Fabrication Of Osteoid-like Collagen Template And Its Regulation On The Biomineralization Of Osteoblasts

Bone is a hierarchical composite material that is constructed via the template mineralization by osteoblasts.In histology,osteoblasts produce the collagen-rich extracellular matrix(ECM)called an osteoid,which is the template for bone development.In osteoid structure,type I collagen(Col I)fibrils produced by osteoblasts assemble at the supra-fibrillar level to form cholesteric liquid crystal(LC).Once osteoid is mineralized,it means the formation of bone.Since it is still difficult to mimic the process of naturally bone formation in vitro,multi-level hierarchical bone tissue is far from being constructed in vitro.In this study,the osteoid structure was mimicked to investigate its regulation on the biomineralization of osteoblasts.It is predicted that the in vitro model systems would enable the bone formation relative to template-guided process and osteogenesis through osteoblasts,with ECM assembly and subsequent mineralization.Data regarding the mineralization mechanisms obtained from the models are valuable for the rational design of bone repair strategies and tissue engineering.In the study,MC3T3-E1 was used and a mineralized medium(m MEM)supplemented with calcium glycerophosphate(Ca-GP)and ascorbic acid(AA)was used for cell differentiation and matrix mineralization.Mineralized medium supplemented with 5 m M Ca-GP and 50 ug/m L AA was selected for the subsequent biomineralization via osteoblasts.Type I collagen(Col I)from rat tail was extracted by an acid-enzymatic method.It was showed that high-concentrated Col I solution could self-assemble into ordered collagen membrane with liquid crystal(LC)structure.In vitro experiments showed that the extracted Col I has the capacity of mineralization and good cell cytocompatibility.The minerlized medium and extracted Col I was used for further study for cell biomineralization.Electrospun collagen templates properly produce the framework of the ECM.1,1,1,3,3,3-hexafluoro-2-propanol(HFIP)/acetic acid(HAc)was used for Col I electrospinning.In this work,by N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride with N-hydroxysulfosuccinimide(EDC-NHS),glutaraldehyde(GTA)and genipin(GP)respectively,electrospun collagen fibers were cross-linked.The crosslinking effectively stabilized the fiber morphology and improved the mechanical properties in both dry and wet state.On the cell assessment,electrospun collagen fibers crosslinked by EDC-NHS,GTA and GP were found to support cell adhesion,spreading and proliferation of MC3T3-E1 and promote the expression of alkaline phosphatase(ALP).Among these crosslinked templates,Es-EDC-NHS template has the best comprehensive performance.Osteoid-like(Os)collagen templates were fabricated by electrospinning(Es),plastic compression and tension(PCT),and shear stress method(SS),and the templates were incubated with MC3T3-E1 for biomineralization.All templates had good cell cytocompatibility and promoted the expression of ALP.The aligned fibers of the osteoid-like templates led to the orderly growth of cells.Reverse transcription-polymerase chain reaction(RT-PCR)results showed that the all osteoid-like templates promoted the osteogenic differentiation and mineralization of osteoblasts by up-regulating the expressions of Col I,runt-related transcription factor 2(Runx2),ALP and osteocalcin(OCN)in different time periods.The SS-template promote the osteogenic differentiation of osteoblasts best.Therefore,SS-template was selected as the optimized osteoid-like(Os)template for following biomineralization via osteoblasts.A collagen Os-template with LC structure was fabricated by SS method and incubated with MC3T3-E1 for biomineralization.The results showed that the ordered fibrils of Os-template collagen fibers promoted the adhesion and proliferation of MC3T3-E1 cells.Osteoblasts were arranged in parallel order on Os-template.During the process of cell mineralization,Os-template promotes the secretion and assembly of cell-secreted Col I fibrils to form ordered dense collagen bundle structure in situ.Further further experimental characterization demonstrated that hydroxyapatite(HAP)compacted into collagen fibrils via both intrafibrillar and interfibrillar mineralization under the regulation of cells.A multi-level hierarchical ordered mineralized bone-like structure was assembly by aligned mineralized collagen bundles.The expressions of Col I,ALP,OCN and Runx2 during cell mineralization were analysed by RT-PCR,and the molecular mechanism of biomineralization via osteoblasts on the Os-template was explored using RT-PCR,ALP activity test,and Col I immunofluorescence staining.At the early stage of the cell culture(3d),cells were on the stage of proliferation and secreted much Col I,which gradually matured and self-assembled into fibrils.Orderly cells guided the Col I fibrils to assemble into micro-scale ordered structure.The cells began to early-stage mineralization after 7 days incubation and ALP expression was up-regulated to dissociate of calcium ions and phosphate ions,which were infiltrated into cell-secreted(CS)collagen fibrils to form HAP.At the middle-stage of mineralization(14d),the cells secreted more Col I and formed a more-dense collagen bundles.ALP,OCN and Runx2 expression reached the highest level and form mineralized collagen fiber-bundles.At the late-stage of mineralization(after 21d),the expression of Col I decreased,but the expressions of other genes were still high.In addition,OCN,as a specific marker of late osteogenic differentiation of osteoblasts,was highly expressed and further regulating bone mineralization deposition in the mineralized bone-like structure.A hierarchical tissue-engineered bone was constructed by cell-mediated mineralization on the Os-template.Based on fabrication of osteoid-like collagen template and ts regulation on the biomineralization of osteoblasts,a hierarchical tissue-engineered bone composed of mineralized collagen fibrils in parallel ordered arrangement was obtained.Osteoblasts were guided by Os-template and produced bone-like mineralized collagen fibril bundles,which is the basic block for constructing hierarchical bone.The assemblied CS collagen fibrils enhanced mineralization,and mineralized collagen fibrils further packed in parallel arrays extending to hundreds of micrometers,where the LC of Os-template regulated the osteoblasts to form an ordered structure similar to bone.Therefore,a nano-to micro-scale hierarchical matrix mimicking bone was produced by the cells’ mineralization in vitro.Such studies also provide reference for bone tissue regeneration and repair,broaden the technical path of tissue engineering bone construction in vitro,and lay a foundation for rapidly implantable synthesized bone.