Investigation On The Construction Of Bone Repair Scaffolds Coupling Vascularization And Osteoginduction Activity

The large segment bone defect has always been a difficult problem in clinical treatment.The application of tissue engineering technology to construct bioactive artificial bone replacement materials is a potential solution.With the progress of advanced materials research and the study of bone microenvironment metabolism mechanism,developing new bone repair materials which can integrate and regulate different biological activities to accelerate the process of bone repair,is currently the key goal in biomedical tissue engineering.Biodegradable soft hydrogel scaffolds and hard polymer scaffolds are the most commonly used artificial bone repair materials at present.However,they have shotcomings of poor biological activity and poor bone repair effect.Physiologically,the process of fracture healing includes a series of cascading biological reactions,and the induction of vascular intrusion and the differentiationof bone progenitor cells into mature osteoblasts are the key points.Therefore,by chemically modifying or combining medicinal small molecular compounds,to constructthe new hydrogel and polymer scaffolds which have osteogeniesis and angiogenesis activity,can greatly promote the clinical translation of bone repair materials.ObjectiveTo construct the soft hydrogel and hard polymer bone repair scaffolds with osteogeniesis and angiogenesis activity.And their effectiveness and mechanism for bone repair were proved by using in vitro experiments,and in different disease models.Methods(1)Based on the rapid gelation of silk protein(silk fibroin,SF)by self-assembly of polypeptide small molecule(NapFFRGD),a new type of injectable hydrogel containing RGD functional groups was constructed.The material characterization and biocompatibility of hydrogel were studied,and the hydrogel loaded with bone marrow mesenchymal stem cells(BMSCs)was applied to the repair of cranial bone defect in mice.The real-time PCR was used to detect bone induction activity and the immunohistochemical staining,histopathological staining and Micro-CT scanning analysiswas used to detect the effect of bone repair in vivo.(2)By using 3D printed and surface modification technology,3D printing polymerization-lactone(Polycaprolactone,PCL)scaffold was constructed to achieve the controlled release of small molecule hypoxic analog compound-DFO.The 3D printedscaffold was tested for material characterization,drug release and biocompatibility.In vitro through cell culture,Alizarin red staining,real-time PCR detection,western-blot detection,immunohistochemical staining and other methods;In vivo,we established a large bone defect model of the distal femur in rats,and tested its repair effect for large bone defect through Micro-CT scanning analysis,immunohistochemical staining,Micro-fil vascular perfusion and so on.Results(1)The introduction of polypeptide small molecules(NapFFRGD)significantly improved the gelation conditions of silk fibroin molecules including lowering of the gelation concentration,shortening of gelation time,improvement of mechanical properties,and enabling the formation of an injectable hydrogel;Introduction of RGD functional group to silk fibroin gel(SF-gel)promoted its biocompatibility and cellular adhesion;In vitro,the new type of silk ibroin hydrogel(SF-RGD gel)can enhance the directional differentiation of bone marrow mesenchymal stem cells(BMSCs)into osteoblasts;In vivo,SF-RGD gel can be used to deliver BMSCs into the bone defect area,which significantly promoted the bone repair in mice.(2)The prepared 3D printing PCL/DFO scaffold has excellent biocompatibility and can achieve the controlled release of DFO;In vitro,it promoted the tube formation of human umbilical vein endothelial cells(HUVECs)by activating the HIF-1α signaling pathway;It also enhanced the mineralization of extracellular matrix and up regulate the expression of osteogenesis related genes,protein and enzyme activity during the induction and differentiation of BMSCs.In vivo,PCL/DFO scaffold significantly promoted new vascular formation,new bone formation and accelerated bone defect healing in the femoral defect site of rats.Conclusion(1)We report on the generation of a bioactive SF and small peptide gelator(NapFFRGD)hydrogel,able to mimic the ECM for cell adhesion and growth.Besides mediating cell adhesion,the self-assembled SF-RGD gel also showed a high potential to induce signal transduction and osteogenic differentiation of MSCs via integrin–RGD interactions.This work suggests that SF could be easily tailored with bioactive peptide gelators to afford bioactive hydrogels with a favorable microenvironment for tissue regeneration applications.(2)We presented a useful and facile method for preparing DFO bridged 3D printed PCL scaffolds with designed macropores andmulti-oriented channels.The prepared PCD scaffolds combined high porosity and surface area with appropriate mechanical strength.Addition DFO did not affect the cell attachment and proliferation of the scaffold,while it showed a significant increase in vascularity regeneration in the scaffolds which was accompanied with more bone growth and osseointegration in a rat weight bearing bone defect model.This indicated that PCD scaffolds can be used for reservation of large bone defects.Given the safety,ease of handling and low expense,translation of this biofunctional scaffolds into clinical trials is of great interest to the orthopedic community.