Osteogenic Properties Of 3D Printed Polycaprolactone/Whitlockite Composite Scaffolds

The incidence of bone defects is high,and there is a great clinical need for bone implants.One of the main challenges for large bone implants to effectively guarantee bone repair is to form a vascular network in the implant.The extent of vascularization is important for the gas exchange of central cells,the supply of nutrients,and the removal of metabolic waste during the formation of new bone tissue.In addition,the nerves distributed in the native bone can also regulate the process of bone remodeling by affecting the function of bone cells.Therefore,the research goal of this thesis is to develop a multifunctional bone tissue engineering scaffold that can not only promote angiogenesis,but also promote osteogenesis,and upregulate the expression of neural function during neo-bone formation.Systematic characterizations were carried out to evaluate the efficacy and safety of the scaffolds in bone repairing.Bone is a tissue composed of organic components（type I collagen,etc.）and inorganic components（calcium phosphate,etc.）,and it is also a tissue with electrophysiological activity.Based on the principles of structural and functional bionic design,this thesis mainly carried out the following researches:1)Based on the fact that the inorganic components of natural bone contain about 20%whitlockite(WH,Ca₁₈Mg₂（HPO₄）₂（PO₄）₁₂),and Mg²⁺has been confirmed to have multiple physiological functions of promoting angiogenesis,improving cell viability and up-regulating the expressions of genes related to neural expression,in this study,we successfully synthesized WH nanoparticles by precipitation method,and furthermore,the nanoparticles were submitted to annealing（650°C,3 h）to made the WH with piezoelectricity（WHE）.As a control,β-tricalcium phosphate（β-TCP）nanoparticles were also synthesized by precipitation method.Then,three-dimensional porous scaffolds were prepared by melt 3D printing using polycaprolactone（PCL）as the organic component,into which,WH,WHE orβ-TCP were added at a mass fraction of10 wt.%or 30 wt.%to prepare organic-inorganic composite scaffolds.To improve their surface hydrophilicity and roughness,these scaffolds were treated with Na OH etching,followed by evaluations of compressive mechanical properties,electrical activity,and cell affinity.It was finally determined that the optimized etching parameters were soaking the scaffolds in 20 M Na OH aqueous solution for 3 hours,while maintaining acceptable compressive strength（approx.10 MPa）to meet the mechanical requirements for bone repairing.After the treatment,the PCL/WHE scaffold still demonstrated the electroactivity.All the prepared scaffolds were noncytotoxic via in vitro cell culture evaluation,that BMSC could attach firmly and spread widely on them with normal morphology and strong viability,showing comparable proliferation behaviors with control.2)Then,the abilities of these scaffolds in promoting the osteogenic and neural differentiation of BMSC,the blood vessel formation in the chicken embryo allantoic membrane angiogenesis model（CAM）,as well as,in inhibiting osteoclast differentiation,were comprehensively evaluated.It was promisingly found that the addition ofβ-TCP,WH and WHE brought an incremental contribution to all the mentioned biological responses.Briefly,compared to PCL scaffold,the PCL/β-TCP composite scaffold had a promoted activity in osteogenesis,which the PCL/WH and PCL/WHE composite scaffolds demonstrated extra benefits in promoting angiogenesis and neurogenesis,at the same time,in inhibiting osteoclasts,due to the release of Mg²⁺.Among them,the electroactive PCL/WHE composite scaffold achieved the most effective enhancement in all these events,thanking to its electroactivity mimicking the electrophysiological nature of bone-related cells.3)Accordingly,by implanting the abovementioned scaffolds into a 5 mm defect using rat calvarial model,their efficiency in modulating bone regeneration were monitored via micro-CT,histological staining and immunohistochemical staining assays at 4 and 8 weeks post-operation.The outcomes of micro-CT examination revealed that limited neo-bone tissue could be detected in the PCL group,and bone regeneration was accelerated in the PCL/β-TCP group.Compared to them,both the PCL/WH and PCL/WHE groups demonstrated more volumes of newly formed bone tissue,which had grown into and integrated with the scaffolds.In accordance with the in vitro results,the electroactive PCL/WHE scaffold achieved the highest efficiency in osteogenesis.Confirmed by both histological and immunohistochemical（CD31,OCN,TRAP）staining results,the efficiency of different scaffold in enhancing bone regeneration was in the order of PCL<PCL/β-TCP<PCL/WH<PCL/WHE,apparently,suggesting that Mg²⁺and electroactivity are the two important factors in the regeneration of vascularized bone tissue.In summary,it can be determined that WH,as a magnesium-containing calcium phosphate,exhibits piezoelectric properties after appropriate annealing treatment,which matches the composition and electrophysiological activity of natural bone.In view of these benefits,the 3D printed organic-inorganic scaffold containing WHE shows significant potentials in promoting bone regeneration as compared with the PCL,PCL/β-TCP and PCL/WH scaffolds,due to its activity in upregulating angiogenesis and neurogenesis,but inhibiting osteoclast maturation.And thus,the developed PCL/WHE scaffold can serve as a multifunctional scaffold for improved bone regeneration and repair.