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 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 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.
ObjectiveTo explore the clinical application and effectiveness of a personalized tissue engineered cartilage with seed cells derived from ear or nasal septal cartilage and poly-glycolic acid (PGA)/poly-lactic acid (PLA) as scaffold in patients with nasal reconstruction. MethodsBetween March 2014 and October 2015, 4 cases of acquired nasal defects and 1 case of congenital nasal deformity were admitted. The patient with congenital nasal deformity was a 4-year-old boy, and the source of seed cells was nasal septal cartilage. The other 4 patients were 3 males and 1 female, aged 24-33 years, with an average of 28.5 years. They all had multiple nasal subunit defects caused by trauma and the source of seed cells was auricular cartilage. The tissue engineered cartilage framework was constructed in the shape of normal human nasal alar cartilage and L-shaped silicone prosthesis with seed cells from cartilage and PGA-PLA compound biodegradable scaffold. The boy underwent nasal deformity correction and silicone prosthesis implantation in the first stage, and the prosthesis was removed and implanted with tissue engineered cartilage in the second stage; the remaining 4 adult patients all used expanded forehead flaps for nasal reconstruction. All 5 patients underwent 1-4 nasal revisions. The implanted tissue engineered cartilage was observed during the operation and taken from 2 patients for histological examination.ResultsAll the incisions healed by first intention after the tissue engineered cartilage implantation, and the expanded forehead flaps survived. Postoperative low fever occurred in 3 patients. No complications such as infection, obvious immune rejection response, and tissue engineered cartilage protrusion were found in all patients. All patients were followed up 9-74 months (mean, 54.8 months). During follow-up, the patients had no obvious discomfort in the nose and the ventilation function were good. All patients were satisfied with the nasal contour. Early-stage histological examination showed the typical cartilage characteristics in 1 patient after the implantation of tissue engineered cartilage. Late-stage histological examination in 1 patient of tissue engineered cartilage showed the characteristics of fibrous connective tissue; and the other showed there was remaining cartilage.ConclusionThe safety of tissue engineered cartilage constructed in vitro for reconstruction is preliminarily confirmed, but the effectiveness still needs further verification.
Objective To study the influence of different mechanical environments on repair cartilage defect with marrow mesenchymal stem cells as seed cells. Methods The rabbit marrow mesenchymal stem cells were isolated and cultured. The cartilage defects were repaired by autologous tissue engineered cartilage with the marrow mesenchymal stem cells as seed cells. Fifteen rabbits with cartilage defect were divided into 3 groups: dislocation group with cell-free scaffold(controlgroup), dislocation group with cartilaginous construct and normal mechanical environment group with cartilaginous construct. The repaired tissue was harvested and examined 6 weeks postoperatively. Results The repair tissue in normal mechanical environment group with cartilaginous construct showed cartilage-like tissue in superficial layer and subchondral bone tissue in deep layer 6 weeks postoperatively. The defect was filled with bone tissue in dislocation group with cartilaginous construct 6 weeks postoperatively. The surrounding normal cartilage tissue showed vascular invasion from subchondral area and the concomitant thinningof the normal cartilage layer. The cartilaginous construct left in the femoral trochlea groove formed hyaline cartilage-like tissue. The defect was repaired byfibrous tissue in control group. Conclusion The repaired tissue by tissue engineered cartilage with marrow mesenchymal stem cells as seed cells showed the best result in normal mechanical environment group, which indicates that it will be essential for the formation and maintenance of tissue engineered cartilage to keep the normal mechanical stress stimulus.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
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 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.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.