Highly Mechanical And Recoverable Double Network Hydrogels Formulated By Poly (Vinyl Alcohol) And Pectin For Bone Regeneration

Objective: Bone fractures are common diseases in orthopedics,affecting millions of people worldwide.Traditional treatments,including autologous bone grafting and allogenic bone grafting,have some drawbacks in effectively repairing bone defects.The challenging aspects are the development of artificial extracellular matrix to improve osteocyte proliferation and mineralization and as an artificial bone graft to avoid bone disease metastasis and immune rejection.In order to solve these problems and better promote the repair of bone defects,the concept of tissue engineering is introduced.Tissue engineering includes the use of associated scaffolds,the introduction of appropriate growth factors and cells,and the more recently used stem cells.With tissue engineering technology,new scaffolds and tissue grafts can be designed to reduce the disadvantages of traditional grafts and improve the engraftment rate,formation rate,osteoconductivity rate,and osteoinduction rate of grafts.And in response to the problem of pan-bone defects,we optimized the design and synthesis of double-network hydrogels as bone tissue engineering scaffold materials for promoting bone regeneration and the use of this material to combat the limitations of recent bone defect treatment.Here,we present a series of double-network(DN)hydrogels constructed from polyvinyl alcohol(PVA)and pectin(PEC)as physical cross-linked composite scaffolds applied to the healing of femoral defects in tissue engineering.Methods:(1)PVA-Pec double-network hydrogel was fabricated,mixed according to the weight ratio of PVA and pectin powder at 8:1 and repeatedly frozen and thawed to form the first layer network structure;then the hydrogel mixture was immersed in calcium chloride solution for ion cross-linking,so as to complete the second layer network structure,and PVA-Pec double-network hydrogel was successfully prepared when equilibrium swelling was achieved.The final sample is defined as PVA-Pec-x,where x represents the total amount of polymer in the volume(w/v)of homogeneous solution.In order to determine the various characteristics of the synthesized PVA-Pec double-network hydrogel,the tests include: electron microscopy to measure the pore size and porosity of the hydrogel scaffold after lyophilization;simulated in vivo environment to measure the swelling rate of the double-network hydrogel;dynamic mechanical analysis(DMA,TAInstrument Q800)to study the mechanical properties of the hydrogel in the "DMA strain rate" mode;and universal tester(Instron 34TM-5(5 k N))for the measurement of compression and cyclic compression cycle tension of the hydrogel scaffold.Osteoblasts were used as seed cells to verify the biocompatibility of double-network hydrogel pairs and the maturation and calcification ability of osteoblasts.Among them,the groups according to different stent materials were: PVA-10,PVA-Pec-5,PVA-Pec-10,and PVA-Pec-15 groups.The biocompatibility of the hydrogels was tested using live-dead cell staining and CCK-8 to verify the proliferative effect of the scaffold materials on the cells.Quantitative fluorescence PCR(q RT-PCR)was used to detect specific genes of osteoblasts: ALP,BMP-2,COL1α1,and Runx-1.Osteoblast calcium nodule precipitation was detected by alizarin red S staining and calcium ion fluorescence staining after osteoblast culture on hydrogel scaffolds.The repairing effect of hydrogel scaffold on femoral condyle defect was verified by in vivo experiments in SD rats.Establish the femoral condyle bone defect model of SD rats,implant the scaffold materials of different groups(blank group,PVA-10 group,PVA-Pec-10 group)into the defect site,collect the samples of repaired femoral condyle when the repair time is 4 weeks and 8 weeks,detect the repair effect of the defect site,and observe the repair of the defect site;observe the repair effect of each group by HE staining,Masson’s trichrome staining and immunohistochemical staining.Results:(1)The hydrogel scaffolds showed a high Young’s modulus of 117.9 ±9.0 k Pa and a compressive modulus of 79.3 ± 20.6 k Pa,and the scaffolds contained 15%(wt)pectin,and the tensile elongation of all scaffolds was above300%.In addition,the scaffolds exhibited enhanced adhesion,spreading,and proliferation of osteoblasts.In addition,the scaffolds significantly promoted osteogenesis by elevating alkaline phosphate activity and calcium biomineralization deposits,as well as osteoblast gene expression.These double-network hydrogel scaffolds also showed great biocompatibility with rat hosts,no immune rejection after transplantation of femoral condyle defects in vivo,good new bone formation and growth and fusion with normal bone tissue.Conclusion: The composite double-network hydrogel synthesized from PVA and Pec materials matches the demand of bone tissue regeneration,and provides support and load for the defect due to its excellent rebound and tensile properties,especially adapted to the irregular shape of the defect,and this hydrogel acts as a synthetic extracellular matrix(ECM)to contribute to the proliferation and differentiation of new cells,and the optimized structural composition and multifunctional characteristics make these hydrogel scaffolds very promising candidates for bone tissue regeneration.