Sustained Release Of Nifedipine From 3D Printed Scaffolds To Block Sympathetic Nerve Activation For Enhancing Bone Regeneration

Bone is a highly vascularized and innervated tissue.Nerve tissue is mainly distributed in the periosteum of the skeletal system,along with blood vessels into the bone tissue to maintain bone homeostasis.In the process of bone repair,nerve tissue regulates the behavior of bone-related cells,and plays an important regulator of bone regeneration.Although 3D bioprinting is widely used in bone tissue engineering,design of 3D bioprinted scaffolds based on innervation effects is rarely reported and still requires extensive investigation.In addition,some researches have shown that traumatic bone defects will activate the damaged sympathetic nervous system in the defect area,it will release catecholamine and affect the bone repair process.Therefore,the development of 3D bioprinted scaffolds with the function of inhibiting catecholamine release is important for the treatment of bone defect.Some researches have shown that the antihypertensive drug nifedipine（NP）would close the calcium channel in nerve cells,thus blocking sympathetic activation and inhibiting the release of catecholamine.This study prepared the bioink（SGLP）composed of calcium channel blocker nifedipine（NP）,sodium alginate（SA）,methacrylate gelatin（GelMA）and laponite（Lap）to construct a hydrogel scaffold for regulating catecholamine secretion and promoting bone repair.Herein,the feeding ratio of Lap in bioink and the printing performance of bioink were inveatigated.The swelling,degradation and drug release as well as the the biocompatibility of scaffolds were studied.The recruitment effect and osteogenic activity on bone mesenchymal stem cells（BMSCs）and inhibition effect on osteoclastic differentiation of bone marrow mononuclear cells（BMMCs）of as-prepard hydrogel scaffolds were explored.Furthermore,the osteogenic capability of hydrogel scaffolds in vivo was evaluated in a skull defect model.The main conclusions include the following parts:（1）The effect of Lap on the rheological features of SA/GelMA bioink and the printing accuracy were defined by regulating the proportion of Lap（0.5%,1% and 2%）in SA/GelMA bioink.Scanning electron microscope（SEM）results showed that the hydrogel scaffold had porous structure,in which the increased content of Lap would cause denser structure of hydrogel network.The results of compression test showed that the increased content of Lap would significantly improve the compression strength of the scaffold,showing the optimal mechanical performance of the SA/GelMA/Lap（SGL）scaffold when the content of Lap was 2%.Degradation assays in vitro showed that the addition of Lap could affect the degradation behavior of the SA/GelMA scaffold and significantly reduce the degradation rate of the scaffold.In vitro ions release curves exhibited that the prepared SGL scaffolds had long-term release of silicon and magnesium ions.In vitro drug release behavior demonstrated that sustained release of NP from SGLP scaffold was achieved.（2）Proliferation assays in vitro showed that addition of 0.5% and 1% Lap had good biocompatibility and could support cell proliferation,but 2% Lap had inhibitory effect on cell proliferation,thus 1% Lap was chosen to prepare the SGLP scaffold.The SGLP scaffold was able to significantly inhibit the catecholamine release behavior in adrenal pheochromocytoma（PC12）cells as compared to the SGL scaffold and control.The transwell migration and scratch healing assays suggested that the SGLP scaffold had more significant promoting effect on BMSCs migration as compared to the SG scaffold and SGL scaffolds.In addition,the SGLP scaffold group significantly enhanced the expression of osteogenic genes,alkaline phosphatase（ALP）activity and mineralized nodules formation in BMSCs.Next,the inhibition ability of SGLP scaffold on osteoclast differentiation was verified by tartrate resistant acid phosphatase（TRAP）staining,measurement of steoclast differentiation-related genes and proteins expression,which revealed that the SGLP scaffold could inhibit the differentiation of BMMCs into osteoclasts.（3）Evaluation of bone regeneration ability of the SGLP scaffold was performed in SpragueDawley（SD）rat skull defect model using micro-CT analysis and histological staining.The results of micro-CT analysis showed that the SGLP scaffold significantly increased new bone tissue formation within the region of the bone defect.Addtionally,the results of immunofluorescence staining indicated that the SGLP scaffold group had better performance in inhibiting osteoclast differentiation and promoting new bone formation as compared to the SGL scaffold group.In conclusion,the SGL bioink had good printability and the 3D bioprinted SGLP scaffold loading with NP@Eth enabled the slow release of NP.The in vitro and in vivo experiments showed that the SGLP scaffold could promote the migration and osteogenic differentiation of BMSCs,while it could inhibit catecholamine release in the nervous system and thereby inhibiting osteoclast differentiation and promoting the migration and osteogenesis of BMSCs.Therefore,based on the regulatory strategy of innervated bone regeneration,our results confirmed that the SGLP scaffold prepared by 3D bioprinting can inhibit osteoclast differentiation and promote bone regeneration,which provides new insights into the regulatory function of innervation on bone regeneration.