Objective To establ ish a porcine model of articular full-thickness cartilage defect characterized byremaining cartilage calcified zone on femoral trochlea, so as to provide a considerable and comparative control group forinvestigating repair effects of tissue engineered scaffolds in articular cartilage defects with cartilage calcified zone remaining.Methods The full-thickness cartilage column defects (6 mm in diameter, 0.2-0.5 mm in depth) without damage on calcifiedcartilage zone were made on the femoral trochlea in 9 clean-grade 6-month-old Guizhou mini pigs by standard cartilage-defectmakingsuites. Microscopical observation was performed after modeling. Scanning were made by 3.0T MRI at 4 weeks. Thengeneral observation, stereomicroscope, and histological staining were used to observe cartilage repair. Results All animals wereal ive. No infection of incisions or patellar dislocations occurred; they were able to walk with partial weight-bearing immediatelyafter surgery and could move freely without limp at 1 week. Obvious signal discontinuity in trochlea and subchondral bone couldbe observed in MRI, without deep signal change in defects surrounding. Microscopical observation showed a few repair tissueand petechia at base of the defect with clear boundary. Nearly intact calcified zone of cartilage and zonal collapse of subchondralbone in defects could be observed with stereomicroscope. Under common microscope, no chondrocytes was found in defects,as well as negative staining of fast green-safranin O and alcian blue. Under polarized microscope, the bottom of defects werefilled with a l ittle of fibrous tissue presenting continuous and b l ight-refraction by sirius red staining. Conclusion Theanimal model of articular full-thickness cartilage defect on femoral trochlea by standard cartilage-defect-making suites can beapplied for the research of cartilage disease in early human osteoarthritis and function of calcified cartilage zone in pig.
Objective To investigate the effect of “two-phase” tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. Methods Thirty-twoNew Zealand white rabbits were involved in the experiment. “Two-phase” allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs wereseparated from 18 New Zealand white rabbits and induced to express chondrocyticphenotype. The chondrocyte precursor cells were seeded onto “two-phase” allogeneic BMG to construct tissue engineering cartilage. Masson’s trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage(3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits(including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n=18), with BMG at the left side(group B, n=18), and without any implant at both sides in the remaining 9 rabbits as a control( group C, n=18). After 1, 3 and6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson’s trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type Ⅱ were carried out to assess the repair efficacy of tissue engineered cartilage. Results The “two-phase” BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 μm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 μm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage graduallythinned, but defect was repaired and the structure of the articular surface ansubchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C(Plt;0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type Ⅱ in group A was also higher than those in groups B and C(Plt;0.01). Conclusion “Two-phase” allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous chondrocyte precursor cells induced from MSCs toconstruct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.
In order to observe the histological changes of the autogenous perichondrium graft from rib in the repair of injured articular cartilage of the condylar process of mandible, 50 rabbits were used, in which 15 were served as control. The articular cartilage with its subchondral bone were resected and an autogenous graft of costal perichondrium was sutured onto the raw surface of the condylar process, and in the controls, only the articular portion of the condylar process was resected without the application of autogenous costal perichondrium graft. The morphological changes of the newly formed cartilage during the process of its development were investigated by hiostological and autoradiog aphic techniques. The result revealed that 10 days after operation, the graft had increased in thickness and was richly populated form the proliferation of mesenchyme-like cells. Twenty to thirty days later, the chondrocytes were matured and the newly formed cartilage had covered the bony surface of mandibular condyle. At 60 days, the newly formed cartilagenous joint surface became glossy, and the morphology and arrangement of cells tended to be regular simulating the morphology of normal articular cartilage. From the experiment, it could be concluded that (1) The autogenous perichondrium graft placed on the condylar surface of mandible could form new articular cartilage which was similar in tissue morphology to the normal condylar cartilage. (2) The process of development of newly formed cartilage was similar to that of the normal cartilage. (3) The motion and loading on the joint could promote the formation of new cartilage and undergo biological reformation, gradually resulting in normal joint morphology. On this basis, the clinical application of autogenous perichondrium graft to repair injured cartilage of the condylar process of the mandible was feasible.
Objective Platelet-rich plasma (PRP) can enhance the chondrocyte prol iferation and repair of cartilage defects. To explore the safety and efficacy of intra-knee-articular injection of PRP to treat knee articular cartilage degeneration by comparing with injecting sodium hyaluronate (SH). Methods Thirty consecutive patients (30 knees) with knee articular cartilage degeneration were selected between January 2010 and June 2010. According to different injections, 30 patients wererandomly divided into PRP group (test group, n=15) and SH group (control group, n=15). There was no significant difference in gender, age, body mass index, and Kellgren-Lawrence grade between 2 groups (P gt; 0.05). Test group received 3.5 mL of PRP intra-knee-articular injections while control group received 2 mL of SH during the same time period. Both treatments were administered in series of 3 intra-knee-articular injections at 3-week intervals. Then, adverse reactions were recorded. International Knee Documentation Committee (IKDC) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, and Lequesne index were used for evaluation of treatment results. Results The patients of 2 groups were followed up 6 months. There were significant differences in IKDC score, WOMAC score, and Lequesne index between pre- and post-injection in 2 groups (P lt; 0.05); no significant difference was found between different time points (3, 4, and 6 months) in test group (P gt; 0.05), while significant differences were found between the postoperative 6th month and the postoperative 3rd and 4th months in control group (P lt; 0.05). There was no significant difference in IKDC score, WOMAC score, and Lequesne index between 2 groups within 4 months (P gt; 0.05), but the effectiveness of test group was significantly better than that of control group at 6 months after injection (P lt; 0.05). Adverse reactions occurred in 12 patients (31 injections) of test group and in 12 patients (30 injections) of control group. No significant difference in onset time, termination time, and duration of adverse reactions were found between 2 groups (P gt; 0.05). Conclusion Intra-knee-articular injection of PRP to treat knee articular cartilage degeneration is safe, which can alleviate symptoms of pain and swell ing and improve the qual ity of l ife of patients; however, further data of large samples and long-term follow-up are needed to confirm the safety and effectiveness.
Objective To review the recent research progress on relationshi p between subchondral bone and cartilage degeneration in osteoarthritis (OA), and to predict future research directions. Methods Recent l iteratures about the pathological changes of subchondral bone in OA were reviewed and analyzed in terms of biomechanics, bone remodel ingand biological factors. Results Subchondral bone sclerosis or softening was the result of osteoarthritis and also closely related to the occurrence and development of OA. Inhibiting the bone metabol ism of subchondral bone could slow the degeneration of articular cartilage. Conclusion For the treatment of OA, it is necessary to pay close attention to cartilage changes and the prevention of subchondral bone degeneration.
Objective To review the latest progress of seeding cells for articular cartilage tissue engineering. Methods The recent original l iteratures on seeding cells for articular cartilage tissue engineering were extensively reviewed. Results The chondrocytes derived from BMSCs’ differentiation would be a main source of seeding cells articular cartilage for tissue engineering. Three-dimensional scaffolds and cultivation surroundings played important roles in the field of articular cartilage tissue engineering. Conclusion The util ization of cytokine and transgenic technology as well as improvements of three-dimensional scaffolds and cultivation surroundings will promote the development of articular cartilage tissue engineering.
OBJECTIVE: To study the gap junction and phenotype of cultured chondrocyte of rabbit, and the gap junction between the chondrocytes in the same cartilage cavities in human femoral head articular cartilage. METHODS: CFDA-AM was added into the medium of the fifth passage of chondrocyte of rabbit in the 96-well plate. The fluorescent in spherical and fibroblast-like chondrocytes was detected separately. The recurrence of the fluorescent in accordant with time in 16 minutes was recorded after blanching the fluorescent with laser. And the fluorescent after blanching of chondrocyte in the cartilage cavities in the proliferative zone of articular cartilage of adult human femoral head was recorded, too. RESULTS: The average fluorescent of the single layer of the fibroblast-like chondrocyte was 83(ranged from 1 to 274), the highest was found in the spherical shaped cell (averaged 2,057, ranged from 340 to 3,538). The recurrence of the fluorescent after the blanching appeared only in the spherical chondrocyte, the gap junctions reappeared only in the spherical chondrocytes, as well as in the cells in the cartilage cavities in the articular cartilage of the human femoral head. CONCLUSION: The appearance of the gap junction is corresponded with the spherical shape, secretion of the cartilage matrix of the chondrocyte. There are gap junctions in the cells in the same cartilage cavities in the articular cartilage of the human femoral head, while no gap junctions in the isolated chondrocytes in the cartilage.
OBJECTIVE To investigate possibility of cartilage cultured in centrifuge tube as graft materials. METHODS: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy. RESULTS: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus. CONCLUSION: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.
OBJECTIVE To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P lt; 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P lt; 0.01). CONCLUSION If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.