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.
【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
OBJECTIVE To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.
To study how to repair the cartilage defect according to the principles of tissue engineering with acellular cartilage matrix as scaffold material. Methods The ear cartilage was obtained from a New Zealand white rabbit(weighing 2.4 kg )and then treated by a modified Courtman’s four-step method to produce the acellular cartilage matrix. Eighteen New Zealand white rabbits (aged 6 months, weighing 2.4-2.6 kg) with no sex l imit were divided into three groups. Forevery rabbit, two pieces of ear cartilage measured 1 cm × 1 cm were excised in each ear. Defects were repaired as follows: group A with the combined graft of acellular cartilage matrix and perichondium, group B with acellular cartilage matrix and group C with perichondium. Three animals in each group were killed 4 and 12 weeks postoperatively, respectively. Tissue samples obtained were analyzed with gross observation, hematoxyl in-eosin stain, Safranine O-alcian blue stain and type II collagen messenger RNA in situ hybridization respectively. Results In gross observation, the repaired sites in groups A and B were not change meaningfully in their shape 4 weeks postoperatively; but they felt a bit of thicker and harder 12 weeks postoperatively. In group C two repaired sites formed scabs at 2 weeks and perforated at 5 weeks. In histological observation, there was a sl ight inflammatory reaction surrounding the acellular cartilage matrix 4 weeks after it was implanted in groups A and B. The inflammatory cells were mainly lymphocytes. The perichondrium graft in group C was collapsed in the defects in HE stain. The defect sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization in all groups. At 12 weeks cells were found in the acellular matrix which arranged in irregular manner in group A in HE stain and was positive for Safranine O-alcian blue stain and type II collagen mRNA in site hybridization. In groups B and C, no new cell was found in HE stain and the repaired sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization. Conclusion Acellular
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.
ObjectiveTo investigate whether subchondral bone microstructural parameters are related to cartilage repair during large osteochondral defect repairing based on three-dimensional (3-D) printing technique. MethodsBiomimetic biphasic osteochondral composite scaffolds were fabricated by using 3-D printing technique. The right trochlea critical sized defects (4.8 mm in diameter, 7.5 mm in depth) were created in 40 New Zealand white rabbits (aged 6 months, weighing 2.5-3.5 kg). Biomimetic biphasic osteochondral composite scaffolds were implanted into the defects in the experimental group (n=35), and no composite scaffolds implantation served as control group (n=5); the left side had no defect as sham-operation group. Animals of experimental and sham-operation groups were euthanized at 1, 2, 4, 8, 16, 24, and 52 weeks after operation, while animals of control group were sampled at 24 weeks. Subchondral bone microstructural parameters and cartilage repair were quantitatively analyzed using Micro-CT and Wayne scoring system. Correlation analysis and regression analysis were applied to reveal the relationship between subchondral bone parameters and cartilage repair. The subchondral bone parameters included bone volume fraction (BV/TV), bone surface area fraction (BSA/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). ResultsIn the experimental group, articular cartilage repair was significantly improved at 52 weeks postoperatively, which was dominated by hyaline cartilage tissue, and tidal line formed. Wayne scores at 24 and 52 weeks were significantly higher than that at 16 weeks in the experimental group (P<0.05), but no significant difference was found between at 24 and 52 weeks (P>0.05); the scores of experimental group were significantly lower than those of sham-operation group at all time points (P<0.05). In the experimental group, new subchondral bone migrated from the surrounding defect to the centre, and subchondral bony plate formed at 24 and 52 weeks. The microstructural parameters of repaired subchondral bone followed a "twin peaks" like discipline to which BV/TV, BSA/BV, and Tb.N increased at 2 and 16 weeks, and then they returned to normal level. The Tb.Sp showed reversed discipline compared to the former 3 parameters, no significant change was found for Tb.Th during the repair process. Correlation analysis showed that BV/TV, BSA/BV, Tb.Th, Tb.N, and Tb.Sp were all related with gross appearance score and histology score of repaired cartilage. ConclusionSubchondral bone parameters are related with cartilage repair in critical size osteochondral repair in vivo. Microstructural parameters of repaired subchondral bone follow a "twin peaks" like discipline (osteoplasia-remodeling-osteoplasia-remodeling) to achieve reconstruction, 2nd week and 16th week are critical time points for subchondral bone functional restoration.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
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.
【Abstract】 Objective The seed cells source is a research focus in tissue engineered cartilage. To observe whether the post-RNA interference (RNAi) chondrocytes could be used as the seed cells of tissue engineered cartilage. Methods Chondrocytes were separated from Sprague Dawley rats. The first passage chondrocytes were used and divided into 2 groups: normal chondrocytes (control group) and post-RNAi (experimental group). Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage. The contents of Aggrecan and Aggrecanase-1, 2 were measured by HE and Masson staining, scanning electron microscope (SEM), and RT-PCR. Results The histological results: no obvious difference was observed in cell number and extracellular matrix (ECM) between 2 groups at 2 weeks; when compared with control group, the secretion of ECM and the cell number increased in experimental group with time. The RT-PCR results: the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group (P lt; 0.05); but the expressions of Aggrecanase-1, 2 mRNA in experimental group were significantly lower than those in control group (P lt; 0.05). The SEM results: the cell number in experimental group was obviously more than that in control group, and the cells in experimental group were conjugated closely. Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage, which can secrete more Aggrecan than normal chondrocytes. But their biological activities need studying further.
Objective To study the effect of platelet lysate (PL) on chondrogenic differentiation of human umbil ical cord derived mesenchymal stem cells (hUCMSCs) in vitro. Methods Umbil ical cords were voluntarily donated by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The surface markers of the cells were detected by flow cytometer. According to different components of inductive medium, the cultured hUCMSCs were divided into 3 groups: group A [H-DMEM medium, 10% fetal bovine serum (FBS), and 10%PL]; group B [H-DMEM medium, 10%FBS,10 ng/mL transforming growth factor β1 (TGF-β1), 1 × 10-7 mol/L dexamethasone, 50 μg/mL Vitamin C, and 1% insul intransferrin- selenium (ITS)]; and group C (H-DMEM medium, 10%FBS, 10 ng/mL TGF-β1, 1 × 10-7mol/L dexamethasone, 50 μg/ mL vitamin C, 1%ITS, and 10%PL). The hUCMSCs were induced in the mediums for 2 weeks. Toluidine blue staining was used to detect the secretion of chondrocyte matrix. Immunofluorescence method was used to identify the existence of collagen trpe II. The expressions of Aggrecan and collagen type II were detected by semiquantitative RT-PCR. Results Flow cytometer results showed that the hUCMSCs did not express the surface markers of hematopoietic cell CD34, CD45, and human leukocyte antigen DR, but expressed the surface markers of adhesion molecule and mesenchymal stem cells CD44, CD105, and CD146. Toluidine blue staining and immunofluorescence showed positive results in group C, weak positive results in group B, and negative results in group A. Semiquantitative RT-PCR showed the expressions of Aggrecan and collagen type II at mRNA level in groups B and C, but no expression in group A. The mRNA expressions of Aggrecan and collagen type II were higher in group C than in group B (P lt; 0.05). Conclusion Only 10%PL can not induce differentiation of hUCMSCs into chondrocytes, but it can be a supplement to the induced mediums. PL can improve hUCMSCs differentiating into chondrocytes obviously in vitro. This study provides new available conditions for constructing tissue engineered cartilage.