Objective To evaluate the immunological reaction and the outcome of allogeneic chondrocyte transplantation in repairing articular cartilage defects in porcins. Methods Full articular cartilage from the knee of two Shanghai white porcins about one-month-old was removed and cut mechanically, digested by 0.25% trypsin and 0.2% type Ⅱ collagenase and cultured in 10％ DMEM medium. Defects of 0.5 cm×0.5 cm involving the subchodral bone were created in both the left and right femur condyloid in 8 two-month-old Yunnai bama porcins. Allogeneic chondrocyte transplantation were implanted in defects at a density of (1.0-2.0)×106,0.2 ml. The lymphocytes from the receivers’ blood were collected before transplantation and after 3, 5, 7 and 12 weeks of transplantation, then mixed with allogeneic chondrocytes to determin the lymphocyte stimulation index(SI) in vitro. The histological observation in vivo was made after 5, 7 and 24 weeks of transplantation. Results Lymphocyte SI at 3, 5, 7 and 12 weeks(1.457±0.062,1.739±0.142,1.548±0.047，1.216±0.028) after transplantation was higher than that before transplantation(1.102±0.034，Plt;0.05). SI began to increase in the 3rd week and reached the peak value in the 5th week, then gradually declined at the 7th and 12th weeks, showing significant differences when compared with in the 5th week (Plt;0.05). Inflammation and lymphocytes infiltration could be seen in subchondral bone and the intergration area between repair tissue and normal cartilage in the 5th week, and then decreased and limited in subchondral bone in the 7th week. Defects were filled with cartilage tissue, which had good intergration with subchondral bone at 24 weeks after transplantation. Conclusion Immunological reactions can be found at early stage of allogeneic chondrocyte transplantation and then decreased with the time, the fullthickness articular cartilage defects could be repaired mainlywith hyaline cartilage by the allogeneic chondrocyte transplantation. This may provide a new method to repair articular cartilage defects clinically.
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
Objective To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods Eight Sprague Dawley rats were selected to obtain the L1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O2 condition (group A), and under 0.5% O2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Results At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B (t=1.026,P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A (t=22.672,P=0.015;t=18.396,P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B (t=0.594,P=0.781;t=1.251,P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C (t=27.143,P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C (t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C (t=37.648,P=0.006;t=16.729,P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C (t=25.583,P=0.011). Conclusion HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.
Objective To sum up the experimental and clinical history as wellas latest development of repair of growth plate injury Methods Recent articles about repair of growth plate injury were extensively reviewed and major reparative methods were introduced, especially including tissue engineering research on growth plate.Results Repair of growth plate injury was a great difficulty inexperimental study and clinical treatment of pediatric orthopedics. Transplantation of free growth plate and cartilage were unfavorably used because of lack ofblood supplement. Although circulation problem was solved by transplantation ofvascularized growth plate, autografts of epiphyseal cartilage were involved in limitation of donor, and allografts of epiphyseal cartilage induced immunological reaction. Noncartilaginous tissue and material could only prevent formation of bony bridge in small defect of growth plate and lacked ability of regenerative repair. Transplantationof tissue engineered cartilage and chondrocytes might be a choice for repair ofgrowth plate injury Conclusion Owing to lack of safe and effective methods ofrepairing growth plate injury, research on chondrocyte and tissue engineered cartilage should be further done.
A solid-liquid two-phase finite element model of articular cartilage and a microscopic finite element model of chondrocytes were established using the finite element software COMSOL in this study. The purpose of the study is to investigate the mechanics environment and the liquid flow field of the host cartilage chondrocytes in each layer by multi-scale method, under physiological load, with the different elastic modulus of artificial cartilage to repair cartilage defect. The simulation results showed that the uniform elastic modulus of artificial cartilage had different influences on the microenvironment of different layer chondrocytes. With the increase of the elastic modulus of artificial cartilage, the stress of the shallow surface layer and the intermediate layer chondrocytes increased and the stress of deep layer chondrocytes decreased. The flow field direction of the middle layer and the bottom layer of cartilage can also be changed by artificial cartilage implantation, as well as the ways of nourishment supply of the middle layer and underlying chondrocytes change. A barrier to underlying chondrocytes nutrition supply may be caused by this, thus resulting in the uncertainty of the repair results. With cross-scale finite element model simulation analysis of chondrocytes, we can quantitatively evaluate the mechanical environment of chondrocytes in each layer of the host cartilage. It is helpful to assess the clinical effect of cartilage defect reparation more accurately.
As a kind of mechanical effector cells, chondrocytes can produce a variety of physical and chemical signals under the stimulation of multiaxial load in vivo, which affect their own growth, development and apoptosis. Therefore, simulating the mechanical environment in vivo has become a research hotspot in the culture of chondrocytes in vitro. Although a large number of reports have fully proved that different mechanical stimulation can regulate the metabolism of chondrocytes, the loading scheme has not been agreed. Starting from different mechanical forms, this review will explore the differences in the regulation of chondrocyte metabolism by different mechanical stimuli, so as to find an advantage scheme to promote the growth and proliferation of chondrocytes and to develop a more stable, effective and reliable experimental strategy.
Objective To investigate the effect of dynamic compression and rotation motion on chondrogenesis of the 3rd passage cell-loaded three-dimensional scaffold in a joint-specific bioreactor in vitro so as to provide theoretical basis of the autologous chondrocyte transplantation in clinical practice. Methods Primary chondrocytes were isolated and cultured from the knee cartilage of 3-4 months old calves. The 3rd passage cells were seeded onto fibrin-polyurethane scaffolds (8 mm × 4 mm). Experiment included 5 groups: unloaded culture for 2 weeks (group A), direct load for 2 weeks (group B), unloaded culture for 4 weeks (group C), direct load for 4 weeks (group D), and unload for 2 weeks followed by load for 2 weeks (group E). The cell-scaffold was incubated in incubator (unload) or in a joint-specific bioreactor (load culture). At different time points, the samples were collected for DNA and glycosaminoglycan (GAG) quantification detect; mRNA expressions of chondrogenic marker genes such as collagen type I, collagen type II, Aggrecan, cartilage oligomeric matrix protein (COMP), and superficial zone protein (SZP) were detected by real-time quantitative PCR; and histology observations were done by toluidine blue staining and immunohistochemistry staining. Results No significant difference was found in DNA content, GAG content, and the ratio of GAG to DNA among 5 groups (P gt; 0.05). After load, there was a large number of GAG in the medium, and the GAG significantly increased with time (P lt; 0.05). The mRNA expression of collagen type I showed no significant difference among 5 groups (P gt; 0.05). The mRNA expression of collagen type II in group B was significantly increased when compared with group A (P lt; 0.01), and groups D and E were significantly higher than group C (P lt; 0.01); the mRNA expression of Aggrecan in groups D and E were significantly increased when compared with group C (P lt; 0.01), and group E was significantly higher than group D (P lt; 0.01); the mRNA expression of COMP in group B was significantly increased when compared with group A (P lt; 0.01), and group E was significantly higher than group C (P lt; 0.01); and the mRNA expression of SZP in group E was significantly increased when compared with groups C and D (P lt; 0.05). The toluidine blue staining and immunohistochemistry staining displayed that synthesis and secretion of GAG could be enhanced after load; no intensity changes of collagen type I and collagen type II were observed, but intensity enhancement of Agrrecan was seen in groups D and E. Conclusion Different dynamic loads can promote chondrogenesis of the 3rd passage chondrocytes. Culture by load after unload may be the best culture for chondrogenesis, while the 3rd passage chondrocytes induced by mechanical load hold less capacity of chondrogenesis.
Objective To explore the effects of the basic fibroblast growth factor（bFGF） gene transfection on the meniscal fibrochondrocytes with the reconstructed lentivirus and to observe the response of the meniscal fibrochondrocytes to the bFGF gene transfection. Methods The cultured meniscal fibrochondrocytes were isolated from the same 3-monthold New Zealand rabbit. The cultured first-generation meniscal fibrochondrocytes were divided into 3 groups:Group A (experimental group), Group B (control group), and Group C (blank group). Each group comprised the cells in a 24hole flask in which each hole contained 2×104 cells. At the confluence of 60%, the fibrochondrocytes in Group A were cultured with the reconstructed lentivirus carrying the bFGF gene. The fibrochondrocytes in Group B were cultured with the lentivirus carrying no bFGF gene. The fibrochondrocytes in Group C were cultured without any intervention. After 48 h, the cell cycle, the collagen synthesis ability, the expression of bFGF, and the cell proliferation ability in each group were investigated. Results In Group A, the bFGF expression of 870±60 pg/ml was detected in the cells 48 h afterthe co-culture; however, in Group B and Group C, no expression of bFGF was found. After the co-culture for 6 days, the results of the MTT colorimetry revealed that the cells in Group A had an absorbtance of 0.427±0.037, which had a significant difference when compared with that in Group B and Group C (0.320±0.042,0.308±0.034,Plt;0.01). The cell cycle was significantly shorter in GroupA than in Group B and Group C (Plt;0.05); The durations of G1, S and G2M of the cells in Group A were 16.28, 12.60 and 11.04 h, but those in Group B and Group C were 23.61, 16.90, 21.33 h and 21.56, 19.80, 21.41 h, respectively. The disintegration per minute of the cells was significantly greater in Group A than in Group B and Group C (7281.69±805.50 vs 5916.40±698.11 and 5883.57±922.63，Plt;0.05). Conclusion The lentivirus vector can transfer the bFGF gene into the meniscal fibrochondrocytes, resulting in an increase of the cell proliferation and the collagen synthesis.
ObjectiveTo study the effect of down-regulated leptin receptor by small interfering RNA (siRNA) in inhibiting the messenger RNA (mRNA) expressions of interleukin (IL)-1β and nitric oxide (NO) of human osteoarthritis chondrocytes, in order to provide reference for basic clinical research. MethodsCartilage was harvested under sterile conditions from osteoarthritis knee joints in patients undergoing total knee arthroplasty. Human articular chondrocytes were isolated and the cells were cultured in vitro. The cells in the 3rd passage were transferred by siRNA Ob-Rb (experimental group) and blank Ob-Rb (control group), respectively. Then mRNA expressions of IL-1β and NO were tested by quantitative polymerase chain reaction at hour 24, 48 and 72 after successful transfection. ResultsThe mRNA expressions of IL-1β increased slightly and that of NO declined slightly at hour 24, 48 and 72 after transfection in the treatment group, but they all were significantly lower than those in the control group (P ＜ 0.05) , and the differences became much larger as time went on. ConclusionLeptin receptor under siRNA technology can significantly inhibit the mRNA expressions of IL-1β and NO in human osteoarthritis chondrocytes.
ObjectiveTo study the effect of chemical extraction of allogeneic tendon and allogeneic chondrocytes for reconstruction of anterior labrum of shoulder joint in rabbits.MethodsThe body weight of 45 adult New Zealand white rabbits ranged from 2.5 to 3.0 kg. The Achilles tendons of 15 rabbits were taken and the allogeneic tendons were prepared by chemical extraction with antigen inactivation. The extracted tendons were compared with untreated tendons by HE and Masson stainings. Chondrocytes were isolated and cultured by trypsin method and identified by immunohistochemical staining of collagen type Ⅱ. The remaining 30 rabbits were used to prepare the model of anterior labrum defect of shoulder joint. After the allogeneic tendon was transplanted to the damaged labrum, the rabbits was randomly divided into two groups (15 in each group). In group A, the allogeneic chondrocytes were injected into the joint immediately after transplantation, while in group B, no treatment was made. At 4, 6, and 8 weeks after operation, 5 transplanted tendons of each group were taken. After general observation, HE staining was used to observe the number of nuclei, Masson staining was used to observe the expression of collagen fibers in muscle fiber tissues, and AB staining was used to detect the glycosaminoglycan level after transplantation, to evaluate the cell growth in the tissues of the two groups of allogeneic tendon.ResultsBy HE and Masson stainings, the allogeneic tendon antigen prepared by chemical extraction method was inactivated and the fibrous tissue structure was intact; collagen type Ⅱ immunohisto-chemistry staining showed that the cultured cells were chondrocytes. After tendon transplantation, the content of glycosaminoglycan in group A was significantly higher than that in group B (P<0.05). At 6 weeks after operation, HE staining showed that the nuclear in tendon tissue of group A was significantly more than that of group B (t=20.043, P=0.000). Masson staining showed that the number of nuclei in tendon tissue of group A was significantly increased, the muscle fibers and collagen fibers were interlaced, the tissue structure was more compact, and the tendon tissue was mainly blue stained; while the number of nuclei in group B was less, mainly collagen fibers of the original graft.ConclusionThe allogeneic tendon inactivated by chemical extraction can be used to reconstruct the defect of anterior labrum of shoulder joint in rabbits, and the combination of allogeneic chondrocytes can promote the healing of tendon transplantation.