Laminated Cell-Collagen Composite Subjected To Moderate Mechanical Stimulation To Promote Bone Repair

Bone defects caused by trauma,tumor,infection,congenital malformations and other causes are common diseases in orthopedics clinics,which seriously affect the quality of life of patients,and its repair and regeneration are still huge challenges for clinicians.In recent years,the great progress made in tissue engineering has brought new hope for the regeneration treatment of bone defects.The new strategy of using scaffolds with bone-mimicking characteristics to promote bone regeneration can create an appropriate microenvironment to promote the rapid repair of bone defects,which has great potential.The construction of bone tissue must integrate structural and mechanical microenvironment characteristics close to natural tissue.However,few studies have thus far focused on recapitulating the microstructural features and physiological environment of native bone.Cortical bone is a composite of collagen fibrils(mainly type I collagen)and mineral phase(carbonated hydroxyapatite),with a highly angle-ply aligned structure of oriented fibers.Meanwhile,under physiological conditions,mechanical stimulation(such as caused by exercise)can promote bone formation and remodeling,increasing bone strength.In order to mimic the above characteristics,in this study,collagen was used as the cell culture substrate,a multi-layer aligned cell-collagen composite with angle-ply structure was developed by using cell micropatterning and layer by layer assembly technique to mimic the microstructure of natural bone,and moderate mechanical stimulation was further applied to the multi-layer aligned cell-collagen composite to investigate the role of cell alignment and mechanical stimulation on osteogenic differentiation of MC3T3-E1 cells and bone formation.In the first part of this study,micropatterning technology was used to construct microgrooved structures with a depth and width of 2 μm and 10 μm on the surface of the collagen membrane to obtain a micropatterned collagen membrane,and cells were seeded on the micropatterned collagen membrane to detect the orientation and proliferation of cells on the collagen membrane.The results showed that,due to the presence of aligned microgrooves,the micropatterned collagen membrane had anisotropic mechanical properties and effectively guided the orientated growth of cellsIn the second part of this study,in order to investigate the effects of cell alignment and mechanical stimulation on cell proliferation and differentiation,we used a customized loaded cell culture system to apply periodic tensile mechanical stimulation to the mono-layer aligned cell-collagen membrane.The results showed that moderate mechanical stimulation could promote MC3T3-E1 cell proliferation without affecting cell adhesion on collagen membranes.Cell alignment and mechanical stimulation could synergistically promote osteogenic differentiation of MC3T3-E1 cells and the aligned cell-collagen membrane had good osteogenic differentiation ability in vitro.In the third part of this study,we further used layer-by-layer assembly technology to construct a multi-layer aligned cell-collagen composite with angle-ply structure characteristics,which was then mechanically stimulated and implanted into the model mouse calvarial defect to investigate its bone-promoting properties in vivo.The results showed that the mechanical stimulation in vitro had no effect on the cell viability of multi-layer aligned cell-collagen composites and significantly promoted the secretion of extracellular matrix.After implanting into the bone defect,the mechanically stimulated multi-layer aligned cell-collagen composites showed the best bone formation,the new bone mass reaches 30%at 8 weeks post-implantation.In conclusion,in this study,a multi-layer aligned cell-collagen composite was constructed by cell micropatterning and layer by layer assembly technique.In vitro and in vivo experiments showed that cell alignment due to the microstructure of the material surface and mechanical stimulation had an important influence on cell behavior and function.Under the synergistic influence of two factors,cell alignment and mechanical stimulation,the cell-collagen composite exhibited excellent bone-promoting ability.The results of this study not only provide a theoretical basis and technical basis for the design of scaffold materials and the selection of mechanical stimulation in bone tissue engineering,have considerable application prospects in bone regeneration,but also provide reference for the repair of other tissues.