Objective To explore the methods of repairing cartilagedefects and to introduce the clinical experience with the autologous osteochondral transplantation. Methods Twenty-five patients with chondral and osteochondral defects of the weight-bearing surfaces were treated by the autologous osteochondral transplantation for the repair of the chondral and osteochondral defects of the unweightbearing surfaces under arthroscope. According to the shape of the defects, the different dimensions of the osteochondral autograft were selected. All the patients began the training of the continuous passive motion after operation. Six weeks after operation, the patients began to walk in the weightbearing habitus. However, in the control group, another 25 patients were retrospectively analyzed, who had chondral and osteochondral defects of the weight-bearing surfaces but were treated only by the cleaning and drilling procedures. The scores evaluated bythe Brittberg-Peterson scoring scale of the 2 group were 98.65±9.87 and 96.98±8.94 respectively. Results The follow-upfor 3-24 months after operation revealed that the treated knee joint had a goodmotion extent. The pain was obviously alleviated. Based on the longitudinal study with the three-dimensional spoiled magnetic resonance imaging (MRI), the signal intensity of the repaired tissues approached to the normal condition. The scores evaluated by the Brittberg-Peterson scoring scale were almost zero 3 monthsafter operation in the experimental group, and the scores were 58.48±6.98 inthe control group. There were significant differences between the experimental group and the control group(P＜0.01). Conclusion Autologous osteochondral transplanation under arthroscope is a good curative method for the cartilage defects, with advantages of minimal invasiveness and avoidanceofrejections resulting from allografts. However, its long-term effect needs to befurther studied. The conventional therapies including cleaning and drilling are useful in alleviating the symptoms.
Objective To investigate the effect of allogeneic chondrocytes-calcium alginate gel composite under the intervention of low intensive pulsed ultrasound (LIPUS) for repairing rabbit articular cartilage defects. Methods Bilateral knee articular cartilage were harvested from 8 2-week-old New Zealand white rabbits to separate the chondrocytes by mechanical-collagen type II enzyme digestion. The 3rd passage chondrocytes were diluted by 1.2% sodium alginate to 5 × 106 cells/mL, then mixed with CaCl2 solution to prepare chondrocytes-calcium alginate gel composite, which was treated with LIPUS for 3 days (F0: 1 MHz; PRF: 1 kHz; Amp: 60 mW/cm2; Cycle: 50; Time: 20 minutes). An articular cartilage defect of 3 mm in diameter and 3 mm in thickness was established in both knees of 18 New Zealand white rabbits (aged 28-35 weeks; weighing, 2.1-2.8 kg), and divided into 3 groups randomly, 6 rabbits in each group: LIPUS group, common group, and model group. Defect was repaired with LIPUS-intervention gel composite, non LIPUS-intervention gel composite in LIPUS group and common group, respectively; defect was not treated in the model group. The general condition of rabbits was observed after operation. The repair effect was evaluated by gross and histological observations, immunohistochemical staining, and Wakitani score at 8 and 12 weeks after operation. Results Defect was filled with hyaline chondroid tissue and white chondroid tissue in LIPUS and common groups, respectively. LIPUS group was better than common group in the surface smooth degree and the degree of integration with surrounding tissue. Defect was repaired slowly, and the new tissue had poor elasticity in model group. Histological observation and Wakitani score showed that LIPUS group had better repair than common group at 8 and 12 weeks after operation; the repair effect of the 2 groups was significantly better than that of model group (P lt; 0.05); and significant differences in repair effect were found between at 8 and 12 weeks in LIPUS and common groups (P lt; 0.05). The collagen type II positive expression area and absorbance (A) value of LIPUS and common groups were significantly higher than those of model group (P lt; 0.05) at 8 and 12 weeks after operation, and the expression of LIPUS group was superior to that of common group at 12 weeks (P lt; 0.05); and significant differences were found between at 8 and 12 weeks in LIPUS group (P lt; 0.05), but no significant difference between 2 time points in common and model groups (P gt; 0.05). Conclusion Allogeneic chondrocytes-calcium alginate gel composite can effectively repair articular cartilage defect. The effect of LIPUS optimized allogeneic chondrocytes-calcium alginate gel composite is better.
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.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.
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 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.
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 manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix (DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. Methods PLGA scaffolds were prepared by low temperature deposition 3D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods. DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques. PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test. Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cell-scaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8 (CCK-8) was used to detect cell proliferation. Results General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores; the surface of the DACECM oriented scaffold was rough, which was a 3D structure with loose pores and interconnected; and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds (P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold (P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance (A) value between the groups at each time point (P>0.05). Conclusion The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.
OBJECTIVE: To study the feasibility of the formation of allogeneic tissue-engineered cartilage of certain shape in immunocompetent animal using the injectable biomaterial. METHODS: Fresh newborn rabbits’ articular cartilages were obtained under sterile condition (lt; 6 hours after death) and incubated in the sterile 0.3% type II collagenase solution. After digestion of 8 to 12 hours, the solution was filtered through a 150 micron nylon mesh and centrifuged, then the chondrocytes were washed twice with phosphate buffered saline (PBS) and mixed with the biomaterial to create a final cell density of 5 x 107/ml. The cell-biomaterial admixture was injected into rabbits subcutaneously 0.3 ml each point while we drew the needle back in order to form the neocartilage in the shape of cudgel, and the control groups were injected with only the biomaterial or the suspension of chondrocytes with the density of 5 x 10(7)/ml. After 4, 6, 8 and 12 weeks, the neocartilages were harvested to analyze. RESULTS: The new nodes could be touched subcutaneously after 2 weeks. In the sections of the samples harvested after 4 weeks, it was found that the matrix secreted and the collagen formed. After 6 weeks and later than that, the neocartilages were mature and the biomaterial was almost completely degraded. The cudgel-shaped samples of neocartilage could be formed by injection. In the experiment group, there was no obvious immune rejection response. On the contrary, there were no neocartilage formed in the control group. CONCLUSION: The injectable biomaterial is a relatively ideal biomaterial for tissue engineering, and it is feasible to form allogeneic tissue engineered cartilage of certain shape by injection in an immunocompetent animal.
ObjectiveTo develop an anti-inflammatory poly (lactic-co-glycolic acid) (PLGA) scaffold by loading xanthohumol, and investigate its anti-inflammatory and cartilage regeneration effects in goats. Methods The PLGA porous scaffolds were prepared by pore-causing agent leaching method, and then placed in xanthohumol solution for 24 hours to prepare xanthohumol-PLGA scaffolds (hereinafter referred to as drug-loaded scaffolds). The PLGA scaffolds and drug-loaded scaffolds were taken for general observation, the pore diameter of the scaffolds was measured by scanning electron microscope, the porosity was calculated by the drainage method, and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared (FTIR) spectrometer. Then the two scaffolds were co-cultured with RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds. Bone marrow mesenchymal stem cells (BMSCs) was obtained from bone marrow of a 6-month-old female healthy goat, cultured by adherent method, and passaged in vitro. The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds, and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8 (CCK-8) assay. The BMSCs-scaffolds were cultured in vitro for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, and biochemical analysis. Finally, the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously, respectively. After 4 weeks, gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, biochemical analysis, and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded scaffolds. Results The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity (P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1β and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold (P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds (P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds (P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds (P<0.05); the relative gene expressions of IL-1β and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds (P<0.05). ConclusionThe drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.