OBJECTIVE This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS The chondrocytes isolated from mini swines’ ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
OBJECTIVE To prevent early closure of growth plate and developmental deformities of limbs by allografts of cultured cartilages into growth plate defects of rabbits. METHODS Chondrocytes isolated from articular cartilage of 1-month rabbits formed cartilage after cultivation in centrifuge tubes. The cartilages cultured for two weeks were implanted into growth plate defects of proximal tibiae of 6-weeks rabbits. At 4th and 16th weeks, X-ray, histologic and immunohistochemical examination were performed. RESULTS The tibiae had no marked deformities after 4 weeks of operation. Histologic examinations showed that the defects were filled with cartilage. Immunohistochemical results of type II collagen were positive. The tibiae with allografts of cultured cartilages had no evident deformities after 16 weeks of operation. Histologic examination showed nearly closure of growth plates. On the contrary, the tibiae on control side formed severe deformities and growth plate were closed. CONCLUSION Allograft of cultured cartilages into growth plate defects may replace lost growth plate tissues, maintain normal growth of limbs and prevent developmental deformity.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
Objective To repair the defects in articular cartilage with collagen complex gradient TCP in vivo andto study the regenerated cartilage histomorphologically. Methods The models of defects in articular cartilage were madeartificially in both condylus lateral is femoris of mature rabbits, male or female, with the weight of 2.0-2.5 kg. The right defects were implanted with the material of Col/TCP as the experimental group and the left defects were untreated as the control group. The rabbits were killed at 4, 6, 8, 12 and 24 weeks after operation, respectively, with 6 ones at each time, and the macroscopic, histological, ultrastructural examinations and semi-quantity cartilage scoring employing Wakitanifa repaired cartilage value system were performed. Results Four weeks after operation, the defects in the experimental group were partly filled with hyal ine cartilage. Twelve weeks after operation, the defects in the experimental group were completely filled with mature hyal ine cartilage. Twenty-four weeks after operation, regenerated cartilage had no ataplasia. However, fibrous tissues were seen in the control group all the time. At 4, 6, 8, 12 and 24 weeks ostoperatively, the Wakitanifa cartilage scores were 7.60 ± 0.98, 5.69 ± 0.58, 4.46 ± 0.85, 4.35 ± 0.12 and 4.41 ± 0.58, respectively, in the experimental group and 10.25 ± 1.05, 9.04 ± 0.96, 8.96 ± 0.88, 8.88 ± 0.68 and 8.66 ± 0.54, respectively, in the control group. At 4, 6, 8, 12 and 24 weeks postoperatively, the collagen II contents were 0.28% ± 0.01%, 0.59% ± 0.03%, 0.68% ± 0.02%, 0.89% ± 0.02% and 0.90% ± 0.01%, respectively, in the experimental group, while 0.08% ± 0.02%, 0.09% ± 0.04%, 0.11% ± 0.03%, 0.25% ± 0.03% and 0.29% ± 0.01%, respectively, in the control group. Differences between the control group and the experimental group were significant (P lt; 0.05). By then, typical chondrocyte was observed by transmission electron microscope in the experimental group and much fiber with less fibrocyte was observed in the control group. Conclusion Three-dimensional scaffold collagen complex gradient TCP may induce cartilage regeneration to repair the defects of articular cartilage in vivo.
A novel structural dynamics test method and device were designed to test the biomechanical effects of dynamic axial loading on knee cartilage and meniscus. Firstly, the maximum acceleration signal-to-noise ratio of the experimental device was calculated by applying axial dynamic load to the experimental device under unloaded condition with different force hammers. Then the experimental samples were divided into non-specimen group (no specimen loaded), sham specimen group (loaded with polypropylene samples) and bovine knee joint specimen group (loaded with bovine knee joint samples) for testing. The test results show that the experimental device and method can provide stable axial dynamic load, and the experimental results have good repeatability. The final results confirm that the dynamic characteristics of experimental samples can be distinguished effectively by this device. The experimental method proposed in this study provides a new way to further study the biomechanical mechanism of knee joint structural response under axial dynamic load.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
ObjectiveTo construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2(BMP-2) and transforming growth factor β3(TGF-β3) genes,to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs. MethodsBMP-2 cDNA and TGF-β3 cDNA were amplified by PCR,and were subcloned into the pEC3.1(+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively.They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3.The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured.The 3rd passage BMSCs were transfered with Ad-BMP-2(group A),Ad-TGF-β3(group B),Ad-BMP-2+Ad-TGF-β3(group C),and untransfected cells served as a control (group D).The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR,immunofluorescence,and Western blot.The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type Ⅱ. ResultsThe Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing.The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully,the titer was 5.6×108 and 1.6×108 pfu/mL respectively.The PCR results showed a light band at 310 bp in group A and at 114 bp in group B,and both 310 bp and 114 bp bands in group C,but no band in group D.The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B,respectively;in group C,both red and green fluorescence expressions were detected,and no red or green fluorescence was detected in group D.The results of Western blot showed that there was a light band at 18×103 in group A and at 50×103 in group B;both 18×103 and 50×103 bands were detected in group C;but no band was detected in group D.The cells were positive for collagen type Ⅱ in groups A,B,and C;group C acquired strong collagen type Ⅱ staining when compared with group A and group B;in group D,the cells were negative for collagen type Ⅱ staining. ConclusionThe recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully.The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection,which could afford modified seeding cells for cartilage tissue engineering.
Objective To explore the relationship of the limited resource of the autologous bone marrow mesenchymal stem cells (MSCs) in articularcavity to the treatment results of full-thickness articular cartilage defect, and to investigate whether the extrogenous sodium hyaluronate(SH) promotes the migration of MSCs cultured in vitro tothe articular defect in vivo. Methods Sixty-six Japan rabbits were made the model of the full-thickness articular cartilage defect (5 mm width and 4 mm depth).The autologous MSCs were extracted from the rabbit femur, cultured in vitro, labeledby Brdu, and injected into the injured articular cavity with or without SH. Theexperiment was divided into 4 groups; group A (MSCs and SH, n=15); group B (MSCs, n=15); group C (SH, n=18); and group D (non-treatment, n=18). The morphologic observation was made by HE staining, Mallory staining and immunohistochemical staining after 5 weeks, 8 weeks and 12 weeks of operation. Results There were significant differences in the thickness of repairing tissue between group A and group B(Plt;0.01); but there were no significant differences between group A and group C, and between group B and group D(P＞0.05). Thehistological observation showed that the main repairing tissue was fibrocartilage in group A and fiber tissue in group B. Conclusion MSCs cultured in vitro and injected into the articular cavity can not improve the treatment results of the articular cartilage defect. Extrogenous SH has effect on repairing cartilage defect. The extrogenous SH has no effect on the chemotaxis of the MSCs, and on the collection of MSCs into the joint defect.
OBJECTIVE To present a simple and reliable method for the reconstruction of metacarpophalangeal joint by the cartilage transplantation of metatarsophalangeal joint. METHODS From 1990, nine cases (11 sides) with traumatic metacarpophalangeal joint defect were treated by the autogenous cartilage transplantation of metatarsophalangeal joint followed by modified treatment. Appropriate biological mechanics was provided by internal fixation and collateral ligament repair. RESULTS Followed up 6 months to 7 years, the range of joint motion was increased 35.1 degrees. The fusion of donor phalanges was fine, and the range of joint motion was decreased, even ankylosis after plastic operation, but no pain and no effect on walk. CONCLUSION The key to successful operation is better matching of cartilage, reliable internal fixation, ligament reconstruction, thin cartilage and little bone of the donor, appropriate biological mechanical surroundings.