| In recent years,the number of bone injuries caused by trauma,disease,accidents,and the aging population has been increasing,and researchers have proposed the concept of "in situ tissue engineering" to solve the problems in traditional bone tissue engineering.In situ tissue engineering advocates the use of self-awareness and active repair by implanting bioactive scaffolds similar to the bone tissue matrix into the patient’s defect site to induce the integration and production of endogenous cells and growth factors in the host tissue.Montmorillonite lamellar structures carry negative charges between them,which can attract free Ca2+to aggregate to saturation and promote the aggregation of inorganic calcium phosphate on its surface,which can be directly absorbed by endogenous cells to participate in the osteogenesis process,and in addition,the trace elements in montmorillonite can afect the differentiation of bone marrow stromal cells and cell signaling pathways,which information suggests that montmorillonite has the potential to influence bone repair at defect sites.With in situ bone tissue engineering as the leading idea,this project has bionically constructed mussel-inspired composite bone repair scaffolds and explored the application of montmorillonite in the field of bone repair.The main research contents and experimental results of the project are as follows:1.Preparation and characterization of montmorillonite(MMT)modified by polydopamine(PDA).The viscous protein secreted by mussels contains catecholic structure of dopamine(DA),which can be automatically oxidized to produce polydopamine(PDA)under natural conditions.Polydopamine can adhere to the surface of any substance,all with good biocompatibility,and can induce nanohydroxyapatite(Nhap)nucleation to promote bone regeneration at the defect site.The specific surface area of montmorillonite was increased after acid modification,the layer spacing was expanded,and the ion permeation was enhanced after activation.During the experiment by acid modification of MMT,dopamine(DA)could be inserted between MMT lamellae and autoxidized to form PDA,thus achieving uniform adhesion of PDA in the internal structure of MMT.FT-IR characterization of the products revealed a weak vibration at 842 cm1.indicating the formation of Al-O-C bond between MMT and PDA,which proved the successful adhesion and intercalation of PDA.2.Preparation and characterization of composite bone repair scaffolds.Guided by the principle of in situ regeneration,the in situ composite bone repair scaffold was constructed bionically using freeze-drying technology.Chitosan(CS)was used as the main structure of the scaffold,and soluble calcium salt and soluble phosphate were used as precursors of nanohydroxyapatite(nHAP),and four different in situ composite bone repair scaffolds were prepared by varying the addition amount of PDA-MMT.It was found that the porosity of all four bone repair scaffolds was greater than 75%,with good water absorption,and the water absorption of each group of scaffolds could reach more than 1800%.The mechanical properties of the scaffolds were gradually enhanced with the increase of PDA-MMT content,and the addition of PDA-MMT helped to enhance the thermal stability of the scaffolds and reduce the degradation rate of the scaffolds(the degradation rates of four groups of scaffolds at day 28 were:C-0:15.63±0.493%,C-1:14.65 ± 0.201%,C-2:14.11 ± 0.392%,C-3:13.11 ± 0.348%).The addition of PDA-MMT was able to provide nucleation sites for nanohydroxyapatite(nHAP)formation,which increased the scaffold’s bionanomineralization properties and protein adsorption capacity(the amount of protein adsorbed by each group of scaffolds were:123.54 ± 1.769μg/10mg(C-0),123.54 ± 13.516 μg/10mg(C-1),129.38±1.561 μg/10mg(C-2),125.62 ±5.810 μg/10mg(C-3).All four groups of stents had good hemocompatibility and did not cause hemolysis(hemolysis rate:0.45%,0.85%,1.28%,and 1.08%,respectively).3.The scaffold material was co-cultured with the infiltrate and cells,and the scaffold was found to have good biocompatibility,no toxic effect on cells,and to promote intracellular actin production by CCK-8 cell proliferation assay,Gimsa staining,AO/EB fluorescence staining,and TRITC/DAPI cell staining.To verify the osteogenic ability of the scaffolds in vitro,cells co-cultured with the scaffold were stained with alizarin red and alkaline phosphatase,and the scaffold was found to promote osteogenesis by differentiation of bone marrow mesenchymal stem cells.The effect of scaffold implantation on femoral repair at the defect site was examined by constructing an SD rat cranial defect model,and bone regeneration was examined using Micro-CT,H&E staining,and Masson staining.The results showed that the repair effect of scaffolds in the C-3 and C-1 groups was significantly better than that in C-0 and blank groups,and the results in the four groups showed that the bone repair effect gradually increased with the increase of PDA-MMT content.The synergistic effect of PDA-MMT could induce the accumulation of endogenous stem cells and active factors to the defect site,and the implantation of scaffolds provided attachment sites for cells and growth factors,which promoted the cell proliferation and differentiation. |
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