 mm (PGA) vs 8.6 ⫻ 7.2 ⫻ 0.7 mm (PGLA). The size of the third pair was 21.5 ⫻ 13.5 ⫻ 0.8 mm (PGA) vs 3.5 ⫻ 3.5 ⫻ 0.2 mm (PGLA). DISCUSSION Effect of Fibrin Glue Coating
In this study, the effects of fibrin glue coating on cell proliferation and collagen synthesis were examined. In vitro, the difference in DNA amount between coated and uncoated constructs was not statistically significant comparing both types of constructs through days 3, 13, and 30. This suggests that fibrin glue did not affect chondrocyte proliferation.
Fig 7. (A) Original PGA construct with fibrin glue coating. (B) Original PGA construct without coating. (C) Coated PGA explant on day 60. (D) Uncoated PGA explant on day 60.
Uncoated
Size (mm)
Wet Wt (mg)
Size (mm)
19.2 ⫻ 16.3 ⫻ 5.0 12.6 ⫻ 11.9 ⫻ 2.7 16.3 ⫻ 12.0 ⫻ 4.9
608 303 101
17.7 ⫻ 14.0 ⫻ 4.4 8.2 ⫻ 4.1 ⫻ 1.2 9.4 ⫻ 7.7 ⫻ 1.4
Cell numbers in uncoated constructs were slightly higher than in coated ones. It may be caused by easier nutrition in uncoated constructs or by the inhibition of cell proliferation by soluble arginine-glycine-aspartate (RGD) peptides in fibrin glue-coated constructs.34 Buckley et al34 reported that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. In PGA constructs, parallel increasing of cell number in both coated and uncoated groups reflected well adhesion and well proliferation of chondrocytes on PGA fibers. In PGLA constructs, the situation is different. DNA content in coated constructs was much higher than in their counterparts on day 30 (13.0 ⫾ 2.9 ng per construct vs 6.5 ⫾ 1.3 ng/mL, P ⬍ .05). The difference indicated that cells were retained in the constructs by the fibrin glue barrier. The difference in collagen content was, however, statistically significant on day 30. This suggests that fibrin glue coating may enhance collagen accumulation or synthesis. Histologic pictures exhibited the higher deposit of ECM in the coated unit than in the uncoated unit. The ECM contained collagen type 1, collagen type II, and GAG. In another experiment, we learned that a greater amount of collagen was detected in culture medium from uncoated constructs (the data are not presented here). We postulate that fibrin glue may play a role in the retention of collagen in constructs. Cell differentiation may be another avenue by which fibrin glue exerts its effects. We noticed that cells adjacent to fibrin glue tended to be spherical, which is the
Fig 8. (A) Original PGLA construct with fibrin glue coating. (B) Original PGLA construct without coating. (C) Coated PGLA explant on day 141. (D) Uncoated PGLA explant on day 141.
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Fig 9. Cluster of chondrocytes adjacent to fibrin glue layer at the periphery of the construct. Notice strong staining with toluidine blue, which indicated high deposition of ECM. The section was taken from a coated chondrocyte-PGA explant on day 60.
more differentiated form capable of synthesizing matrix as was reported by Zanetti and Solursh.35 However, the precise correlation between the cell shape and differentiation is not fully understood. ECM condensation is necessary for durability and the mechanical strength of tissue-engineered cartilage. Cell-cell interactions,36,37 composition of the ECM,38 – 40 changes in cell shape,35 and several specific cytokines41 play a role in this process. In this study, a fibrin glue envelope, resulting in less leakage of cells and ECM, enhanced ECM condensation. Chondrocyte aggregates were formed and the life span
of tissue-engineered cartilage was increased in vitro and in vivo. Effect of the Polymer Cores
Initially, we tried injecting chondrocytes in homogenous fibrin glue units. However, only a part of cells could be retained due to lack of void spaces. We observed that cartilaginous tissue did form in cell-fibrin glue constructs. Polymer cores with fibrin glue coating mimic native condition of chondrocyte aggregation. Fibronectin is the main component of fibrin glue. Tavella et al42 reported that
Fig 10. Cluster of chondrocytes in the center of the construct. Notice weak staining with toluidine blue, which indicated low deposition of ECM. The section was taken from a coated chondrocyte-PGA explant on day 60.
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FEI, TAN, LEE ET AL Table 2. Physical Data of Chondrocyte-Polymer Explants From Rabbits On Day 278 PGA
Pair 1 Pair 2 Pair 3
PGLA
Cell Number Per Unit
Wet Wt (mg)
Size (mm)
Wet Wt (mg)
Size (mm)
2.4 ⫻ 10 17 ⫻ 105 17 ⫻ 105
296 293 400
11.1 ⫻ 15.4 ⫻ 2.1 16.6 ⫻ 9.8 ⫻ 1.2 21.5 ⫻ 13.5 ⫻ 0.8
130 123 105
3.9 ⫻ 3.9 ⫻ 0.4 8.6 ⫻ 7.2 ⫻ 0.7 3.5 ⫻ 3.5 ⫻ 0.2
5
fibronectin plays a role in the early aggregation of dedifferentiated chondrocytes. Fibronectin expression is down regulated during chondrocyte differentiation. Fibronectin is deposited at the periphery of the cell aggregates, whereas laminin is localized in the central region.
synthesis of ECM processed faster in PGA than that in PGLA. Besides the property of the fibers, the pore size in the PGA cores is larger than in PGLA cores. It may be the other reason for the difference in collagen amount between PGA and PGLA cores.
Affinity Between Chondrocytes and Polymer Fibers
Antigenicity of Fibrin Glue
Affinity between chondrocytes and polymer fibers is closely related to cartilage formation. Some investigators4,6,9,10 recommended the use of PGA, PGLA, or both fibers as scaffolds. The evidence from this study is that chondrocytes grew healthier in PGA fibers than in PGLA fibers and the
Fibrinogen is a soluble plasma protein. The molecule appears as a trinodular rod. After thrombin cleavage, it is converted to fibrin monomers; these self-associate to form an insoluble homopolymeric structure, the fibrin clot.43 The fibrin has strong antigenicity. In the histologic sections of a chondrocyte-fibrin glue explant (70 days after transplantation), many lymphocytes and neutrocytes surrounded by void spaces gathered in the periphery area of the explant. The holes may have resulted from enzyme secretion. In order to reduce antigenicity of allogeneic proteins, we have tried to purify autologous fibrinogen from plasma using differential precipitation methods. However, the content of such fibrinogen from the self-blood plasma was too low to form feasible constructs. In the present study, bovine fibrin glue was used. Comparing construction of the whole units, less fibrin glue was needed for coating, so less antigenicity was caused. In conclusion, cartilage formation on PGA and PGLA fibers with or without fibrin glue coating was studied and compared. The effects of fibrin glue coating on cell-polymer constructs were increased collagen content, increased number of spherical-shaped chondrocytes, and increased mass retention after transplantation. Cartilage formation is better in PGA cores than in PGLA cores. ACKNOWLEDGMENTS We thank the director and staff of the Department of Experiment Surgery for their assistance in the animal experiments.
REFERENCES
Fig 11. (A) Original PGA construct without fibrin glue coating. (B) Original PGLA construct without coating. (C, E, G) Uncoated PGA explant on day 278. (D, F, H) Uncoated PGLA explant on day 278.
1. Brittberg M, Lindahl A, Nilsson A, et al: J Med 331:889, 1994 2. Breinan HA, Minas T, Hsu HP, et al: J Bone Joint Surg [Am] 79:1439, 1997 3. Minas T, Nehrer S: Orthopedics 20:525, 1997 4. Vacanti JP, Morse MA, Saltzman WM, et al: J Pediatr Surg 23:3, 1988 5. Cima LG, Ingber DE, Vacanti JP, et al: Biotechnol Bioeng 38:145, 1991 6. Miko AG, Bao Y, Cima LG, et al: J Biomed Mater Res 27:183, 1993 7. Langer R, Cima LG, Tamada JA, et al: Biomaterials 11:738, 1990
FIBRIN GLUE COATING 8. Sims CD, Butler PEM, Casanova R, et al: Plast Reconstr Surg 98:843, 1996 9. Bursac PM, Freed LE, Biron RJ, et al: Tissue Eng 2:141, 1996 10. Singhal AR, Agrawal CM, Athanasiou KA: Tissue Eng 2:197, 1996 11. Quarto R, Campanile G, Cancedda R, et al: Endocrinology 138:4966, 1997 12. Lee JD, Hwang O, Kim SW, et al: Life Sci 61:293, 1997 13. Woodard JC, Donovan GA, Fisher LW: Bone 21:171, 1997 14. Sullivan TA, Uschmann B, Hough R, et al: J Biol Chem 269:22500, 1994 15. Lee DA, Salih V, Stockton EF, et al: Clin Orthop 228, 1997 16. Yellowley CE, Jacobs CR, Li Z, et al: Am J Physiol 273:c30, 1997 17. Tavella S, Bellese G, Castagnola P, et al: J Cell Sci 110:2261, 1997 18. Camper L, Heinegard D, Lundgren-AE: J Cell Biol 138: 1159, 1997 19. Qi WN, Scully SP: J Orthop Res 15:483, 1997 20. Nehrer S, Beinan HA, Ramappa A, et al: Biomaterials 18:769, 1997 21. Baker TL, Goodwin TJ: In vitro Cell Dev Biol Anim 33:358, 1997 22. Glowacki J, Trepman E, Folkman J: Proc Soc Exp Biol Med 172:93, 1983 23. Jackson MR, Macphee MJ, Drohan WN, et al: Blood Coagul Fibrinolysis 7:737, 1996 24. Zarge JI, Husak V, Huang P, et al: Am J Surg 174:188, 1997 25. Ruvolo G, speziale G, Voci P, et al: Ann Thorac Surg 63:570, 1997 26. Greisler HP, Gosselin C, Ren D, et al: Biomaterials 17:329, 1996
217 27. Fortunato G, Marini E, Valdinucci F, et al: J Craniomaxillofac Surg 25:124, 1997 28. Vacanti A, Langer R, Schloo B, et al: Plast Reconstr Surg 88:753, 1991 29. Freed LE, Marquis JC, Nohria A, et al: J Biomed Mater Res 27:11, 1993 30. Kim WS, Vacanti JP, Cima L, et al: Plast Reconstr Surg 94:233, 1994 31. Ronfard V, Broly H, Mitchell V, et al: Burns 17:181, 1991 32. Kim YJ, Sah RLY, Doong JYH, et al: Anal Biochem 174:168, 1988 33. Woessner JF: Arch Biochem Biophys 93:440, 1961 34. Buckley CD, Pilling D, Henriquez NV, et al: Nature 397:534, 1999 35. Zanetti NC, Solursh M: J Cell Biol 99:115, 1984 36. Solursh M, Reiter RS: Dev Biol 78:141, 1980 37. Tavella S, Raffo P, Tacchetti C, et al: Exp Cell Res 215:354, 1994 38. Dessau W, von der Mark K, von der Mark H, et al: J Embryol Exp Morphol 57:51, 1980 39. Mackie EJ, Thesleff L, Chiquet-Ehrismann R: J Cell Biol 105:2569, 1987 40. Maleski MP, Knudson CB: Exp Cell Res 225:55, 1996 41. Chang SC, Hoang B, Thomas JT, et al: J Biol Chem 269:28227, 1994 42. Tavella S, Bellese G, Castagnola P, et al: J Cell Sci 110:2261, 1997 43. Kreis T, Vale R: Guidebook to the Extracellular Matrix and Adhesion Proteins. Oxford: Oxford University Press; 1993, p 52
A
RTICULAR cartilage has little capacity to regenerate itself. Pain and disability associated with cartilage damage in joints are difficult to treat and this has prompted the search for means of restoring hyaline cartilage on articular surfaces. One promising method entails implanting autologus chondrocytes on denuded joint surfaces.1–3 To obtain sufficient quantities of chondrocytes for this purpose, researchers have employed a variety of substances to promote the growth of chondrocytes in vitro.4 –10 Examples include the use of serum-free media,11 growth factors,12 vitamin A, C, and D,13,14 synovial fluid,15 and calcium.16 Others analyzed the components of extracellular matrix (ECM) such as collagen type II, fibronectin, and laminin and investigated their effects on chondrocyte activity.17,18 Reports indicate that these interact with integrin receptors on cell membranes. Qi and Scully19 showed that type II collagen regulated chondrocyte activity by mediating the response of chondrocytes to transforming growth factor-beta 1. Similarly, Nehrer et al20 reported that the type and pore size of collagen influenced the behavior of canine chondrocytes. Furthermore, it was reported that chondrocytes in suspension produced more ECM than cells seeded on flat surfaces.21,22 Fibrin glue is widely used as a sealant in surgery,23 commonly after dural repair, liver resection, and in burn surgery. Reports suggest that fibrin glue promotes neovascularization and connective tissue regeneration.24 –27 In this study, fibrin glue was used to coat cell-polymer constructs consisting of chondrocytes seeded on polyglactin (PGA) and poly-L-lactic acid (PLLA) polymers. The aim was to investigate the effect of fibrin glue coating on chondrocyte growth and extracellular matrix production in vitro and in vivo.
cartilage was used principally for in vitro studies, whereas rabbit cartilage was used for in vivo experiments.
Polymer Constructs PGA fibers were obtained from unbraided surgical sutures (0-0 Vicryl suture material, Ethicon, Somerville, NJ). Random tangles of PGA fibers were placed in cylindrical moulds and bonded with 2% of PLLA in methylene chloride.30 Scaffolds consisting of a mixture of PGA and PLLA (50% to 50%) (PGLA) (Medisorb Technologies International LP) were created using the solventcasting particulate-leaching technique.29 The constituents of fibrin glue were bovine fibrinogen (Sigma) in aprotinin solution and thrombin (Sigma) in phosphate-buffered solution containing calcium chloride.31 Fibrin glue was cast in cylindrical moulds to produce semisolid disks. To create coated constructs, polymer scaffolds were cast with fibrin glue in similar moulds. All constructs were irradiated with 2.5 ⫻ 106 rads for sterilization. Cultured chondrocytes were injected into the constructs and the composite units were used for the in vitro and in vivo studies.
Laboratory Study
MATERIALS AND METHODS Chondrocytes
Ninety units, 0.3 cm in diameter and 0.5 cm in thickness, were divided into three groups of 30 units each and cultured in glass bottles for 3, 13, and 30 days. Each bottle contained a magnetic stir bar and was maintained in a CO2 incubator. Each group of 30 constructs was subdivided into six subgroups (5 units per subgroup). For in vitro culture, the units were placed in individual pockets on a cotton sheet. This customized carrier was in turn suspended in culture by copper suspension wires. The six subgroups were chondrocyte-PGA with fibrin glue coating (group I), without fibrin glue coating (group II), chondrocyte-PGLA with fibrin glue coating (group III), without coating (group IV), PGA-fibrin glue without cells (group V), and PGLA-fibrin glue without cells (group VI). Each cell-polymer unit contained approximately 1.5 ⫻ 105 chondrocytes. At predetermined time intervals, the cell-polymer constructs were extracted for analysis. They were weighed, frozen, and lyophilized. The units were then digested with 1 mL papain buffer solution (3 mg/mL) at 60°C for 16 to 20 hours. The cell number per sample was estimated by DNA quantification using
Chondrocytes were derived from human cartilage and rabbit pinna. Cartilage harvest was performed under aseptic conditions. The cells were isolated by digestion with type II collagenase as previously described.28,29 These were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY) containing 4500 mg/L glucose and 584 mg/L glutamine, 10% fetal calf serum (FCS; GIBCO), proline (46 g/mL), ascorbic acid (50 g/mL), insulin (5 g/mL), transferrin (125 g/mL), penicillin (100 U/mL), and streptomycin (100 g/mL) (Sigma, St Louis, Mo). Human
From the Departments of Clinical Research (X.F., D.-F.S., S.-E.A.) and Plastic Surgery (B.-K.T., C.-L.F., S.-T.L.), Singapore General Hospital, Singapore. Supported by the Department of Clinical Research, Singapore General Hospital, Singapore. Address reprint requests to Dr Xun Fei, Scientific Officer, Department of Clinical Research, Singapore General Hospital, Outram Rd, Singapore 169608.
0041-1345/00/$–see front matter PII S0041-1345(99)00939-2 210
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 210–217 (2000)
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Fig 1. Histologic appearance of fibrin glue-coated cell-polymer constructs cultivated for 3 days (A), 13 days (B), and 30 days (C). (Toluidine blue staining, magnification ⫻200). Notice the chondrocyte attached to the PGA fiber (small arrow). Cross-section of PGA fiber, which stained positively for ECM, is indicated by the big arrow.
Hoechst 33258 dye.32 Total collagen content was determined based on the hydroxyproline content (determined by reaction with chloramine T and P-dimethylaminobenzaldehyde33). For histologic analysis, the constructs were fixed in 10% formalin, frozen sectioned (5 m thick), and stained with H&E. Toluidine blue was used to stain glycosaminoglycan (GAG). Type I, type II, and type IV collagens were detected by immunohistochemistry.
Animal Study The cell-polymer constructs were transplanted in three rabbits. Each rabbit received four constructs: (1) chondrocyte-PGA, (2) chondrocyte-PGA with fibrin glue coating, (3) chondrocyte-PGLA, and (4) chondrocyte-PGLA with fibrin glue coating. Each unit contained approximately equal number of autologous chondrocytes: rabbit I, 0.8 ⫻ 106 cells each; rabbit II, 5 ⫻ 106 cells each; and rabbit III, 0.3 ⫻ 106 cells each. The animals were euthanized on days 10, 60, and 141, respectively. Prior to transplantation, each construct was encircled with a steel wire for easy identification during retrieval. These cell-polymer constructs were placed subcutaneously (SC) on paravertebral fascia. At harvest, the explants were weighed and measured. Sections were obtained for histologic analysis. Cell survival was evaluated with apoptosis fluorescence immunostaining (Promega, Madison, Wis). Type II collagen was detected with anti-type II collagen immunostaining (green colored) and cell nuclei were labeled with propidium iodide (orange colored). GAG expression was detected with toluidine blue.
Statistical Evaluation The DNA concentration and collagen content were reported as mean ⫾ standard error (M ⫾ SD). Independent t tests were performed to determine statistical significance.
RESULTS Laboratory Study
Histomorphology. At day 3, sections of fibrin glue-coated PGA constructs showed an intact fibrin glue envelope, which appeared bright pink with H&E and bright blue with toluidine blue. Chondrocytes were concentrated at the center of the construct. Dividing cells were found mainly attached to the PGA fibers, whereas nondividing cells were scattered in void spaces. Both spherical and spindle-shaped cells were present. At day 13, the ECM was present and distributed throughout the construct. Uniform staining of the PGA fiber surface was seen, indicating the existence of ECM on the fiber surface. The fiber cores were not stained. On day 30, cross-sections of the fibers were distinctly stained, suggesting that the components of ECM, such as collagen and GAG, had penetrated the core of the fibers. Degraded PGA material with refractive shine was noted (Fig 1). Type 1 and type II collagen can be detected by immunohistochemistry. Sections from uncoated PGA constructs on days 3, 13, and 30 presented with less ECM than in the coated counterparts. The deposition of ECM on the surface of the fibers was observed on day 13 and the penetration of ECM in the cores of the fibers was seen on day 30. The morphology of chondrocytes and the degraded PGA materials were similar to those in the coated units (Fig 2). Physical Properties of Cell-Polymer Constructs
Over 30 days of culture, the wet weight of the coated PGA constructs decreased to 70% of the wet weight on day 3,
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Fig 2. Histologic appearance of uncoated cell-polymer constructs cultivated for 3 days (A), 13 days (B), and 30 days (C). (Toluidine blue staining, magnification ⫻200). Notice the chondrocyte attached to the PGA fiber (small arrow). Cross-section of PGA fiber, which stained positively for ECM, matrix, is indicated by the big arrow.
whereas the wet weight of uncoated counterparts decreased to 50%. These data suggest that coated constructs retained mass better than uncoated constructs. Biochemical Components
Measured profiles of DNA and collagen content in the fibrin glue-coated and uncoated units are given in Figures 3 and 4. There was a time-dependent increase in DNA content over 4 weeks of cultivation. There was a timedependent increase in DNA content in coated and un-
Fig 3. DNA content in coated and uncoated PGA units (ng/mL). M ⫾ SD; n ⫽ 4, P ⬎ .05.
coated constructs. The collagen content was slightly higher in the uncoated constructs than that in the coated ones. The difference of the amount between the two groups was not significant statistically (P ⬎ .05; Fig 3). The collagen content increased more rapidly in the coated groups than in the uncoated groups. Collagen
Fig 4. Collagen content in coated and uncoated PGA units (g/mL). M ⫾ SD; n ⫽ 4, group t test, P ⬍ .01 on day 30.
FIBRIN GLUE COATING
Fig 5. DNA content in coated and uncoated PGLA units. M ⫾ SD; n ⫽ 4, P ⬍ .05 on day 30.
content in each coated construct on day 30 was 1.00 ⫾ 0.09 g, about 1.37 times that of the uncoated units (0.73 ⫾ 0.05 g). The difference between coated and uncoated groups was statistically significant (P ⬍ .01; Fig 4). In the PGLA samples, the cell numbers increased more rapidly in the coated constructs than in the uncoated ones. The DNA amount on day 30 was 13.0 ⫾ 2.9 ng per construct, approximately twice that of the uncoated ones (6.5 ⫾ 1.3 ng per construct; Fig 5). Similarly, the collagen content was higher in the coated constructs than in the uncoated ones. On day 30, collagen content in the coated constructs was 0.23 ⫾ 0.02 g, about 1.96 times the average collagen content of the uncoated ones (0.12 ⫾ 0.03 g). The difference between the two groups was statistically significant (P ⬍ .01; Fig 6). Animal Study
Cell-polymer constructs buried SC in three rabbits were explanted on days 10, 60, and 141. The four constructs (chondrocytes-PGA, chondrocytes-PGA with fibrin glue coating, chondrocytes-PGLA, and chondrocytes-PGLA with coating) did not demonstrate any obvious difference in volume on day 10. The physical dimensions of the constructs are given in Table 1. On day 60, the size difference was noticeable. The coated PGA construct was thicker and larger (14.1 ⫻ 14.1 ⫻ 6.1 mm), whereas the uncoated was thinner and smaller (7.0 ⫻ 7.0 ⫻ 1.7 mm; Fig 7). The wet weight of the coated PGA construct was approximately 64% of the weight on day 10, whereas its counterpart was only 26%. Similar results were observed for day 141 samples. The coated PGLA construct appeared more solid and was
213
Fig 6. Collagen content in coated and uncoated PGLA units (g/unit). Mean ⫾ SD; n ⫽ 4, group t test, P ⬍ .05 on day 30.
thicker than its counterpart, which appeared membranous (Table 1, Fig 8). The wet weight of the coated PGLA construct was approximately 40% of the weight on day 10, whereas its counterpart was only 16%. Only three units were harvested on day 141. The missing one (PGA with fibrin glue) had extruded due to infection. Histologic sections of coated constructs appeared significantly more compact and homogenous than the uncoated counterparts on day 60. Connective tissue was seen extending centripetally, distributed among clusters of chondrocytes. Chondrocytes at the periphery adjacent to the fibrin glue layer were spherical in shape, closely resembling well-differentiated chondrocytes. However, those located at the core of the unit were spindle shaped. In addition, higher levels of matrix were detected at the periphery, evidenced by deep staining with toluidine blue (Figs 9, 10). Most cells in both groups with or without coating were chondrocytes evidenced by type II collagen labeling. The majority of cells were viable, confirmed by apoptosis immunostaining. Both groups had neovascular ingrowth. The presence of polymer fractions was noted. Sections of the day 141 and coated construct showed grossly the same features, but with less cartilage component and more neovascular ingrowth. In the uncoated construct, the cartilaginous matrix was reduced to a narrow strip. Comparison of PGA and PGLA Cores
The results in this study demonstrated that the PGA cores were better than the PGLA cores in terms of size and chemical components in both fibrin glue-coated and uncoated conditions. For the in vitro study, in the coated
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FEI, TAN, LEE ET AL Table 1. Physical Data of Chondrocyte-Polymer Explants From Rabbit PGA Coated
Rabbits
Duration of Implant (d)
Wet Wt (mg)
1 2 3
10 60 141
1030 666 —
Uncoated
Size (mm)
Wet Wt (mg)
16.1 ⫻ 15.6 ⫻ 5.9 14.1 ⫻ 14.1 ⫻ 6.1 —
790 210 97
PGLA Coated
Size (mm)
Wet Wt (mg)
17.7 ⫻ 16.6 ⫻ 5.4 7.0 ⫻ 7.0 ⫻ 1.7 12.9 ⫻ 10.8 ⫻ 5.1
1053 393 424
constructs with the same initial cell number, the collagen amount in PGA units on day 30 was 3.57 ⫾ 0.26 g/mL, whereas that in the PGLA units was 1.30 ⫾ 0.12 g/mL. In the uncoated constructs, the collagen amount in PGA units was 2.05 ⫾ 0.23 g/mL, whereas that in PGLA counterparts was 0.58 ⫾ 0.05 g/mL. For the in vivo study, in the coated constructs with the same initial size, the size in PGA explant on day 60 was 14.1 ⫻ 14.1 ⫻ 6.1 mm, whereas the size of the PGLA counterpart was 12.6 ⫻ 12.6 ⫻ 4.9 mm. As a further example of an in vivo experiment (Table 2 and Fig 11), three paired cell-polymer constructs without fibrin glue coating, with the same initial cell number and the same size in the operation day, were harvested from two rabbits on day 278. The size of the first pair was 11.1 ⫻ 15.4 ⫻ 2.1 mm (PGA) vs 3.9 ⫻ 3.9 ⫻ 0.4 mm (PGLA). The size of the second pair was 16.6 ⫻ 9.8 ⫻ 1.2 mm (PGA) vs 8.6 ⫻ 7.2 ⫻ 0.7 mm (PGLA). The size of the third pair was 21.5 ⫻ 13.5 ⫻ 0.8 mm (PGA) vs 3.5 ⫻ 3.5 ⫻ 0.2 mm (PGLA). DISCUSSION Effect of Fibrin Glue Coating
In this study, the effects of fibrin glue coating on cell proliferation and collagen synthesis were examined. In vitro, the difference in DNA amount between coated and uncoated constructs was not statistically significant comparing both types of constructs through days 3, 13, and 30. This suggests that fibrin glue did not affect chondrocyte proliferation.
Fig 7. (A) Original PGA construct with fibrin glue coating. (B) Original PGA construct without coating. (C) Coated PGA explant on day 60. (D) Uncoated PGA explant on day 60.
Uncoated
Size (mm)
Wet Wt (mg)
Size (mm)
19.2 ⫻ 16.3 ⫻ 5.0 12.6 ⫻ 11.9 ⫻ 2.7 16.3 ⫻ 12.0 ⫻ 4.9
608 303 101
17.7 ⫻ 14.0 ⫻ 4.4 8.2 ⫻ 4.1 ⫻ 1.2 9.4 ⫻ 7.7 ⫻ 1.4
Cell numbers in uncoated constructs were slightly higher than in coated ones. It may be caused by easier nutrition in uncoated constructs or by the inhibition of cell proliferation by soluble arginine-glycine-aspartate (RGD) peptides in fibrin glue-coated constructs.34 Buckley et al34 reported that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. In PGA constructs, parallel increasing of cell number in both coated and uncoated groups reflected well adhesion and well proliferation of chondrocytes on PGA fibers. In PGLA constructs, the situation is different. DNA content in coated constructs was much higher than in their counterparts on day 30 (13.0 ⫾ 2.9 ng per construct vs 6.5 ⫾ 1.3 ng/mL, P ⬍ .05). The difference indicated that cells were retained in the constructs by the fibrin glue barrier. The difference in collagen content was, however, statistically significant on day 30. This suggests that fibrin glue coating may enhance collagen accumulation or synthesis. Histologic pictures exhibited the higher deposit of ECM in the coated unit than in the uncoated unit. The ECM contained collagen type 1, collagen type II, and GAG. In another experiment, we learned that a greater amount of collagen was detected in culture medium from uncoated constructs (the data are not presented here). We postulate that fibrin glue may play a role in the retention of collagen in constructs. Cell differentiation may be another avenue by which fibrin glue exerts its effects. We noticed that cells adjacent to fibrin glue tended to be spherical, which is the
Fig 8. (A) Original PGLA construct with fibrin glue coating. (B) Original PGLA construct without coating. (C) Coated PGLA explant on day 141. (D) Uncoated PGLA explant on day 141.
FIBRIN GLUE COATING
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Fig 9. Cluster of chondrocytes adjacent to fibrin glue layer at the periphery of the construct. Notice strong staining with toluidine blue, which indicated high deposition of ECM. The section was taken from a coated chondrocyte-PGA explant on day 60.
more differentiated form capable of synthesizing matrix as was reported by Zanetti and Solursh.35 However, the precise correlation between the cell shape and differentiation is not fully understood. ECM condensation is necessary for durability and the mechanical strength of tissue-engineered cartilage. Cell-cell interactions,36,37 composition of the ECM,38 – 40 changes in cell shape,35 and several specific cytokines41 play a role in this process. In this study, a fibrin glue envelope, resulting in less leakage of cells and ECM, enhanced ECM condensation. Chondrocyte aggregates were formed and the life span
of tissue-engineered cartilage was increased in vitro and in vivo. Effect of the Polymer Cores
Initially, we tried injecting chondrocytes in homogenous fibrin glue units. However, only a part of cells could be retained due to lack of void spaces. We observed that cartilaginous tissue did form in cell-fibrin glue constructs. Polymer cores with fibrin glue coating mimic native condition of chondrocyte aggregation. Fibronectin is the main component of fibrin glue. Tavella et al42 reported that
Fig 10. Cluster of chondrocytes in the center of the construct. Notice weak staining with toluidine blue, which indicated low deposition of ECM. The section was taken from a coated chondrocyte-PGA explant on day 60.
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FEI, TAN, LEE ET AL Table 2. Physical Data of Chondrocyte-Polymer Explants From Rabbits On Day 278 PGA
Pair 1 Pair 2 Pair 3
PGLA
Cell Number Per Unit
Wet Wt (mg)
Size (mm)
Wet Wt (mg)
Size (mm)
2.4 ⫻ 10 17 ⫻ 105 17 ⫻ 105
296 293 400
11.1 ⫻ 15.4 ⫻ 2.1 16.6 ⫻ 9.8 ⫻ 1.2 21.5 ⫻ 13.5 ⫻ 0.8
130 123 105
3.9 ⫻ 3.9 ⫻ 0.4 8.6 ⫻ 7.2 ⫻ 0.7 3.5 ⫻ 3.5 ⫻ 0.2
5
fibronectin plays a role in the early aggregation of dedifferentiated chondrocytes. Fibronectin expression is down regulated during chondrocyte differentiation. Fibronectin is deposited at the periphery of the cell aggregates, whereas laminin is localized in the central region.
synthesis of ECM processed faster in PGA than that in PGLA. Besides the property of the fibers, the pore size in the PGA cores is larger than in PGLA cores. It may be the other reason for the difference in collagen amount between PGA and PGLA cores.
Affinity Between Chondrocytes and Polymer Fibers
Antigenicity of Fibrin Glue
Affinity between chondrocytes and polymer fibers is closely related to cartilage formation. Some investigators4,6,9,10 recommended the use of PGA, PGLA, or both fibers as scaffolds. The evidence from this study is that chondrocytes grew healthier in PGA fibers than in PGLA fibers and the
Fibrinogen is a soluble plasma protein. The molecule appears as a trinodular rod. After thrombin cleavage, it is converted to fibrin monomers; these self-associate to form an insoluble homopolymeric structure, the fibrin clot.43 The fibrin has strong antigenicity. In the histologic sections of a chondrocyte-fibrin glue explant (70 days after transplantation), many lymphocytes and neutrocytes surrounded by void spaces gathered in the periphery area of the explant. The holes may have resulted from enzyme secretion. In order to reduce antigenicity of allogeneic proteins, we have tried to purify autologous fibrinogen from plasma using differential precipitation methods. However, the content of such fibrinogen from the self-blood plasma was too low to form feasible constructs. In the present study, bovine fibrin glue was used. Comparing construction of the whole units, less fibrin glue was needed for coating, so less antigenicity was caused. In conclusion, cartilage formation on PGA and PGLA fibers with or without fibrin glue coating was studied and compared. The effects of fibrin glue coating on cell-polymer constructs were increased collagen content, increased number of spherical-shaped chondrocytes, and increased mass retention after transplantation. Cartilage formation is better in PGA cores than in PGLA cores. ACKNOWLEDGMENTS We thank the director and staff of the Department of Experiment Surgery for their assistance in the animal experiments.
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Fig 11. (A) Original PGA construct without fibrin glue coating. (B) Original PGLA construct without coating. (C, E, G) Uncoated PGA explant on day 278. (D, F, H) Uncoated PGLA explant on day 278.
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