| Critical-sized bone defects caused by trauma or disease require surgical treatment.However,autografts are restricted by issues of donor tissue availability,while both allografts and xenografts present shortcomings with immune response complications.With the advent of tissue engineering science and technology,tissue-engineered bone scaffolds are promising strategies for treatment of bone defects.During the last decade,great progress has been achieved using 3D printing technologies to construct macroporous bioceramic scaffolds,thereby revolutionizing the traditional treatments of bone defects.Various 3D printing methods,have been used for bone scaffold fabrication,while most of them are not suitable for printing scaffolds with small size and high precision.Digital light processing(DLP)-based 3D printing is suitable to fabricate bone scaffolds with regular shape and high precision,which is expected to be an effective way to solve the above problems.In this work,a novel bone tissue-engineered macroporous scaffold was constructed by DLP-based 3D printing technique and subsequent sintering approach using biphasic calcium phosphate(BCP).Furthermore,bone morphogenetic protein-2(BMP-2)was facilely incorporated into this scaffold through a self-assembled polydopamine(PDA)modification process.The resultant scaffold presents an interconnected porous structure with pore size of～570 μm,sufficient compressive strength(up to ～3.6 MPa),suitable surface roughness and hydrophilicity,and the self-assembly Ca-P/PDA composite nanolayer exhibited excellent BMP-2 delivery properties,sustained-release property of BMP-2 for 35 d.Notably,this BMP-2/PDA-BCP scaffold presents favorable effects on the adhesion,proliferation,osteogenic differentiation,and extracellular matrix(ECM)mineralization of bone marrow stromal cells(BMSCs).Furthermore,in vivo bone tissue regeneration experiments conducted on rats demonstrated that the scaffolds could induce a significant improvement of osteogenetic ability,with cell layer aggregation adjacent to the scaffolds and continuous new bone generation within the scaffold.In the animal model of beagle,the scaffold showed strong ability to induce angiogenesis and ectopic osteogenesis.Collectively,it is anticipated that the BMP-2/PDA-BCP scaffold is of great potential in the treatment of bone defects,which provides a feasible method for the bone tissue regeneration scaffolds and clinical transformation of high accuracy personalized repair.Methods(1)Designed precisely and produced personalized and implantable BCP bioceramics scaffold by DLP 3D printing.(2)PDA-BCP scaffold with Ca/P-PDAlayer was made from BCP scaffold by the selfassembled PDAmodification process.(3)BMP-2/PDA-BCP was prepared by loading BMP-2 through PDA coating.(4)The physicochemical properties of scaffolds were evaluated systematically,including: macroscopic and microscopic morphology,hydrophilicity,roughness,Ca,P ion release,BMP-2 load and release,compressive strength/stiffness and shrinkage,etc.(5)The biological properties of the scaffolds in vitro were tested by using BMSCs of rats,including cytotoxicity,cell adhesion and proliferation,cell apoptosis,and osteogenic differentiation of cells.(6)Experiments on biocompatibility testing and bone defect repairing were carried out in vivo,including subcutaneous implantation and skull defect repair in rats.(7)Vasculogenesis,ectopic osteogenesis and special site bone defect repairing experiments were carried out,including intramuscular implantation,subcutaneous implantation and hard palatal bone defect repair experiments in beagles.Results(1)A BCP ceramic scaffold with large and micro-porous structure and personalized shape was designed and prepared by DLP 3D printing.(2)Ca-P/PDA nanocomposite layer on the surface of the scaffold was formed by PDA modification,which enriched the microstructure of the scaffold surface.(3)BMP-2 protein can be loaded with low dose and released slowly through PDA layer.(4)The results of material characterization showed that the PDA coating changed the macroscopic and microscopic morphology of the BCP scaffold,affected the release of Ca and P ions in the microenvironment,the surface roughness and hydrophilicity of the scaffold,and maintained good mechanical properties.(5)The extracts of the scaffolds had no obvious cytotoxicity,and the modified scaffolds had no actual effect on cell apoptosis.All the scaffolds showed preferable biocompatibility,and the spread morphology of cells on the surface of scaffold in each group was stable.Meanwhile,the scaffolds of PDA coating group showed better cell adhesion promotion and proliferation.(6)The BMP-2 loading improved the osteogenic ability of the scaffold,and the BMP-2/PDA-BCP scaffold had the best ability to promote osteogenic differentiation of BMSCs in vitro.(7)The scaffolds in each group showed exactly biocompatibility in vivo,and the scaffolds with PDA coating showed stronger cell adhesion,and there were cell aggregation layers around the scaffolds.(8)BV/TV and Tb.N in BMP-2/PDA-BCP group were the largest among all groups.HE and Masson staining images showed that the number of new bone formation was the largest in the BMP-2/PDA-BCP group among the four scaffold groups,and the expression level of OCN was the highest.(9)Beagle muscle implantation and ectopic bone formation assays showed that the BMP-2/PDA-BCP group had the largest number of blood vessels and the largest vascular area,which were significantly different from the other scaffolds.The area of new bone formed in BMP-2/PDA-BCP group was the largest,and the mature bone was the most.Conclusion(1)Various shapes and high precision structures of BCP ceramic scaffolds were prepared by using DLP 3D printing technology.PDA-BCP scaffold with Ca-P/PDA hybrid layer was obtained through PDA surface modification,which changed the surface roughness and hydrophilicity of the scaffolds,and promoted the adhesion and proliferation of BMSCs on the scaffolds.(2)The BMP-2/PDA-BCP scaffold was prepared,which could load and sustainedrelease BMP-2,and showed better biological characteristics and osteogenic differentiation of BMSCs in vitro.(3)It was found that the BMP-2/PDA-BCP scaffolds still showed good biocompatibility and stronger osteogenic ability in vivo,which was verified in rat model of subcutaneous implantation and skull defect.(4)The results of experiments in beagle model showed that the BMP-2/PDA-BCP scaffolds showed strong ability to induce angiogenesis and ectopic osteogenesis. |
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