| Purpose:The objective of this study was to compare biological properties of a native articular cartilage extracellular matrix (ACECM)-derived oriented scaffold and the Chondro-Gide bilayered scaffold. We compare the recovery effects after implanting the bionic ACECM orientated scaffolds and Chondro-Gide bi-layer scaffolds compounded rabbit knee chondrocytesrespectively into rabbit articular cartilage defects, in order to provide improved selection and clinical application of tissue-engineered cartilage.Methods:The ACECM oriented scaffold was made by grinding porcine articular cartilage using the wet method, performing differential centrifugation, and then collecting the acellular cartilage extracellular matrix (ECM). The ACECM-derived oriented scaffold was made through directional crystallization and freeze-drying, followed by physical and chemical cross-linking to form a 3-D porous, white, spongy, oriented scaffold. The Chondro-Gide scaffold is a bilayered scaffold made from porcine skin collagen and was purchased from Switzerland. (I) The physical properties of the surfaces of the two scaffolds were assessed using light microscopy, and scanning electron microscopy was used to examine the pore size and porous channel structure, porosity, water absorption expansion coefficient, and biomechanical properties. (II) The scaffolds were qualitatively observed after being seeded with rabbit cartilage cells for cell viability using FDA/PI staining, and frozen sections were stained with toluidine blue, safranin O, and alcian blue, as well as immunohistochemical staining for collagen types I and II. (Ⅲ) Quantitative analyses were also performed after seeding rabbit cartilage cells onto the scaffolds, including the MTT cytotoxicity assay, detection of cell adhesion, assessing total collagen via hydroxyproline content, determining the glycosaminoglycan (GAG) and DNA contents, and the expression of col I, col II, col X, aggrecan, and SOX-9 via RT-PCR. (IV) The two scaffolds were implanted subcutaneously in rats and pathological grading was performed at 1,2, and 4 week after implantation using HE-stained paraffin sections. The pathological grading examined inflammatory cells, lymphocytes, and the thickness of the cyst wall formed in the tissues after implantation. (V) Taking 30 white rabbits from New Zealand and dividing into 5 groups at random, the animal models were established with the articular cartilage defects through operation and then implanted the orientated scaffoldsand Chondro-Gide bi-layer scaffolds compounded isolated and cultivated third-generation ACECM of the rabbit’s knee into the rabbit’s articular cartilage defects. Taking the materials 3 and 6 months after the operation, verify the feasibility of two kinds of scaffolds to recover the articular cartilage defects through general observation, histological staining, histological scoring and GAG detection of the new cartilage tissue. (Ⅵ)Statistical analysis.Results:The ACECM oriented scaffolds were generally round, white, spongy, and porous channels were structured in a longitudinal porous arrangement with pore diameters of 150-260μm, porosity of 91.75±3.73%; water absorption expansion coefficient of 35-45%, biomechanical compression stiffness of 6.674±0.470 N/mm, and elastic modulus of 3.303±0.233 MPa. Three days after cell seeding, the qualitative FDA/PI staining indicated the presence of a large number of viable cells. The scaffolds were positive for toluidine blue, safranin O, alcian blue, type II collagen and negative for type I collagen. The MTT toxicity of the scaffold was Grade I, and the adhesion rate was 68% at 3 hours and 86% at 6 hours. The content of hydroxyproline was 10.42±2.73 μg/mg, of GAG was 25.12± 4.12 μg/mg, and of DNA was 10.52± 5.79 μg/mg. By RT-PCR, Col I expression was 0.412±0.1421, Col II was 4.312± 0.5412, aggrecan was 3.761±0.4123, Sox-9 was 0.576±0.0812, and Col X was 0.132 ±0.0541. After subcutaneous implantation in rats, the sections were pathologically graded as’qualified’.The Chondro-Gide scaffolds were white, generally square, and the collagen on the superficial layer was dense and transversely arranged with a pore size of 40-100 μm. Loose hair-shaped coarse collagen fibers were found in the exterior layer and the pore size was about 50-180 μm with a porosity of 67.75±2.73%, water absorption expansion coefficient of 10-15%, biomechanical compression stiffness of 2.738±0.409 N/mm and elastic modulus of 1.355±0.202 MPa. Three days after cell seeding, viable cells were observed on the surface of the pores, though the cells at the dense areas were loosely attached. This scaffold was negative for toluidine blue, safranin O, alcian blue, and type Ⅱ collagen, but positive for type Ⅰ collagen. The MTT toxicity of the scaffold was Grade Ⅱ, and the cell adhesion rate was 53% at 3 hours and 74% at 6 hours. The content of hydroxyproline was 2.09±1.05μmg, of GAG was 21.74±5.79μg/mg, and of DNA was 7.31±4.37μg/mg. By RT-PCR, Col I expression was 1.63±0.2145, Col II was 0.242±0.1163, aggrecan was 1.261± 0.1321, Sox-9 was 3.075±0.4121, and Col X was 7.561±1.2145. After subcutaneous implantation in rats, the sections were pathologically graded as’qualified’. The scoring results of the general observation show that the recovery results of the control group and single-scaffold group are obviously inferior to those of the ACECM orientated scaffold group and ACECM Chondro-Gide group. Carry out the statistical analysis for the general scoring results of each group with the material taking time in 3 month and 6 month time points using the software of SPSS 10.0 and analysis of variance completely designed at random, the results showing that:the difference has the sinificant meaning (F=37.1, P=0.00).It means that the recovery results of the samples in different periods have statistical meaning. After the comparison in pairs between the ACECM orientated scaffold group and ACECM Chondro-Gide group in 6 month, the difference P>0.05, which has no statistical meaning. The histological scoring results show that:carry out the statistical analysis for the histological scoring results of 5 scaffold groups with the material taking time in 3 month and 6 month using the software of SPSS 10.0 and analysis of variance completely designed at random, the results showing that:the difference has the sinificant meaning (F=100.06, P=0.00), which means that the recovery results of the samples in different periods have statistical meaning. After the comparison in pairs, the effect of the ACECM orientated scaffold group is the best (P<0.05). The GAG detection results show that the results of the analysis of variance completely designed at random show that the ACECM orientated scaffold group, ACECM Chondro-Gide group and normal cartilago articularis group have different content of GAG of samples for which the materials are taken at different time points, thus the difference has the statistical meaning (P<0.05). The GAG content of the ACECM orientated scaffold group is higher than that of the ACECM Chondro-Gide group (both of them are lower than that of the normal cartilago articularis group).Conclusions:The two scaffolds have different characteristics. The ACECM sponge porous scaffolds are vertically arranged, and the transverse surface pore size, porosity, water absorption expansion coefficient, cell adhesion rate, and the contents of GAG, aggrecan and type II collagen were higher than those of the Chondro-Gide scaffold. In addition, it is more aadvantageous as a cartilage tissue engineering repair material for soft tissues in the joint. The smaller surface layer pores, dense arrangement of collagen, and villiform shape of the collagen fibers on the exterior surface of the Chondro-Gide scaffold suggest that it could be applied to prevent cell leakage and protect the adherent cells in the exterior layers by first suturing the scaffold in place and then injecting suspensions of cells during surgery to repair joint cartilage tissue defects. Among all recovery groups, the ACECM orientated scaffold group has the best effect to recover the cartilage defects, while the recovery effects of the controlgroup and single scaffold implantation group are worst. Although the general observation results show that the difference between the ACECM orientated scaffold group and ACECM Chondro-Gide group has no statistical meaning, their recovery tissues have significant difference, which can be shown in the histological scoring results and GAG quantitative study. |
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