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.
ObjectiveTo investigate the effect of three-dimensional cultivation with dynamic compressive stimulation on promotion of cartilage growth in vitro, by constructing tissue engineered cartilage with three-dimensional porous articular cartilage extracellular matrix (ECM) scaffolds laden with rabbit chondrocytes and performing mechanical stimulation by compressive stress in bioreactor. MethodsChondrocytes of healthy adult New Zealand rabbits were isolated, and passage 2 chondrocytes were seeded onto three-dimensional porous articular cartilage ECM scaffolds for 5 days pre-cultivation, and then were divided into 2 groups:Group A continued static culture as control; group B (dynamic culture condition) underwent dynamic compressive strain stimulation (compressive strain of 15%, frequence of 1 Hz) in a bioreactor. Cell viability and distribution in scaffolds were observed; the glycosaminoglycan (GAG) content, collagen content, and total DNA content were measured after 3 weeks of culturing; and elastic modulus was evaluated by mechanical test. ResultsLaser scanning confocal microscopy indicated that cells grew well and evenly distributed in the scaffold of group B, while poor cells growth and loss of staining in the central region of the scaffolds were observed in group A. Scanning electron microscopy showed that chondrocytes possessed good adhesion, proliferation, and growth on the scaffolds of group B; while the number of chondrocytes was significantly reduced, and cells scattered in group A. Biochemical composition analysis showed that collagen, GAG, and DNA contents of cell-scaffold constructs were (675.85±27.93) μg/mg, (621.72±26.75) μg/mg, and (16.98±3.23) μg/sample in group B, and were (438.72±6.35) μg/mg, (301.63±30.51) μg/mg, and (10.18±4.39) μg/sample in group A respectively, which were significantly higher in group B than in group A (t=18.512, P=0.000;t=17.640, P=0.000;t=2.790, P=0.024). Mechanical testing indicated that the elastic modulus of group B[(0.67±0.09) MPa] was significantly higher than that of group A[(0.49±0.16) MPa] and cell-free scaffolds[(0.43±0.12) MPa] (P < 0.05). ConclusionMimetic compressive stress with three-dimensional dynamic conditions created in the bioreactor is superior to the ordinary static three-dimensional cultivation, it can provide the optimal environment for chondrocytes on the ECM scaffolds, which may be a good way to construct tissue engineered cartilage in vitro.
【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 introduce the application of polymer material, chitosan, in the cartilage tissue engineering. Methods The recent original articleson the application of chitosan in cartilage tissue engineering were extensivelyreviewed. The biocompatibility and biodegradation characters of chitosan and its application were analysed.Results Chitosan has a high degree of biocompatibility and a favorable chondrogenic characteristic. It can support the maintenance of the phenotypic morphology of chondrocytes besides being used as a scaffold for cell growth. Conclusion The perspect of the application of chitosan in cartilage tissue engineering is hopeful.
【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 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.
Objective To evaluate collagen（Col）hyaluronan (HA) chondroitin sulfate (CS) tri-copolymer as a new biomimetic biodegradable polymer scaffold for application of the articular cartilage tissue engineering. Methods The Col-HACS tricopolymer was prepared by freezing and lyophilization and was cross-linked by 1-ethyl-3(3-dimethy inaminoproyl) carbodiimide (EDC). The morpholog icalcharacteristics of the matrices were evaluated by the SME and HE stainings. The rabbit chondrocytes were isolated and seeded in the tricopolymer scaffold. Morphology, proliferation and differentiation of glycosaminoglycan (GAG), and phenotypic expression of the rabbit articular chondrocytes cultured within the tricopolymer scaffold were indicated by the histological examination, SEM, biochemica l analysis, and reverse transcriptase PCR for collagen typeⅡ（ColⅡ）. Results The chondrocytes proliferated and differentiated well, and th ey preserved the phenotypic expression of ColⅡ in the Col-HA-CS scaffold. After the 21day cell culture within the Col-HA-CS scaffolds, the cartilage-specific morphologyand the structural characteristics such as lacunae appeared，and DNA and GAG conten ts increased with the time. In addition, DNA and GAG contents were significantly higher in the Col-HA-CS matrix than in the collagen matrix alone (Plt;0.05 ). Conclusion These results show that the Col-HA-CS tri-copolymer matrices can provide an appropriate environment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.
Objective To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-t ransforming growth factor-β1 (TGF-β1) gene and to evaluate the feasibility of selfinduction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further “gene enhanced tissue engineering” research. Methods The rabbit MSCs was transfected with the reconstructed PGL3-TGF-β1gene by the Liposo mesMethod, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evalua ted by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-β1 and collagen Ⅱ were examined at 2 and 7 days of the cell culture afte r transfe ction with PGL3-TGF-β1gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was perfor med with the software of the SPSS 11.0, compared with the control group and the blank group. Results Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-β1gene by the Liposomes Method achie ved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (Plt;0.01). Tested by MTT, the living acti vity of the transfected cells was proved to be significantly decreased (Plt;0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF-β1 positive particles were detected in the experimental group，but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the ttest (Plt;0.01). By the immunohistochemistry me thod (SABC) to study collagen Ⅱ, there were more positive particles in the transfected cells in t he experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (Plt;0.01). Conclusion Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-β1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfecte d BMS cells with PGL3-TGF-β1 gene can express and excrete TGF-β1when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
Objective To evaluate the feasibility and the value of the layered cylindric collagenhydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affe cts the cell behaviors. Methods The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen／h ydro xyapatite composite scaffold and were cultured in a three-dimensional environme n t for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron micros copy, and immunohistochemistry. Results The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 μm. and the porosity was 89%; the pore diameter of the bottom layer was about 85 μm and the porosity was 85%. The layered cylindric collagenhydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytesattached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. Conclusion The layered cylindric collagenhydroxyapatite composite scaffold has a good cellular compatibility, and it is ber in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue enginee ring.
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.