Cellprep [Biotecnología Celular]

LEESA M. BARONE, PhD
Genzyme Tissue Repair, Cambridge, Massachusetts

Abstract

It is generally agreed that damage involving the articular cartilage surface and confined to the cartilage undergoes little restoration. Cartilage has little intrinsic ability to heal itself.
The disability and pain that result from damage to articular cartilage have stimulated
the serch for ways of facilitating cartilage repair.
For repaired or regenerated cartilage to perform satisfactorily as a joint tissue, it must
restore normal pain-free motion of the synovial joint. Therefore, the repaired tissue must have the structure, compsition, mechanical properties, and durability similar to natural articular cartilage. A number of methods for promoting cartilage repair for chondral defects have been explored. These include debridement and lavage, subchondrial bone drilling, microfracture, abrasion arthroplasty, high tibial osteotomy, periosteal/perichondrial grafting, and mesenchymal stem cell implantation. However, with these procedures, the resulting repaired tissue is inferior to the original articular cartilage.
Brittberg et al. reported a method for repairing deep cartilage defects in the femorotibial articular surface of the knee joint in humans.1
Cultured autologous chondrocytes, cells isolated from an individual's own cartilage, can be expanded in vitro and returned to the damaged site for repair of their damaged cartilage. This remarkable process is characterized by modulation of gene expression during proliferation and subsequent re-differentiation of cultured chondrocytes. This report will provide biological and molecular evidence that human chondrocytes isolated from articular cartilage and expanded in monolayer culture, retain their ability to re-express cartilage-specific phenotypic markers when inducedto differentiate.

Introduction

Damage to articular cartilage is an exceedingly common problem affecting the joints of millions of people. This is major problem considering the poor regenerative capacity of adult articular cartilage. Injuries of the articular cartilage that do not penetrate the subchondral bone do not heal and eventually progress to the degeneration of the articular surface.
The disability and pain that results from damage to articular cartilage have stimulated the search for ways of facilitating cartilage repair. For repaired or regenerated cartilage to perform satisfactorily as a joint tissue, it must restore normal pain-free motion of the synovial joint. For this to occur, the repaired tissue must have the structure, composition, mechanical properties, and durability similar to the natural articular surface. A number of methods for promoting repair of cartilage defects have been explored. Surgical options to treat the damaged cartilage include: debridement and lavage, subchondral bone drilling, microfracture, abrasion arthroplasty, and high tibial osteotomy.2-8 Each of these procedures may help to relieve clinical symptoms of pain, locking, and swelling, but none result in the restoration of hyaline articular cartilage.
The repair tissue stimulated by these procedures is fibrocartilage, which lacks the appropriate biochemical and biomechanical properties of normal hyaline articular cartilage. Treatment of cartilage defects with autologous tissue or mesenchymal cells has also been explored. Transplantation of periosteal/perichondrial grafts into articular cartilage defects as a means of inducing cartilage regeneration is a recent approach in articular cartilage repair.9-13 An advantage of this grafting procedure is that large areas of articular surface can be covered. The "hyaline-like" repaired tissue that initially forms tends to calcify and is replaced with bone through endochondral ossification. Similar results are obtained with mesenchymal stem cell implantation.14-16 Mesenchymal stem cells are pluripotent cells derived from bone marrow that have the capacity to form many tissue types. In animal models, it has been shown that the hyaline-like cartilage that forms also calcifies, with bone resulting. Another and more promising method for repairing deep cartilage defects in the femorotibial articular surface of the knee joint in humans has been reported.1 Cultured autologous chondrocytes, cells isolated from an individual's own cartilage, can be expanded in vitro and returned to the damaged site for repair of their damaged cartilage. This remarkable process is characterized by modulation of gene expression during proliferation and subsequent re-differentiation of cultured chondrocytes.
This report will focus on the biological and molecular characterization of human articular chondrocytes during proliferation and differentiation.

Articular Cartilage Biology

Articular cartilage (also known as hyaline cartilage) is a thin layer that covers the ends of bones in movable joints. This thin layer of tough, opaque tissue ranges from about 1 mm to 5 mm in thickness in the human knee. It permits practically frictionless motion of the bones forming the joint and is capable of absorbing load forces that are in the range of five times the body weight. While cartilage should last a lifetime, the constant wear and occasional trauma it receives can result in the degenerative process leading to osteoarthritis. This is due to the fact that articular cartilage has a poor intrinsic capacity for repair.
Articular cartilage is neither innervated nor vascularized. It receives its nutrient requirements from the synovial fluid, which bathes the articular surface, or from the underlying subchondral bone.
Articular cartilage is a complex, highly organized tissue (Fig. 1). The cellular component of cartilage is the chondrocyte. The chondrocytes form 1% or less of the tissue volume, however, they are the only living element in the tissue. The chondrocytes are encased within the articular cartilage matrix, which they have produced during development. The matrix consists of collagens and proteoglycan aggregates (i.e., aggrecan). Articular cartilage contains a number of different collagens: types II, XI, and IX (Fig. 2). Type II collagen is the main building block of the fibril; it provides the basic architectural structure of the cartilage. These fibers are extremely strong and have a great capacity for resisting stress. Type XI collagen is integrated throughout type II collagen and serves as a core to the fibrils. Type IX collagen is linked to type II collagen through specific covalent bonds and appears to be an intermediate between the collagen fibers and the proteoglycans. Collagen comprises about 65% of the dry weight of cartilage. The collagens provide cartilage with its tensile and biomechanical strength. An individual proteoglycan molecule consists of a central protein core to which are attached many negatively charged sulfated glycosaminoglycan (GAG) side chains like chondroitin sulfate and keratan sulfate (Fig. 3). A proteoglycan aggregate is composed of many such molecules attached to a long hyaluronic acid chain. This interaction is stabilized by link protein. Since the GAG chains are negatively charged, water molecules become trapped, forcing the proteoglycan aggregates to become distended with the water molecules. Water can account for 60-80% of the total cartilage weight. The proteoglycans and collagen types can be used as phenotypic markers for articular cartilage. The properties of articular cartilage can, therefore, be accounted fo by the collagen, which provides structure and strength, and by the proteoglycans engorged with water, which provide resistance to compression.

Figure 1. Schematic of Articular Cartilage.
The cartilage matrix consists of long collagen fibers (blue-green) which surround and constrain the hydrated proteoglycan aggregate (yellow); a chondrocyte lies in the background

Figure 2. Schematic of a Collagen Fibril of Articular Cartilage.
The main collagen in cartilage is type II collagen.
These fibers provide the basic architectural structure of cartilage. They are
extremely strong and have a great capacity for resisting stress. Type IX and
XI collagens are minor collagens.

Figure 3. Schematic of a Proteoglycan Aggregate of Articular Cartilage.
An individual proteoglycan molecule consists of a central protein core to which many sulfate glycosaminoglycan side chains (chondroitin sulfate and keratan sulfate) are attached. A proteoglycan aggregate is composed of many such molecules attached to a long hyaluronic acid chain. Link protein stabilizes this interaction.


	Cell Biology of Human Chondrocytes Environmental factors influence chondrocyte morphology and the matrix that they produce. Normal adult articular cartilage consist of round chondrocytes in their lacunae arranged in a columnar organization (Fig. 4). The chondrocytes are surrounded by their matrix, mainly type I collagen and proteoglycan. To culture human adult chondrocytes in vitro, the chondrocytes have to be freed from their matrix (Fig. 5). The cartilage biopsy is removed and put through an enzymatic digestion to release the cells from their matrix. The cells are then placed in a monolayer culture dish for expansion. The cells attach to the substratum of the dish, spread, and proliferate rapidly. Healthy chondrocytes grown on monolayer culture on plastic have a fibroblastic appearance and produce predominantly type I collagen (Fig. 6). This process, termed de-differentiation. is reversible. Monolayer chondrocytes will re-express type II collagen and the large aggregating proteoglycans once placed in the proper environment, whether in suspension in vitro or in the joint. The reversibility of this process is key to the successful repair of articular cartilage with cultured autologous chondrocytes.

Figure 4. Immunohistological Section Through the Joint.
Normal adult articular cartilage consist of round chondrocytes in their lacunae arranged in a columnar organization. Type II collagen (brown) surrounds the chondrocytes.

Figure 5. Cultured Chondrocyte Processing and Implantation Technique.
A healthy cartilage biopsy is removed and put through an enzymatic digestion. The cells are then expanded on a monolayer culture. The cells are then released from the substratum of the dish. These chondrocytes can be placed in either an in vitro suspension culture or in the joint where they will redifferentiate and re-express the cartilage phenotype.

Figure 6. Articular Chondrocytes Grown in Monolayer Culture.
Chondrocytes attached to substratum of culture dish. They differentiate and appear as dermal fibroblasts.

Biochemical Characteristics of Human Chondrocytes

To ensure that cultured chondrocytes subjected to proliferative monolayer culture in tissue culture flasks retain their ability to differentiate and form a matrix that closely approximates the character of native articular cartilage, several differentiation models can be used. Two in vitro systems used to examine the ability of chondrocytes to re-express the differentiated cartilage phenotype are the culturing of chondrocytes in agarose and alginate. These are semi-solid mediums which prevent the cells from attaching to a substratum. When chondrocytes are suspended in agarose for 3 to 4 weeks following expansion on monolayer culture flasks, they form colonies of cells with "halos" of matrix surrounding the cells, characteristic of chondrocytes (Fig. 7).16 These differentiated colonies re-express cartilage markers such as sulfated proteoglycan, as visualized by Safranin O stain (which stains sulfated proteoglycan orange; Fig. 8). The culturing of chondrocytes in alginate offers an advantage over culturing the cells in agarose to examine the re-expression of the cartilage phenotype. Differentiated chondrocyte colonies from the alginate can be recovered at different time points and examined for their ability to produce normal biochemical and molecular cartilage markers. To examine the
proliferative ability of chondrocytes on monolayer cultures and cells grown in alginate, chondrocytes can be isolated from the two systems and examined by immunohistochemical analysis using BrdU labeling (Fig. 9). BrdU is a DNA marker. Cells isolated from monolayer cultures are actively proliferating and dividing. Cells isolated from alginate cultures form clusters and only cells along the periphery of the nodule are dividing and proliferating while cells in the center are encased in matrix and are no longer proliferating. Therefore, as the levels of proliferation drop, the levels of differentiation increase. To examine the collagen and proteoglycan that the cells are producing, human chondrocytes are seeded into uspension cultures using alginate. Following 2 weeks, 4 weeks, and 6 weeks in the alginate, the differentiated colonies are recovered and analyzed (Fig. 10). Texas red conjugated to type I collagen antibody (red color) and the nucleus of the chondrocyte using Hoech dye (blue color) are analyzed at the different time points with immunofluorescence.
Results reveal that type I collagen is "turned on" by 2 weeks, with additional increase in colony size and further deposition of type II collagen by 4 and 6 weeks in alginate. When chondroitin sulfate (specific GAG for articular cartilage) is analyzed at 2 and 6 weeks, similar pattern is observed (Fig. 11).

Molecular Markers for Chondrocytes

Gene expression during proliferation and subsequent re-differentiation of cultured human chondrocytes is also being examined (Fig. 12). Total mRNA is probed with specific reverse transcribed probes for the presence of the following genes: aggrecan, type II collagen, type I collagen, and 18S rRNA. To control for RNA integrity and yield, the RNA samples are tested for hybridization to 18S rRNA cDNA. Chondrocytes grown in monolayer cultures express type I collagen mRNA. Following anchorage independent culture in alginate, the chondrocytes switch their collagen gene expression to predominantly type I collagen mRNA. All normal adult articular chondrocyte strains examined demonstrate activation of type II collagen mRNA by
one to two weeks in suspension culture. Types I and II collagen are expressed at similar levels during early stages in suspension culture. However, by ten weeks in alginate, the cells express only type II collagen mRNA. Another specific marker of hyaline cartilage aggrecan, was significantly enhanced by suspension culture.

Figure 7. Chondrocytes Grown in Semi-solid Agarose Media.
Chondrocytes differentiate and form colonies in agarose. A halo of matrix surrounds the chondrocytes.

Figure 8. Chondrocytes Grown in Semi-solid Agarose Media and Stained with Safranin O/ Fast Green.
Chondrocytes differentiate and form colonies in agarose. They re-express cartilage phenotypic markers such as sulfated proteoglycan. Safranin O stains the sulfated proteoglycan produced in the matrix by the chondrocytes orange and Fast Green stains the cells.


	Figure 9. Immunohistochemical Analysis of Chondrocytes Grown in Monolayer Culture and Alginate for 4 Weeks. Chondrocytes differentiate and form colonies in agarose. They re-express cartilage phenotypic markers such as sulfated proteoglycan. Safranin O stains the sulfated proteoglycan produced in the matrix by the chondrocytes orange and Fast Green stains the cells.


	Figure 10. Immunofluorescence of Chondrocytes Grown in Alginate for 2, 4, and 6 Weeks. Cultured human chondrocytes were seeded into suspension culture using alginate. Following 2 weeks (a), 4 weeks (b), and 6 weeks (c) in the alginate the differentiated colonies were recovered and analyzed. Texas red conjugated type II collagen antibody (red color) and the nucleus of the cell using Hoechst dye (blue color) were analyzed at the different time points. Immunohistochemical analysis revealed that type II collagen was turned on by 2 weeks, with additional increase in colony size and further deposition of type II collagen by 4 and 6 weeks.


	Figure 11. Immunofluorescence of Chondrocytes Grown in Alginate for 2 and 6 Weeks. Cultured human chondrocytes were seeded into suspension culture using alginate. Following 2 weeks (a) and 6 weeks (b) in the alginate, the differentiatied colonies were recovered and analyzed. Texas red conjugated chondroitin sulfate antibody (red color) and the nucleus of the cell using Hoechst dye (blue color) were analyzed at the different time points. Immunohistochemical analysis revealed that chondroitin sulfate was turned on by 2 weeks, with additional increase in colony size and further production of chondroitin sulfate by 6 weeks.

Figure 12. Time course of Chondrocytes Differentiation in Alginate.
Total RNA, isolated from monolayer cultured chondrocytes and alginate cultured chondrocytes at timed intervals, was probed with specific transcribed probes for the presence of the following genes: aggrecan, type II collagen, type I collagen, 18S rRNA. Chondrocytes grown in monolayer cultures express type I collagen RNA. Following anchorage independent culture in alginate the chondrocytes switch their expression to predominately type II collagen RNA. Another specific marker of hyaline
cartilage, aggrecan was not turned off in monolayer culture, and was significantly enhanced by suspension culture.

Cultures were also examined for expression of other collagen genes: type X collagen and type IX collagen (Fig. 13). Chondrocytes grown in monolayer cultures express type I collagen mRNA, as do dermal fibroblasts. Following anchorage-independent culture in alginate, the chondrocytes switch their gene expression to predominantly type II collagen, with some type IX collagen (a minor component of hyaline cartilage). In contrast, type X collagen, an indicator of chondrocyte hypertrophy and eventual bone formation, was not turned on by adult articular chondrocytes grown in monolayer or anchorage-independent cultures out to 5 months. This demonstrates that the adult articular chondrocytes are committed
cells that differentiate into adult articular cartilage when implanted into
chondral defects and are not capable of differentiating further into bone. In
contrast, cells derived from periosteum, perichondrium, or mesenchyme express type X collagen and have been shown to form bone upon implantation into chondral defects.17-19

Cultured Autologous Chondrocytes to Repair Chondral Defects in a Canine

Model

This study was designed to evaluate the role of cultured autologous chondrocytes in the repair of full-thickness articular cartilage defects in a weight-bearing animal model, the adult canine.
Focal cartilage defects (4 mm diameter) were surgically created on the femoral condyles of adult dogs (Fig. 14). Animals were separated into three treatment groups: (1) untreated defects; (2) untreated defects covered with periosteum alone; or (3) defects covered with periosteum and implanted with cultured autologous articular chondrocytes. Animals were sacrificed, and joints harvested for analysis at 6 weeks, 3 months, 6 months and 1 year. Cartilage defects were harvested at predetermined time intervals, analyzed for gross appearance. and processed for histological analysis. Results indicate that upon introduction of the chondrocytes into the defect, the cells begin the process of re-differentiation, turning on cartilage specific genes, like type II collagen and aggrecan. By 6 weeks, chondrocyte-treated defects have more cell fill than untreated controls. Also visible in treated defects are chondrocytes within lacunac (a small cavity in which chondrocytes are encircled) surrounded by a matrix of type II collagen (Fig. 15). To ensure that the chondrocytes that were implanted into the defect were facilitating the repair, autologous chondrocytes were transfected in vitro with the b-galactosidase gene and implanted into defects created in the model and analyzed at 6 weeks (Fig. 16).
Immunohistochemical analysis revealed that the implanted cells remain within the defects primarily along the periphery of the defect and integrate along the edges with the host adjacent cartilage. Type II collagen matrix surrounds the b-galactosidase labeled chondrocytes within the filled defect. By six months, the reparative tissue has developed, in some cases to hyaline articular cartilage (Fig. 17). Varying levels of spontaneous cartilage repair in the absence of chondrocyte implantation were also apparent, complicating the interpretation of the results. However, blinded histological analysis revealed a general trend toward more cartilage repair with chondrocyte
implantation at 6 months. By one year this trend was no longer apparent (Fig. 18). All defect types: untreated, untreated with periosteum, and treated with cells and periosteum, were similar. The canine model provided many important observations: autologous chondrocytes implanted in the defect remain in the site and are capable of facilitating the repair to a hyaline-like articulating cartilage with type II collagen and sulfated proteoglycan surrounding chondrocytic cells. However, several problems were noted in the canine model: 1) the subchondral bone plate is thinner than in humans, leading to more frequent fracture and bleeding into the defect, resulting in fibrous fill; 2) the canine periosteum is thicker than the cartilage to be repaired: 3) canine cartilage is only 0.5 - 1.0 mm thick, whereas human cartilage is 3-5 mm thick; and 4) there is evidence of endogenous regeneration of articular cartilage within the control untreated defects in dogs.

Figure 13. Collagen expression during the in vitro differentiation of articular chondrocytes.
Total RNA, isolated from fibroblasts, monolayer cultured chondrocytes and alginate cultured chondrocytes at timed intervals, was probed with specific transcribed probes for the presence of the following genes: type X collagen, type II collagen, type I collagen, and type IX collagen. Chondrocytes grown in monolayer cultures express type I collagen RNA, as do dermal fibroblasts. Following anchorage independent culture in alginate the chondrocytes switch their expression to predominately type II collagen RNA, with some type IX collagen (a minor component of hyaline cartilage). Type X collagen, an indicator of chondrocyte hypertrophy and eventual bone formation, is never turned on by adult articular chondrocytes.

	Figure 14. Macroscopic view of defect created in the canine model. Two cylindrical focal cartilage defects (4 mm diameter) were created in the femoral condyle in the canine. Articular cartilage was removed down to but not through the subchondral bone plate.


	Figure 15. Immunohistochemical section of type II collagen of the defect treated with chondrocytes in the canine model at 6 weeks. Autologous chondrocytes were implanted into focal defects created in the canine model. Animals were sacrificed at 6 weeks and analyzed for type II collagen. Type II collagen is a specific phenotypic marker for articular cartilage. a) Differentiating chondrocytes expressing type II collagen with developing lacunae are apparent in chondrocyte-treated defects. b) Close-up of nest.


	Figure 16. Chondrocytes genetically tagged with b-galactosidase reporter gene in the canine model at 6 weeks. To ensure that the chondrocytes that were implanted into the defect are facilitating the repair autologous chondrocytes were transfected in vitro with the b-galactosidase gene and implanted into defects created in the canine model. Animal were sacrificed at 6 weeks and analyzed by histology and immunohistochemistry. a) Cross-section of defect treated with labelled cells and covered with periosteum. Chondrocytes labelled with the gene will turn blue when reacted with the x-ga substrate. b) Immunohistochemical section of the defect containing the b-galactosidase treated chondrocytes and stained with type II collagen (brown). Note: type II collagen matrix surrounds the b-galactosidase treated cells.


	Figure 17. Histological sections of defects stained with hematoxylin and eosin in the canine model at 6 months (26 weeks). Defects were created in the femoral condyles in the canine model. Defects were either treated with no cells in the left knee (a, c, e) or treated with cultured autologous chondrocytes in the right knee (b, d, f) in three separate animals (ab, cd, ef). Animals were sacrificed at 6 months and defects were analyzed by Hematoxylin and Eosin histology. Results revealed that there was hyaline-like cartilaginous fill in both treated and untreated defects. There was a general trend toward more hyaline, articular cartilage fill in cell treated defects (b, d, f).


	Figure 18. Histological sections of defects stained by safranin O in the canine model at 1 year (52 weeks). Focal cartilage defects were surgically created in the femoral condyles of the canine model. Animals were separated into three treatment groups: untreated defects (a, d); untreated defects covered with periosteum (b, e); or defects implanted with cultured autologous chondrocytes and covered with periosteum (c, f). Animals were sacrificed at 1 year and defects analyzed for Safranin O. At one year, the trend seen at 6 months was not apparent. Levels of fibrous tissue, articular cartilage, and subchondral bone penetration in all treatment types were virtually identical. Significant levels of spontaneous regeneration of hyaline-like cartilage were noted in those defects having an uncompromised subchondral bone plate.

Conclusions

True committed articular chondrocytes mantain their ability to reversibly augment collagen gene expression, going from type II in the biopsy sample to type I in the monolayer and back to type II once implanted in the defect. The reversibility of this process is key to the successful repair of damaged articular cartilage with cultured autologous chondrocytes, and is characterized by the re-expression of hyaline articular cartilage markers upon implantation into focal defects. The finding that type X collagen, an indicator of chondrocyte hypertrophy and eventual bone formation, is not turned on by adult articular chondrocytes indicates that adult articular
chondrocytes are committed cells that are not capable of differentiating
further into bone. To date, the use of cultured autologous chondrocyte
implantation appears to be the most effective technique for restoring articular cartilage to chondral defects because it promotes long-term tissue repair and returns a hyaline-like articulating cartilage surface to damaged joints.

References

1. Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Engl J Med. 1994:331(14):889-895.

2. Baumgaertner MR, Cannon WD, Vittori JM, et al. Arthroscopic debridement of the arthritic knee. Clinical Orthopaedics Related Res. 1990; 253:197-202.

3. Jackson RW. Arthroscopic treatment of degenerative arthritis. In: McGinty JB, ed. Operative Arthroscopy. New York, NY: Raven Press; 1991: 319-323.

4. Jackson RW, Marans HJ, Silver RS. Arthroscopic treatment of degenerative arthritis of the knee. J Bone Joint Surg.1988; 70B:332.

5. Chang RW, Falconer J, Stulberg SD, et al . A randomized, control trial of arthroscopic surgery versus closed-needle joint lavage for patients with osteoarthritis of the knee. Arthritis Rheum. 1993; 36(3):289-296.

6. Johnson LL. Arthroscopic abrasion arthroplasty. In: McGinty JB, ed. Operative Arthroscopy. New York, NY: Raven Press; 1991: 341-360.

7. Tippet JW. Articular cartilage drilling and osteotomy in osteoarthritis of the knee. In: McGinty JB, ed. Operative Arthroscopy. New York, NY: Raven Press; 1991: 325-339.

8. Coventry MB, Ilstrup DM, Wallrichs SL. Proximal tibial osteotomy. J Bone Joint Surg. 1993;75A(2): 196-201.

9. Homminga GN, Bulstra SK, Bouwmeester PSM , Van Der Linden AJ. Perichndrial grafting for cartilage lesions of the knee. J Bone Joint Surg Br. l990;72B: 1003-1007.

10. Skoog T, Johansson SH. The formation of articular cartilage from free perichondrial grafts. Plastic Recons Surg. 1976;57(1):1-6.

11. O'Driscoll SW, Keeley FW, Salter RB, et al. Durability of regenerated cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. J Bone Joint Surg.1988;70A:595-606.

12. Rubak JM, Poussa M, Ritsila V. Chondrogenesis in repair of articular cartilage defects by free periosteal grafts in rabbits. Acta Orthop. 1982;53:181-186.

13. Ritsula VA, Santavirts S, Alhopuro S, et al. Periosteal and perichondral grafting in reconstructive surgery. Clin Orthop. 1994;302:259-265.

14. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg. 1994;76A(4):579-592.

15. Grande DA, Southerland SS, Manji R, et al. Repair of articular cartilage defects using mesenchymal stem cells. Tiss Eng. 1995;1(4):345-353.

16. Benya PD, Schaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. 1982;30:215-224.

17. Hsich PC, ThanapipatsiriS, Anderson P, et al. Repair of articular cartilage defects with periosteum. 41st Annual Meeting ORS Orlando FL. 1995;P172-29.

18. Topping RE, Anderson P, Goss D, Balian G. Effect of growth factors influencing type X collagen expression in periosteum. 41st Annual Meeting ORS Orlando FL. 1995; P29-5.

19. Johnstone B, Yoo JU, Barry FP. In vitro chondrogenesis of bone marrow-derived mesenchymal cells. 42nd Annual Meeting ORS Atlanta GA. 1996;P65-11.

Acknowledgments

The research presented in this report was directed by Ross Tubo, PhD, Director of Cell Biology at Genzyme Tissue Repair. The research was performed by Leesa M. Barone, PhD, Francois Binette, PhD, Todd Gagne, David McQuaid, and Courtney Wrenn.

Genzyme tissue repair

Genzyme
Tissue Repair
64 Sidney Street
Cambridge, MA 02139
USA
Telephone: 800-232-7546
Fax: 617-494-6561

Genzyme B.V.
Gooimeer 3-30
1411 DD Naarden
The Netherlands
Telephone: 31 (0)35 699 1200
Fax: 31 (0)35 694 3214