3D Printed Scaffold Enables Sequential Release Of Dual Factors To Guide Immunoregulation For Promoting Vascularization And Bone Repair

During the bone repair process,the microenvironment in the site of bone injury will be changed.In order to promote bone repair,the favorable bone microenvironment can be regulated by the implants.Specially,the early formation of a well-developed vascular network plays a significant role in accelerating the repair of bone defects.The in vivo vascularization of bone grafts is a complex process involving multiple factors.The delivery of bioactive factors by the scaffold materials that promote angiogenesis have been regarded as an effective way to accelerate neovascularization in vivo.However,to achieve controlled release of pro-angiogenic factors for improving the efficacy of bone repair is still a challenge.In addition,bacterial infection of bone implants can seriously affect the effect of osteogenesis and eventually lead to the failure of bone remodeling.Therefore,the construction of 3D printed bone scaffold with antibacterial activity and pro-angiogenic ability for bone regeneration can meet the clinical requirements of bone defect repair.Studies have shown that strontium(Sr)ions can promote the transition of the inflammatory stage to the anti-inflammatory stage and induce the formation of type H vessels by regulating the phenotype of macrophages,thereby providing a more conducive microenvironment to bone repair and promoting angiogenesis and bone formation.In addition,SLIT3 has been demonstrated to promote the formation of type H vessels,thus promoting bone repair.Therefore,the synergistic effect of SLIT3 and Sr may couple the angiogenesis and osteogenesis,which can regulate the bone microenvironment and improve the neovascularization of newly formed bone tissue,thereby promoting bone tissue regeneration.Based on these findings,this study aimed to construct a bone scaffold with controllable release of SLIT3 and Sr ions.Sr-doped hydroxyapatite(Sr HA)was incorporated into the 3D-printed poly(ε-caprolactone)(PCL)scaffold using 3D printing technology.Then 3D-printed scaffold was modified with carboxymethyl chitosan/hyperbranched polylysine(CCS/HBPL)using self-assembly method to obtain polyelectrolyte-modified composite scaffolds(Sr HA@PCH).The physicochemical properties and antibacterial activity of the composite scaffolds were tested,and the potentials to promote vascularization and bone regeneration were evaluated both in vitro and in vivo.The main research contents and results are included as follows:(1)The Sr HA@PCL scaffold was prepared by 3D printing technology.The oppositely charged CCS and HBPL were modified onto the surface of Sr HA@PCL scaffold by layer-bylayer self-assembly method.The results showed that the antibacterial performance of the composite scaffold was increased with the increasing number of polyelectrolyte layers.The composite scaffold with 10 layers of polyelectrolytes modification had better antibacterial effect,showing that the growth inhibition rates against E.coli and S.aureus were 84.27 ± 4.81%and 89.82 ± 5.29%,respectively.The mechanical test showed that the addition of Sr HA can increase the compressive strength of Sr HA@PCH(61.52 ± 0.97 MPa)and the compressive modulus(10.16 ± 0.58 MPa).(2)SLIT3 protein was adsorbed on the Sr HA@PCH scaffold to prepare a dual-factor delivery scaffold(SLIT3/Sr HA@PCH).In vitro release results of SLIT3 protein and Sr ion showed that SLIT3 protein was released quickly,while Sr ion kept sustained release profile.Thus,a sequential release mode of SLIT3 and Sr ion was achieved and was conducive to the early vascularization and long-term osteogenesis.In vitro cell experiments showed that SLIT3/Sr HA@PCH scaffold had good biocompatibility,which could induce macrophage polarization from M1 to M2-type and thus increasing the expression of angiogenesis-related genes,as well as promoting cell migration and tubular formation of human umbilical vein endothelial cells(HUVECs).Furthermore,the immunoregulation on macrophage also promoted osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).(3)The in vivo immunoregulation ability of SLIT3/Sr HA@PCH scaffold was evaluated by subcutaneous embedding model and femur defect model in SD rats.Immunofluorescence staining results showed that the scaffold could improve the infiltration of M2-type macrophages,and the measurement of inflammation-related cytokines showed that the scaffold could improve the expression level of anti-inflammatory factors.The in vivo results also showed that SLIT3/Sr HA@PCH scaffolds could create a better microenvironment for bone regeneration.The osteogenic ability of the composite scaffold was evaluated by micro-CT,histological staining and immunofluorescence staining.The results showed that the SLIT3/Sr HA@PCH scaffold significantly promoted more new bone tissue at the bone defect site and induced the formation of type H vessels,thus presenting better performance of bone repair.In summary,based on the 3D printing and layer-by-layer self-assembly method,we have constructed a dual-factor delivery scaffold for achieving good antibacterial property and vascularized bone regeneration ability.The results of in vitro and in vivo studies showed that the SLIT3/Sr HA@PCH scaffold can promote the formation of type H vessels through the synergistic effect of SLIT3 protein and Sr ion,and thus promoting in vivo vascularization and bone regeneration,which could be a promising scaffold for bone defect repair.