| ObjectiveTo explore the feasibility of constructing tissue-engineered cartilage by ECM/SF biomimetic scaffold.Materials and Methods1.Preparation of cartilage extracellular matrix?ECM?:Fresh hyaline cartilage from goats was extracted,and cartilage extracellular matrix powder was prepared by homogenization-decellularization-pulverization procedure.2.Chondrocyte extracellular matrix/silk fibroin?ECM/SF?preparation:four groups of scaffolds were prepared by freeze-drying method.?1?1:1 ECM/SF scaffold:made by mixed extracellular matrix suspension and silk fibroin solution at 1:1.?2?2:1 ECM/SF scaffold:made by mixed extracellular matrix suspension and silk fibroin solution 2:1.?3?4:1 ECM/SF scaffold:made by mixed extracellular matrix suspension and silk fibroin solution at 4:1.?4?ECM scaffold:made of extracellular matrix suspension alone.3.The compressive modulus of the scaffold was measured,the secondary structure of the protein was analyzed by Fourier transform infrared spectroscopy?FTIR?, the microstructure was observed by scanning electron microscopy?SEM?,and the pore size and porosity were preliminarily calculated.CCK-8 cytotoxicity test was carried out on the scaffold.The live-dead staining was used to observe the viability of BMSCs by fluorescence microscopy.4.Isolation and culture of bone marrow mesenchymal stem cells?BMSCs?.5.Biochemical assay was used to evaluate the chondrogenic differentiation and matrix secretion of bone marrow mesenchymal stem cells in scaffolds.6.We investigated the chondrogenic differentiation of BMSCs in scaffolds by quantifying the mRNA relative expression levels of chondrogenic-related genes such as SOX-9,COL I and COL II and hypertrophic-related genes COL X.7.After 14 and 28 days of subcutaneous implantation in nude mice,cartilage-like tissues were taken out for paraffin section.Histological observation was used to evaluate the chondrogenic differentiation and matrix secretion ability of bone marrow mesenchymal stem cells.Results1.The diameter and height of ECM/SF scaffolds were 6 mm and 2 mm respectively.2.Fourier transform infrared spectroscopy showed that the absorption peaks of amide I and amide II of crosslinked ECM/SF scaffolds shifted to 1620-1622 and 1526-1528 cm-1.3.The pore size of ECM scaffolds,4:1 ECM/SF scaffolds,2:1 ECM/SF scaffolds and 1:1 ECM/SF scaffolds were as follows:179.96±12.62?m,112.87±12.12 ?m,87.29±14.21?m and 55.79±9.45?m,porosity of which were as follows:94.23%±2.36%,95.52%±3.14%,95.31%±1.76%and 97.54%±3.82%.4.The water uptack of ECM,4:1 ECM/SF,2:1 ECM/SF and 1:1 ECM/SF scaffolds were respectively as follows:2046%±89.34%,2070%±112%,2132%±124% and 2142%±125.4%.The degration rates were as follows:ECM scaffolds>4:1 ECM/SF>2:1 ECM/SF>1:1 ECM/SF.5.Compressive modulus of ECM scaffolds,4:1 ECM/SF scaffolds,2:1 ECM/SF scaffolds and 1:1 ECM/SF scaffolds were as follows:0.07219±0.06429 Mpa,0.1180±0.04633 Mpa,0.1638±0.01161 Mpa and 0.1871±0.01323 Mpa respectively.6.CCK-8 cytotoxicity test and dead cell staining test proved that silk fibroin/extracellular matrix scaffold was non-toxic to bone marrow mesenchymal stem cells.the OD values?optical densities?of ECM scaffolds,4:1 ECM/SF scaffolds,2:1 ECM/SF scaffolds and 1:1 ECM/SF scaffolds at 450 nm were 2.08 ±0.1140,2.14±0.1156,2.16±0.0875 and 2.07±0.1157,respectively.7.The results of biochemical assay showed that the four scaffolds could promote the chondrogenesis and matrix secretion of bone marrow mesenchymal stem cells.Among them,2:1 ECM/SF scaffolds had the strongest chondrogenic differentiation and matrix secretion ability of bone marrow mesenchymal stem cells.8.The COL II/COL I expression ratio in the 2:1 ECM/SF scaffold was approximately two fold higher than that in the ECM scaffold9.Histological observation of ECM/SF scaffolds showed that Saffron O and Masson staining were positive in all groups It was observed that cells adhered well and distributed evenly in 2:1 ECM/SF scaffolds,with the most extracellular matrix secreted.10.Immunohistochemical results showed that type II collagen staining was the most obvious in 2:1 ECM/SF compared with other groups,while type I collagen staining was not obvious compared with other groups.ConclusionsIn the present study,biomimetic composite scaffolds with different ratios of SF and ECM were fabricated by lyophilization.These ECM/SF scaffolds of different component ratios varied in size and structure and possessed ideal porosity and water-uptake capacities.They also possessed greater mechanical strength than the ECM scaffold.In vitro results demonstrated that these ECM/SF scaffolds possessed low toxicity and promoted chondrogenic differentiation of BMSCs.In vivo results demonstrated the biocompatibility of the ECM/SF scaffolds,as well as their ability to form cartilaginous matrix in subcutaneous regions in nude mice.In conclusion,according to our results,scaffolds containing optimal concentrations of ECM and SF are potential candidates for application in cartilage tissue engineering.The ECM/SF scaffold made by adding SF into ECM mimics the micro-environment and mechanical properties of normal cartilage.It can directly induce BMSCs into chondrocytes and construct ideal tissue-engineered cartilage. |
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