Construction Of PCL-TCP Composite Scaffold Modified With Porous Surface And Nano-DBM Coating In Bone Graft Substitute

Research backgroundIn the clinical work of orthopedics,the bone defects caused by trauma,tumor,deformity,degeneration and other diseases during the occurrence,development,and treatment have always been one of the difficult problems for surgeons.The treatment of bone defects is inseparable from the reconstruction of bone tissue,and in the reconstruction of the bone tissue structure,a suitable filling material is needed to replace the original missing bone.Autograft,allograft or other filling materials used in the clinic always have some disadvantages.With the emergence of bone tissue engineering and 3D printing technology,the development of bone graft substitutes has got a new direction.The ideal bone graft substitute should have four characteristics,which are osteogenic,osteoinductive,osteoconductive and osseointegration.Polycaprolactone(PCL)and Tricalcium phosphate(TCP)are materials with good biocompatibility and biodegradability.As composite materials,they have good osteoconduction and osteoinduction properties as well as a certain mechanical strength.The research on composite materials of these two is also one of the current hot topics.The change of surface morphology directly affects the proliferation and differentiation behavior of cells attached to the scaffold.The porous scaffold with surface roughness can promote the osteogenic differentiation of cells.Demineralized bone matrix(DBM),as a kind of allograft,contains 5% of most osteogenic induction factors in addition to 93% collagen.Nanoscale DBM,which may minimize its influence on the original scaffold morphology,may be a feasible coating modification method for bone tissue engineering scaffold.In this study,through 3D printing technology,the composite scaffolds of PCL and TCP were subjected to roughening modification,and coating with nanoscale DBM.After a series of physical property characterization,in vitro and in vivo experiments,the ability of osteogenesis of the new scaffold to repair the bone defect was evaluated.Research methodsFirstly,the PCL-TCP composite scaffold was made by fused deposition modeling(FDM)technology,which is one of the 3D printing technologies,and then the roughening modification of the scaffold fiber was completed by organic solvent etching to prepare a porous surface of the scaffold.The morphology,porosity and mechanical properties were tested.After that,the nanoscale DBM was loaded onto the fiber surface of PCL-TCP composite scaffold by vacuum freeze-drying method to complete the coating modification.Subsequently,human bone marrow mesenchymal stem cells(BMSCs)were seeded on the PCL-TCP composite scaffold with a porous surface and nano-DBM-coated scaffold,and the PCL-TCP composite scaffold without any modification was used as a control.The CCK-8 assay,the alkaline phosphatase activity assay,the calcium content assay,the real-time fluorescent quantitative PCR,and the Western Blot were used to evaluate the adhesion,proliferation and osteogenic differentiation of BMSC on different composite scaffolds.Finally,a critical defect model of the middle tibia of New Zealand rabbits was established.The PCL-TCP composite scaffold with a porous surface and nano-DBM-coated scaffolds were used as filling materials,and calcium phosphate artificial bone was used as a control.Through radiologic and histological examination at different time points after the operation,a preliminary study on the in vivo osteogenesis of the new scaffold was done.Research resultsThe PCL-TCP composite scaffold made by FDM technology has a pre-configured porous structure,and the pores connect with each other.The porous surface is achieved by an organic solution etching method,which created macro-and micro-pores and increase of the surface roughness and porosity.The original pores of the scaffold were still maintained at about 500μm,and the newly formed larger pores were about 50~300μm,and the smaller pores were smaller than 10μm.The mechanical strength of the PCL-TCP composite scaffold with the porous surface was close to that of the human cancellous bone.After the DBM coating,the original porous structure could still exist,and the pore diameter ranged from 50 to 200 μm.The surface of the fiber was coated with lots of nanoscale DBM agglomerates with irregular shapes and sizes,and the minimum particle size was 200-300 nm.For in vitro test,compared with PCL-TCP composite scaffolds and PCL-TCP composite scaffolds with a porous surface,the nanoDBM-PCL-TCP composite scaffold was more effective in adhesion and osteogenic differentiation of BMSC.For in vivo experiment,the nanoDBM-PCL-TCP composite scaffold showed better osteogenesis than the PCL-TCP composite scaffold with the porous surface.The bone volume fraction and bone mineral density of the former were significantly higher than the latter after implantation;compared with the commercial β-TCP artificial bone,the early in vivo osteogenesis of nanoDBM-PCL-TCP composite scaffold was slightly better,but the results at 12 weeks were basically the same.The results indicate that the nanoDBM-PCL-TCP composite scaffold with had good osteogenic properties and can effectively promote new bone formation.Research conclusionsThe nanoDBM-PCL-TCP composite scaffold made by the present research had excellent pore structure and mechanical properties,which is similar to cancellous bone and can effectively promote the adhesion proliferation and osteogenic differentiation of BMSC in vitro.It could also promote the repair of critical bone defects.The novel scaffold had good osteogenesis in vitro and in vivo and was expected to be a promising bone substitute.