ObjectiveTo summarize the research progress of microRNA (miRNA) in the osteoarthritis (OA) cartilage matrix degradation. MethodsThe domestic and foreign related literature about the miRNA in the OA cartilage matrix degradation was reviewed, summarized, and analyzed. ResultsOA is a common chronic joint disease characterized by cartilage degeneration, its etiology and pathogenesis are still not completely clear. miRNA, a kind of small single stranded non-coding RNA molecule, is closely correlated with inflammatory mediators and various cytokines during the cartilage matrix degradation, suggesting that miRNAs have important regulatory functions at the molecule and cellular levels. ConclusionmiRNA can serve as potential biomarkers and will give new insight into diagnosis and therapeutic strategies in OA.
ObjectiveTo investigate the relationships between the expression of integrin β1 and activated cells in a partial-thickness articular cartilage injury model of adult rats. MethodForty-five male Sprague Dawley rats (aged 10 weeks and weighing 300-400 g) were randomly divided into operated group (n=15) , sham-operated group (n=15) , and control group (n=15) . Partial-thickness articular cartilage injury model was made by scarification in operated group, direct suture after opening of the knee joint was performed in sham-operated group, and no operation was done in control group. Five rats were sacrificed at 1, 7, and 14 days after operation respectively for macroscopic evaluation, HE staining, Safranin O staining, CD105, BrdU, CD105/integrin β1 immunofluorescence and double labeling staining. The histological score of HE staining, gray value of Safranin O staining and CD105-positive cells count were compared among groups at each time point. ResultsMacroscopic evaluation showed chondromalacia and cartilage fibrosis around the linear injury with aggravating tendency with time in operated group, but no chondromalacia and cartilage fibrosis in sham-operated and control groups. HE staining demonstrated a number of activated cells accumulating around the linear injury with nonuniform distribution in operated group, and uniform size and distribution in sham-operated and control groups. The histological scores at each time point in operated group were significantly higher than those in sham-operated group and control group (P<0.05) , but no significant difference was found between different time points in 3 groups (P>0.05) . Safranin O staining was nonuniform with hypochromasia around linear injury in operated group, but the staining was uniform in sham-operated group and control group. Gray value of Safranin O staining had no significant difference among groups and among different time points in the same group (P>0.05) . BrdU-positive and CD105-positive cells distributed unevenly around the linear injury in operated group, uniform distribution was observed in sham-operated group and control group. CD105-positive cells count in operated group was significantly higher than those in sham-operated group and control group at each time point (P<0.05) ; CD105-positive cells increased significantly with time in operated group (P<0.05) . CD105/integrinβ1-positive cells were observed around the linear injury in operated group, but was not observed in sham-operated group and control group. ConclusionsThe partial-thickness articular cartilage injury model is successfully established in rats, and cartilage injury could not be repaired completely in the model. The activated cells aggregation around the linear injury can be observed, but there is no obvious relationships between activated cells and cartilage matrix. These activated cells are in proliferation and could express both CD105 and integrin β1.
Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
The small molecule nutrients and cell growth factors required for the normal metabolism of chondrocyte mainly transport into the cartilage through free diffusion. However, the specific mass transfer law in the cartilage remains to be studied. In this study, using small molecule rhodamine B as tracer, the mass transfer models of cartilage were built under different pathways including surface pathway, lateral pathway and composite pathway. Sections of cartilage at different mass transfer times were observed by using laser confocal microscopy and the transport law of small molecules within different layers of cartilage was studied. The results showed that rhodamine B diffused into the whole cartilage layer through surface pathway within 2 h. The fluorescence intensity in the whole cartilage layer increased with the increase of mass transfer time. Compared to mass transfer of 2 h, the mean fluorescence intensity in the superficial, middle, and deep layers of cartilage increased by 1.83, 1.95, and 3.64 times, respectively, after 24 h of mass transfer. Under lateral path condition, rhodamine B was transported along the cartilage width, and the molecular transport distance increased with increasing mass transfer time. It is noted that rhodamine B could be transported to 2 mm away from cartilage side after 24 h of mass transfer. The effect of mass transfer under the composite path was better than those under the surface path and the lateral path, and especially the mass transfer in the deep layer of cartilage was improved. This study may provide a reference for the treatment and repair of cartilage injury.
Cartilage with limited self-repairing ability is a kind of tissue with relatively hypocellular structure, low nerve distribution and vascular nutrient. Cartilage tissue engineering provides a new therapeutic idea for cartilage injured cartilage repairing in clinical practice. Electrospinning fibrous scaffold with three-dimensional structure like extracellular matrix is suitable for cell growth and bioactive factor loading for cartilage tissue engineering. This paper introduces studies of the application of electrospinning technology in repairing damaged cartilage by simulating highly hierarchical structures and mechanical features from the aspects of composition optimization, structure optimization and multi-technology combination.
ObjectiveTo investigate the ability to repair osteochondral defect and the biocompatibility of porous tantalum loaded with bone morphogenetic protein 7 (BMP-7) by observing the effect of porous tantalum loaded with BMP-7 in repairing articular cartilage and subchondral bone defect. MethodsThe cartilage defect models of medial femoral condyle were established in 48 New Zealand white rabbits, which were randomly divided into 3 groups (n=16): porous tantalum material+BMP-7 (group A) and porous tantalum material (group B) were implanted into the right side of the medial femoral condyle; and no material was implanted as control (group C). The general condition of animals was observed after operation, then the specimens were harvested for gross observation, histological observation, and scanning electron microscope (SEM) observation at 4, 8, and 16 weeks after implantation, micro-CT was used to observe the cartilage and bone ingrowth and bone formation around porous tantalum at 16 weeks after implantation. ResultsNo animal died after operation and wound healed well. Gross observation showed that defects of groups A and B were covered with new cartilage with time, but earlier new cartilage formation and better repair were observed in group A than group B, no repair occurred at the site of bone defects, and defect surface was filled with fibrous tissue in group C. Cartilage repair gross score of group A was significantly higher than that of group B at 8 and 16 weeks (P < 0.05) but no significant difference was found between groups A and B at 4 weeks (P>0.05). SEM observation showed that the number of new cartilage and osteoblasts increased gradually with time, and the implanted material was gradually covered with the extracellular matrix, and the new bone tissue grew into the pores of the material; the neonatal bone tissue and extracellular matrix secretion of group A were significantly more than those of group B. The toluidine blue staining results showed that new cartilage and bone tissue gradually increased in the porous tantalum interface, and new bone trabecula formed and grew in the pores, the bone and the porous tantalum contact tended to close, and cartilage defect was gradually covered with cartilage like tissue, cartilage tissue and porous tantalum combined more closely in groups A and B at 4, 8 and 16 weeks. New cartilage and bone tissue of group A was more than that of group B. Micro-CT analysis indicated that the bone mineral density, trabecular thickness, trabecular number, and bone volume fraction of group A were significantly higher than those of group B at 16 weeks (P < 0.05), but the trabecular bone space was significantly lower than that of group B (P < 0.05). ConclusionThe domestic porous tantalum has good biocompatibility, domestic porous tantalum loaded with BMP-7 can promote the formation of a stable connection with the host and has a good effect on cartilage and subchondral bone defect repair.
Objective To know the basic research and the clinical application of cartilage-derived retinoic acid-sensitive protein (CD-RAP) in orthopedic and in other clinical fields. Methods The literature related to CD-RAP in basic research and clinical application were extensively reviewed. Results CD-RAP, as a protein, which is cartilage-specific,could be a marker of the joint diseases. It also can be used to monitor metastsais of melanoma. Conclusion CD-RAP test provides a new way to study repair of cartilage and metastsais of melanoma.
ObjectiveTo isolate and identify the cartilage progenitor cells (CPCs) from normal cartilage, and to explore the influence of interleukin 1β (IL-1β) in different concentrations on its chondrogenesis. MethodsCPCs were isolated from normal cartilage of adult New Zealand white rabbit with the fibronectin adhesion assay;the cell phenotype was identified;and the cloning and differentiation of CPCs were observed. CPCs were incubated with H-DMEM in group A, with chondrogenic induced medium in group B, with chondrogenic induced medium+0.1 ng/mL IL-1β in group C and chondrogenic induced medium+1.0 ng/mL IL-1β in group D for 3 weeks. The histology, biochemistry, and real-time fluorescence quantitative PCR were performed to observe the effect of IL-1β on the chondrgenic differentiation. ResultsThe CPCs from normal cartilage expressed positively stem cell phenotype, which have similar ability of cloning and differentiation to stem cells. The cell pellets in groups C and D were significantly smaller than those in group B, and cell showed hypertrophic morphology change. There were more expressions of collagen type Ⅱ and collagen type X in group B than in group A, in group B than in groups C and D, and in group C than group D with Safranin O staining. The biochemistry results showed that collagen type Ⅱ content, glycosaminoglycan (GAG) content, and the ratio of GAG/DNA were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05);but the DNA content was significantly higher in groups C and D than in group B (P<0.05), and no significant difference between groups C and D (P>0.05). The real-time fluorescence quantitative PCR results showed that the relative mRNA expressions of collagen type Ⅱ, collagen type X, and Sox-9 were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05), but the relative mRNA expressions of Runx-2 and matrix metalloproteinase 13 were significantly higher in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05). ConclusionThere are CPCs having the character of stem cells in normal cartilage, and they have the capability of cloning and potential differentiation. IL-1β can inhibit the chondrogenesis of CPCs, and possibly promote the osteogenic differentiation.
Objective To evaluate the synergistic effect of bone morphogenetic protein 14 (BMP-14) and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells (ADSCs) so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits (weighing, 1.5 kg and 2.0 kg). The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction (alizarin red staining), chondrogenic induction (alcian blue staining), and adipogenic induction (oil red O staining). The optimum multiplicity of infection (MOI) of transfection of adenovirus-cytomegalovirus (CMV)-BMP-14-internal ribosome entry site (IRES)-human renilla reniformis green fluorescent protein 1 (hrGFP-1) was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups: group A, co-culture of ADSCs transfected by BMP-14 and chondrocytes (1 ∶ 1 in Transwell chambers); group B, co-culture of ADSCs and chondrocytes (1 ∶ 1 in Transwell chambers); group C, culture of ADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan (GAG) content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C (P lt; 0.05), and groups B and D were significantly higher than group E (P lt; 0.05), but no significant difference was found between groups B and D (P gt; 0.05). Conclusion It can promote differentiation of ADSCs into chondrocytes by BMP-14 co-culture with chondrocytes, and they have a synergistic effect.
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.