TISSUE ENGINEERING Volume 10, Number 5/6, 2004 © Mary Ann Liebert, Inc.
Regeneration of Hyaline Articular Cartilage with Irradiated Transforming Growth Factor 1-Producing Fibroblasts SUN U. SONG, Ph.D.,1 YOUNG-JIN HONG, M.D.,1 IN-SUK OH, M.D.,2 YOUNGSUK YI, Ph.D.,3 KYOUNG BAEK CHOI, M.S.,1 JUNG WOO LEE, M.S.,1 KWANG-WON PARK, B.S.,1 JEOUNG-UK HAN, M.D.,1 JUN-KYU SUH, M.D.,1 and KWAN HEE LEE, M.D.1
ABSTRACT The regeneration of hyaline articular cartilage by cell-mediated gene therapy using transforming growth factor 1 (TGF-1)-producing fibroblasts (NIH 3T3-TGF-1) has been reported previously. In this study, we investigated whether TGF-1-producing fibroblasts irradiated with a lethal dose of radiation are still capable of inducing the regeneration of hyaline articular cartilage. NIH 3T3TGF-1 fibroblasts were exposed to doses of 20, 40, or 80 Gy, using a irradiator, and then injected into artificially made partial defects on the femoral condyle of rabbit knee joints. The rabbits were killed 3 or 6 weeks postinjection and hyaline articular cartilage regeneration was evaluated by histological and immunohistochemical staining (n 5 per each group). Irradiated NIH 3T3-TGF1 fibroblasts started to die rapidly 3 days after irradiation; moreover, the kinetics of their viability were similar regardless of the radiation intensity. TGF-1 expression, measured by ELISA, showed that the TGF-1 protein produced from the irradiated cells peaked 5 days after irradiation and thereafter declined rapidly. Complete filling of the defect with reparative tissue occurred in all the groups, although variations were observed in terms of the nature of the repair tissue. Histological and immunohistochemical staining of the repair tissue showed that the tissue newly formed by irradiated NIH 3T3-TGF-1 fibroblasts after exposure to 20 Gy had hyaline cartilage-like characteristics, as was observed in the nonirradiated controls. On the other hand, the repair tissue formed by NIH 3T3-TGF-1 fibroblasts irradiated with 40 or 80 Gy showed more fibrous cartilage-like tissue. These results suggest that TGF-1-producing fibroblasts irradiated up to a certain level of lethal dose (i.e., 20 Gy) are able to induce normal-appearing articular cartilage in vivo. Therefore, irradiated heterologous cell-mediated TGF-1 gene therapy may be clinically useful and an efficient method of regenerating hyaline articular cartilage. INTRODUCTION
H
YALINE ARTICULAR CARTILAGE is a unique tissue, a complex mixture of chondrocytes and extracellular matrix proteins. Chondrocytes play an essential role in maintaining normal joint function by synthesizing and
degrading cartilage extracellular matrix. Hyaline articular cartilage is a unique tissue that contains neither a blood supply nor lymphatic drainage. The limited regenerative capacity of hyaline articular cartilage prevents the healing of traumatic injuries1 and other types of defects. Moreover, untreated cartilage defects could eventually
1Clinical
Research Center, College of Medicine, Inha University, Inchon, South Korea. of Orthopedic Surgery, College of Medicine, Inha University, Inchon, South Korea. 3TissueGene, Gaithersburg, Maryland. 2Department
665
SONG ET AL.
666
develop into osteoarthritis. Osteoarthritis is the most frequently encountered orthopedic disease associated with cartilage damage, and the degeneration of hyaline articular cartilage is the main pathogenesis of osteoarthritis.2 Most joints in the body are susceptible to cartilage damage. As the average human life span increases, the need to repair regenerative cartilage is also rapidly growing. However, no satisfactory treatment is available as yet.3 Novel strategies have been developed to repair or stimulate the regeneration of articular cartilage. These include the transplantation of osteochondral grafts, periosteum, cultured autologous chondrocytes, mesenchymal stem cells, and mixtures of biodegradable polymer and cytokine proteins.4–14 Despite some promising results, these methods have yielded regenerated cartilage tissue varying from fibrous to hyaline-like. Transplantation cells and tissues can be autologous or heterologous, and each procedure has advantages and disadvantages.3 Transplantation of autologous chondrocytes presents no problems of host immune rejection, but it takes at least 2 to 3 weeks to grow and expand chondrocytes from a biopsy specimen of cartilage and requires two open surgeries. On the other hand, transplantation of heterologous chondrocytes, whether allogeneic or xenogeneic, into the joint may induce a host immune reaction even though articular cartilage is avascular; however, the technique requires neither a biopsy specimen to grow chondrocytes ex vivo nor open surgery. We previously reported that the injection of mouse fibroblasts containing the transforming growth factor 1 (TGF-1) transgene into a partial hyaline articular cartilage defect produced hyaline-like cartilage within 6 weeks of transplantation in rabbits.15 Histological staining results showed that regenerated tissue was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline articular cartilage. Although the previous approach was xenogeneic, that is, the transplantation of mouse fibroblasts to a rabbit knee joint, no significant immune reactions were observed, probably because of the avascular nature of the cartilage and the immunosuppressive activity of TGF-1 protein produced from the injected cells. For clinical application, major concerns with allogeneic or xenogeneic cell transplantations include the immunogenicity and tumorigenicity induced by the transplanted cells. In this study, mouse fibroblasts containing TGF-1 were irradiated to minimize the possibility of immunogenicity and injected into a partial cartilage defect on the femoral condyle of a rabbit to find out whether these irradiated cells can still stimulate the regeneration of hyaline articular cartilage. Here we report that some irradiated NIH 3T3-TGF-1 fibroblasts (e.g., irradiated up to 20 Gy), are able to repair a partial hyaline cartilage defect in 6 weeks, much like nonirradiated NIH 3T3-TGF1 fibroblasts.
MATERIALS AND METHODS Cell culture and radiation Generation of NIH 3T3-TGF-1 cells was described previously.13 Irradiated NIH 3T3-TGF-1 cells and nonirradiated control NIH 3T3-TGF-1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and G418 (200 ng/mL). Cultured cells were treated with trypsin and 3 104 cells per milliliter in a sterile Eppendorf tube were irradiated with a dose of 20, 40, or 80 Gy, using a irradiator (IBL437; CIS biointernational, Saclay, France).
Animals and intraarticular injection New Zealand White rabbits weighing 2.0–2.5 kg were selected for the animal study. Animals were mature and had a tidemark line. The knee joint was exposed and a partial cartilage defect (3 6 mm, 1–2 mm deep) was made on the hyaline cartilage layer of the femoral condyle with a surgical knife. Control nonirradiated NIH 3T3TGF-1 cells or irradiated NIH 3T3-TGF-1 cells (15–20 L, 2 106 cells/mL) were loaded to the top of the defect. The cells were left in the defect for 15–20 min to allow the cells to permeate the wound before suturing. This study was approved by the Institutional Animal Care and Use Subcommittee of the Inha University College of Medicine (Inchon, South Korea).
MTT assay Nonirradiated control cells or irradiated NIH 3T3-TGF1 cells (1–2 104) were plated in a 6-well plate. After 1, 3, 5, 7, 9, 11, 13, and 15 days, 200 L of MTT solution (5 mg/mL in phosphate-buffered saline [PBS], sterilized by filtration) was added to the cells growing in 2 mL of medium and then incubated for an additional 4 h at 37°C. The reaction was stopped after the incubation by adding 0.1 mL of 10% sodium dodecylsulfate (SDS) in 0.01 M HCl and incubation was continued overnight at 37°C. Absorbance at 595 nm was measured with a DU650 spectrophotometer (Beckman Coulter, Fullerton, CA).
ELISA Nonirradiated control cells or irradiated NIH 3T3TGF-1 cells (1–2 104) were plated in a 6-well plate. After 1, 3, 5, 7, and 9 days, 200 L of supernatant from each well was added to an enzyme-linked immunosorbent assay (ELISA) plate coated with TGF- soluble receptor type II; subsequent steps were performed in accordance with the manufacturer’s instructions (Quantikine kit; R&D Systems, Minneapolis, MN).
Histology and immunohistological staining After harvesting the knee joints, specimens were fixed in formalin and decalcified in nitric acid. Blocks of the
REGENERATION OF HYALINE ARTICULAR CARTILAGE specimens were embedded in paraffin and cut into 0.5- to 0.8-nm-thick slices. Sections were stained with hematoxylin–eosin, safranin-O, toluidine blue, and Masson’s trichrome for microscopic examination of the repair tissue. Sections, prepared as described above, were deparaffinized and hydrated by sequential incubations in xylene and ethanol. After washing in 1 PBS for 2 min, the sections were blocked with 3% H2O2 for 10 min. The primary antibody against TGF-1, type II collagen (SouthernBiotech, Birmingham, AL), or mouse proliferating cell nuclear antigen (PCNA) (Serotec, Raleigh, NC) was applied to the sections and incubated for 1 h. The control sections were incubated in 1 PBS without the primary antibody at this stage. Sections were washed and blocked with 5% milk in 1 PBS for 20 min before incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. The chromogen reaction was performed with 0.05% diaminobenzidine (DAB) in 1 PBS for 5 min, and sections were subsequently stained with hematoxylin and mounted.
RESULTS Effect of irradiation on viability and TGF-1 productivity of NIH 3T3-TGF-1 fibroblasts in vitro TGF-1-expressing fibroblasts, called NIH 3T3-TGF1 cells, were generated by transduction with a retroviral vector carrying the TGF-1 gene, as described previously.13 To observe the effect of irradiation on the
667
viability of NIH 3T3-TGF-1 fibroblasts, cells were irradiated with 20, 40, or 80 Gy of irradiation, and grown in a standard medium. Their viabilities were measured by MTT assays performed 1, 3, 5, 7, 9, 11, 13, and 15 days postirradiation. Results of the MTT assays showed that the irradiated NIH 3T3-TGF-1 fibroblasts started to die rapidly 3 days after irradiation and that more than half the cells were dead after 9 days (Fig. 1A). All cells had died 2 to 3 weeks after irradiation. This demonstrated that all the doses used here were lethal to the NIH 3T3-TGF1 fibroblasts, and that the overall cell death kinetics of the various doses were similar. However, it was noticed that cells irradiated with 20 Gy showed a slower rate of cell death from 5 days after irradiation than did cells exposed to 40 or 80 Gy. To determine how long irradiated cells produced TGF-1 protein after irradiation, we measured the amount of TGF-1 protein present by the ELISA method 1, 3, 5, 7, and 9 days postirradiation. Results of the ELISA indicated that the irradiated cells produced TGF-1 proteins up to 5 days after irradiation, and that TGF-1 protein secretion and radiation doses were inversely related (Fig. 1B). Compared with the level of TGF-1 proteins produced by nonirradiated NIH 3T3TGF-1 fibroblasts, TGF-1 proteins produced by the cells irradiated with 20, 40, or 80 Gy was about 50, 25, or 10% of that of the nonirradiated cells, respectively.
Repair of hyaline articular cartilage defect with irradiated NIH 3T3-TGF-1 fibroblasts To investigate whether TGF-1-producing fibroblasts irradiated with a lethal dose of radiation are still able
FIG. 1. Effect of irradiation on the viability and TGF-1 productivity of NIH 3T3-TGF-1 fibroblasts in vitro. (A) Viability of NIH 3T3-TGF-1 cells was measured by MTT assay 1, 3, 5, 7, 9, 11, 13, and 15 days after irradiation. Each point represents the average of three independent experiments, with error bars representing the standard deviations. All values were normalized to the relative viability of the corresponding nonirradiated controls from the same experiment. Cell viabilities were relatively similar after the cells were exposed to doses of 20, 40, or 80 Gy of radiation. (B) Amount of TGF-1 protein was measured by ELISA 1, 3, 5, 7, and 9 days postirradiation. Compared with the level of TGF-1 proteins produced by nonirradiated NIH 3T3TGF-1 fibroblasts, TGF-1 protein production by cells irradiated with 20, 40, or 80 Gy was about 50, 25, or 10%, respectively.
668
to induce the repair of hyaline cartilage, cells were exposed to doses of 20, 40, or 80 Gy with a irradiator and then injected into an artificially made partial defect on the femoral condyle of a rabbit knee joint as previously described.13 The cartilage defects were filled with reparative tissues 6 weeks after injecting irradiated fibroblasts containing the TGF-1 transgene, regardless of irradiation intensity (Fig. 2). However, it was noticed that the quality of the repair tissue and the relative per-
SONG ET AL. centage of hyaline cartilage repair varied significantly and that these depended on the intensity of the radiation (Fig. 2). For example, the nature of reparative tissue formed with NIH 3T3-TGF-1 cells irradiated with 20 Gy was similar to that formed with nonirradiated cells (Fig. 2B and D). On the other hand, reparative tissue formed when using NIH 3T3-TGF-1 cells irradiated with 40 or 80 Gy showed a mixture of fibrous cartilagelike tissue (Fig. 2F and H). In addition, the tidemark lines
FIG. 2. Regeneration of hyaline articular cartilage with irradiated NIH 3T3-TGF-1 fibroblasts. (A, C, E, and G) Femoral condyle of rabbit knee 6 weeks after injection with either nonirradiated NIH 3T3-TGF-1 cells (A) or NIH 3T3-TGF-1 cells irradiated with 20 Gy (C), 40 Gy (E), or 80 Gy (G). (B, D, F, and H) Overview of defects stained with Masson’s trichrome from knee joints injected with nonirradiated NIH 3T3-TGF-1 cells (B) or cells irradiated with 20 Gy (C), 40 Gy (E), or 80 Gy (G). NIH 3T3-TGF-1 fibroblasts irradiated with 20 Gy were able to induce regeneration of hyaline articular cartilage in the condyle defects, whereas cells irradiated with 40 or 80 Gy generated fibrous cartilage-like tissue. Arrows indicate edges of defect.
REGENERATION OF HYALINE ARTICULAR CARTILAGE
669
FIG. 3. Relative percentage of repair tissue regeneration with irradiated NIH 3T3-TGF-1 fibroblasts. The relative percentage of repair tissue regeneration 6 weeks after injection of irradiated NIH 3T3-TGF-1 fibroblasts was measured as the ratio of regenerated cartilage height to that of normal cartilage. Standard deviations are represented by error bars. The relative percentage of repair tissue regeneration by NIH 3T3-TGF-1 fibroblasts irradiated with 20 Gy was similar to that achieved with nonirradiated cells.
in reparative tissues formed with cells exposed to 40 or 80 Gy were not as clear as those in reparative tissues formed with nonirradiated cells or cells irradiated with 20 Gy. The relative percentage of cartilage repair, measured as the ratio of repair tissue height to that of adjacent normal cartilage 6 weeks after transplantation, also decreased as the intensity of the radiation increased (Fig. 3). The average percentage of cartilage repair with cells irradiated with 0, 20, 40, and 80 Gy was 98, 96, 84, and 80%, respectively (Fig. 3). These statistical data also indicated that the relative percentage of cartilage repair by NIH 3T3-TGF-1 cells irradiated with 20 Gy was similar to that with nonirradiated cells.
Histological staining of repair cartilage tissue To measure the integrity and characteristics of the reparative tissue further, sections of repair tissue were stained with safranin-O and toluidine blue. Histological staining of reparative tissue formed with irradiated NIH 3T3-TGF1 cells was compared with that of tissue formed by nonirradiated control cells. Compared with the staining of control sections (Fig. 4A and B), the intensity of both safranin-O and toluidine blue staining of reparative tissues formed with irradiated cells was reduced, and this was reduced further as the strength of the irradiation was increased (Fig. 4C–H). These results suggest that NIH 3T3TGF-1 cells exposed to higher doses of radiation form less hyaline cartilage-like tissue during repair.
Immunohistochemical staining of repair cartilage tissue To show that reparative tissue was generated by injected irradiated NIH 3T3-TGF-1 cells and that is possessed hyaline articular cartilage characteristics, immunohistochemical staining was performed on sections of reparative tissue formed by NIH 3T3-TGF-1 cells irradiated with 20 Gy 3 weeks after transplantation. Immunohistochemical staining of sections of reparative tissue, using TGF-1 and PCNA antibodies, showed that TGF-1 protein expression occurred only in reparative tissue and that PCNA-positive cells were markedly increased in the same tissue (Fig. 5A and B). Type II collagen immunohistochemical staining proved that the reparative tissue had characteristics of hyaline articular cartilage (Fig. 5C). Mouse MHC class I staining indicated that some of the injected mouse fibroblasts cells remained in the regenerated cartilage tissue, although the staining is rather weak (Fig. 5E). In contrast, rabbit and goat secondary antibody controls showed no immunohistochemical staining in the same reparative tissues (Fig. 5D and F, respectively). These immunohistochemical staining results, along with the other histological staining data shown in Figs. 2 and 4, suggest that the generation of reparative tissue is stimulated by TGF-1 expression from the injected cells irradiated with 20 Gy, and that the newly formed tissue has the characteristics of hyaline articular cartilage.
SONG ET AL.
670
FIG. 4. Histological staining of cartilage regenerated with irradiated NIH 3T3-TGF-1 fibroblasts. (A, C, E, and G) Regenerated articular cartilage was stained with safranin-O 6 weeks after injecting either nonirradiated NIH 3T3-TGF-1 cells (A) or NIH 3T3-TGF-1 cells irradiated with 20 Gy (C), 40 Gy (E), or 80 Gy (G). (B, D, F, and H) Regenerated articular cartilage was stained with toluidine blue 6 weeks after injection with nonirradiated NIH 3T3-TGF-1 cells (B) or NIH 3T3-TGF-1 cells irradiated with 20 Gy (D), 40 Gy (F), or 80 Gy (H). Regenerated hyaline-like articular cartilage formed using cells irradiated with 20 Gy has the characteristics of normal hyaline cartilage, whereas that formed using cells exposed to 40 or 80 Gy consists of a mixture of fibrous and hyaline-like cartilage. Arrows indicate edges of defect.
DISCUSSION This study demonstrates that irradiated NIH 3T3 fibroblasts containing the TGF-1 transgene can be used to stimulate the repair of hyaline articular cartilage in a partial cartilage defect model. The integrity of the structure of newly formed repair tissue, using NIH 3T3-TGF1 cells irradiated with 20 Gy, was similar to that of normal cartilage tissue, whereas structures formed by cells irradiated with 40 or 80 Gy showed a mixture of fibrous connective tissue and fibrous cartilage-like tissue. These results suggest that fibroblasts containing TGF-1 and irradiated with doses of up to at least 20 Gy are still able
to produce sufficient TGF-1 protein to stimulate the remaining chondrocytes and/or chondrocyte precursors residing in the wound base, inducing them to secrete extracellular matrix proteins in vivo. TGF-1 expression measured in vitro by ELISA after irradiation showed that the production of active TGF-1 protein increased up to 5 days after irradiation and declined thereafter (Fig. 1B), indicating that transgene expression was not totally blocked for the first several days after irradiation. The results of histological staining with safranin-O and toluidine blue demonstrated that the nature of the newly generated repair tissues depends on the intensity of irradiation. As shown in Fig. 4, the intensity of safranin-
REGENERATION OF HYALINE ARTICULAR CARTILAGE
671
FIG. 5. Immunohistochemical staining of regenerated cartilage formed by irradiated NIH 3T3-TGF-1 fibroblasts. A–F) Regenerated tissue stained with PCNA (A), TGF-1 (B), type II collagen (C), or mouse MHC class I (E) antibody, or with rabbit or goat secondary antibody alone (controls; D and F), 3 weeks after injection with NIH 3T3-TGF-1 cells irradiated with 20 Gy (original magnification, 40). Expression of PCNA, TGF-1, and mouse MHC class I proteins was detected in regenerated cartilage (RC). Expression of type II collagen proteins was detected in both regenerated cartilage (RC) and normal cartilage (NC) at similar intensity. Regeneration of hyaline-like articular cartilage was induced by injecting NIH 3T3-TGF-1 fibroblasts irradiated with 20 Gy. Regenerated cartilage areas marked by arrows are also shown in insets at higher original magnification (200).
O and toluidine blue staining of reparative tissue produced by irradiated NIH 3T3-TGF-1 cells decreased as the irradiation intensity increased, versus the intensity of staining of reparative cartilage formed by nonirradiated NIH 3T3-TGF-1 fibroblasts. Therefore it is likely that irradiation decreases the synthesis of extracellular matrix components, such as keratan and chondroitin sulfate chains. The results of immunohistochemical staining suggest that TGF-1, type II collagen, and PCNA proteins were present in reparative tissue 3 weeks after injection of the fibroblasts irradiated with 20 Gy (Fig. 5A–C). These results suggest that the injected irradiated cells produce TGF-1 protein in vivo after transplantation and that they also synthesize hyaline cartilage-specific extracellular matrix proteins in the newly formed repair tissue. However, it should be noted that the amount of these proteins detected by immunohistochemical staining seems to be lower than that associated with nonirradiated cells, described previously.13 Our previous study suggested that cell-mediated gene therapy might be a novel way of treating orthopedic diseases in which hyaline articular cartilage damage has oc-
curred. Transplantation of autologous chondrocytes expanded in vitro is widely accepted as a clinical approach.16–18 However, the disadvantages of this mode of transplantation, such as the need for open surgery and its high cost, limit its widespread clinical application. It would be clinically useful if allogeneic cells could be used readily without subsequent immunogenicity or tumorigenicity of the transplanted allogeneic cells. The results of the present study suggest that such irradiated heterologous cells could possibly be transplanted to regenerate hyaline articular cartilage in vivo, because the irradiation of such cells before transplantation reduces the immunogenicity and tumorigenicity of the injected heterologous cells. We hope that the results of this study advance the feasibility of cell-mediated gene therapy using allogeneic or xenogeneic heterologous cells. A cell-mediated TGF-1 cytokine gene therapy approach might be more beneficial for cartilage regeneration, because cartilage tissue is avascular and TGF-1 has immunosuppressive activity. Therefore, irradiated allogeneic cell-mediated TGF-1 gene therapy may be clinically useful and an efficient method for treating orthopedic diseases involving hyaline articular damage.
SONG ET AL.
672
ACKNOWLEDGMENTS These studies were partially supported by a grant from TissueGene (Gaithersburg, MD) and by an Inha University research grant (INHA-21107).
REFERENCES 1. Bentley, G., and Greer, R.B. Homotransplantations of isolated epiphyseal and articular cartilage chondrocytes into joint surfaces of rabbits. Nature 230, 385, 1971. 2. Bentley, G., Smith, A.V., and Mukerjhee, R. Isolated epiphyseal chondrocyte allograft into joint surface: An experimental study in rabbits. Ann. Rheum. Dis. 37, 449, 1978. 3. Brittberg, M., Lindahl, A., and Nilsson, A. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N. Engl. J. Med. 331, 889, 1994. 4. Brittberg, M. Autologous chondrocyte transplantation. Clin. Orthop. 367(Suppl.), 147, 1999. 5. Buckwalter, J.A., and Mankin, H.J. Articular cartilage: Tissue design and chondrocyte–matrix interactions. Instr. Course Lect. 47, 477, 1998. 6. Chen, F.S., Frenkel, S.R., and DiCesare, P.E. Chondrocyte transplantation and experimental treatment options for articular cartilage defects. Am. J. Orthop. 26, 396, 1997. 7. Chesterman, P.J., and Smith, A.U. Homotransplantation of articular cartilage and isolated chondrocytes: An experimental study in rabbits. J. Bone Joint Surg. Am. 50, 184, 1968. 8. Gillogly, S.D., Voight, M., and Blackburn, T. Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J. Orthop. Sports Phys. Ther. 28, 241, 1998. 9. Grande, D.A., Singh, I.J., and Pugh, J. Healing of experimentally produced lesions in articular cartilage following chondrocyte transplantation. Anat. Rec. 218, 142, 1987. 10. Hunziker, E.B., and Rosenberg, L.C. Repair of partialthickness defects in articular cartilage: Cell recruitment
11.
12.
13.
14. 15. 16.
17.
18.
from the synovial membrane. J. Bone Joint Surg. Am. 78, 721, 1996. Johnson, B., and Yoo, J.U. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin. Orthop. 367(Suppl.), 156, 1999. Kimura, T., Yasui, N., and Ohsawa, S. Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin. Orthop. 186, 231, 1984. Lee, K.H., Song, S.U., Hwang, T.S., Yi, Y., Oh, I.S., Lee, J.Y., Choi, K.B., Choi, M.S., and Kim, S.-J. Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor 1-producing fibroblasts. Hum. Gene Ther. 12, 1805, 2001. Mankin, H.J. The reaction of articular cartilage to injury and osteoarthritis. N. Engl. J. Med. 291, 1285, 1974. Mankin, H.J. The response of articular cartilage to mechanical injury. J. Bone Joint Surg. Am. 64, 460, 1982. O’Driscoll, S.W., Keeley, F.W., and Salter, R.B. Durability of regenerated articular 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. Am. 70, 595, 1988. O’Driscoll, S.W. Current Concepts Review: The healing and regeneration of articular cartilage. J. Bone Joint Surg. Am. 80, 1795, 1998. Wakitani, S., Goto, T., and Young, R.G. Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Eng. 4, 429, 1998.
Address reprint requests to: Kwan Hee Lee, M.D. Clinical Research Center College of Medicine Inha University 7-206, 3-Ga, Shinheung-Dong Chung-Gu, Inchon 400-711, South Korea E-mail:
[email protected]
This article has been cited by: 1. Elizaveta Kon, Roberto Buda, Giuseppe Filardo, Alessandro Di Martino, Antonio Timoncini, Annarita Cenacchi, Pier Maria Fornasari, Sandro Giannini, Maurilio Marcacci. 2010. Platelet-rich plasma: intra-articular knee injections produced favorable results on degenerative cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 18:4, 472-479. [CrossRef] 2. Youngsuk Yi , Kyoung Baek Choi , Chae-Lyul Lim , Jong-Pil Hyun , Hyeon-Youl Lee , Kun Bok Lee , Lillian Yun , Asli Ayverdi , Sally Hwang , Vivian Yip , Moon Jong Noh , Kwan Hee Lee . 2009. Irradiated Human Chondrocytes Expressing Bone Morphogenetic Protein 2 Promote Healing of Osteoporotic Bone Fracture in RatsIrradiated Human Chondrocytes Expressing Bone Morphogenetic Protein 2 Promote Healing of Osteoporotic Bone Fracture in Rats. Tissue Engineering Part A 15:10, 2853-2863. [Abstract] [Full Text] [PDF] [PDF Plus] 3. Jordan Wicker, Kenneth Kamler. 2009. Current Concepts in Limb Regeneration. Annals of the New York Academy of Sciences 1172:1, 95-109. [CrossRef] 4. Charles A. Gersbach, Jennifer E. Phillips, Andrés J. García. 2007. Genetic Engineering for Skeletal Regenerative Medicine. Annual Review of Biomedical Engineering 9:1, 87-119. [CrossRef] 5. Takatomi Oku, Satoshi Iyama, Tsutomu Sato, Yasushi Sato, Maki Tanaka, Tamotsu Sagawa, Kageaki Kuribayashi, Tetsuya Sumiyosi, Kazuyuki Murase, Takuro Machida, Tetsuro Okamoto, Takuya Matsunaga, Tetsuji Takayama, Minoru Takahashi, Junji Kato, Hirofumi Hamada, Yoshiro Niitsu. 2006. Amelioration of Murine Dextran Sulfate Sodium-induced Colitis by Ex Vivo Extracellular Superoxide Dismutase Gene Transfer. Inflammatory Bowel Diseases 12:7, 630-640. [CrossRef] 6. A. J. Salgado, J. E. Figueiredo, O. P. Coutinho, R. L. Reis. 2005. Biological response to pre-mineralized starch based scaffolds for bone tissue engineering. Journal of Materials Science: Materials in Medicine 16:3, 267-275. [CrossRef]
