Enzymatic Reaction Generates Magnesium-based Mineralized Microspheres With Superior Bioactivity For Enhanced Bone Regeneration

ObjectiveBone is a unique organ that retains the inherent ability for controllable growth with remodeling in response to damage.However,in the cases of large bone defect caused by high-energy trauma,tumor resection,osteoporosis or complications of diabetes,the inherent self-repair capacity is not enough to reach the goal of full recovery.Despite the autologous bone has been considered as the“gold standard”clinically,the demand for tissue-engineered bone grafts is increasing greatly because of the well-defined chemistry and architecture,unlimited availability without any risk of donor site morbidity or infection transmission compared with autogenous or allogeneic bone grafts.In recent years,the role of magnesium-based materials in bone tissue engineering have been extensively studied.Magnesium ions are involved in bone healing by activating osteoblast differentiation,enhancing human bone-derived cell adhesion and promoting angiogenesis.Moreover,magnesium ions are also involved in the mineralization process,regulating bone formation and resorption.However,the ultra-high concentration of magnesium ions may have negatively effects on the osteoblast differentiation and bone mineralization,leading to correlated bone diseases.In addition,the long-term exist of magnesium ions may affect bone remodeling.Therefore,the functions of magnesium ions in bone tissue regeneration are strongly concentration and time dependent.At present,the existing magnesium matrix composites have complex structural design,poor homogeneity and insufficient mass production capacity.Therefore,it is necessary to explore a new method to prepare magnesium-incorporated composite scaffolds.Methods and ResultsSection 1:Enzymatic reaction preparation and characterization of magnesium phosphate mineralsMethods:The product was synthesized using Na₂ATP and Mg Cl₂·6H₂O as raw materials and ALP as catalyst.X-ray diffraction（XRD）,Fourier transform infrared spectroscopy（FTIR）,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and high-performance liquid chromatography（HPLC）were used to characterize the physicochemical characteristics of the products.The biocompatibility of the product was verified by CCK8 assay.And then,ALP staining and alizarin red staining were performed to evaluate whether it had superior osteoinductive ability compared to magnesium phosphate control samples synthesized by common hydrothermal method.Results:The precipitation was successfully synthesized enzymatically.XRD andFTIR confirmed that the main component of the product was magnesium phosphate（Mg P）hydrate（JCPDS:49-0752）.SEM and TEM results showed that the synthesized Mg P was flower-like spherical particles formed by lamellar self-assembly.EDS suggests that O,P and Mg elements are evenly distributed over the sample.HPLC demonstrated that Mg P released a small amount of ATP and AMP in PBS,suggesting that the Mg P mineral contains ATP and AMP biomolecules and is organic-inorganic hybrid mineral.CCK8 experiments demonstrated that the biocompatibility of the product was similar to that of ordinary Mg P.The results of ALP staining and alizarin red staining showed that Mg P prepared by enzymatic method had better osteoinductive ability compared with Mg P synthesized by common hydrothermal method.Section 2:Preparation and characterization of magnesium phosphate mineralized microspheresMethods:In this experiment,the methylacryloylated silk fibroin（Sil MA）microspheres were prepared by microfluidics combined with photo-crosslinking.Magnesium-based mineralization microspheres（Sil MA@Mg P）were then prepared by introducing the Sil MA microspheres into the enzymatic mineralization system described previously.Finally,the physical and chemical properties of the microspheres were characterized in detail by XRD,FTIR,SEM,TG,degradation experiments,and ion release assay.Results:Sil MA microspheres were successfully prepared in this experiment.Sil MA@Mg P microspheres containing Mg P mineral crystals were then prepared by biomimetic mineralization using the microspheres as mineralized matrice.The prepared mineralized microspheres were observed to be uniform in size and well dispersed under a light microscope,and mineral crystals on the microsphere surface can be observed at high magnification.XRD and FTIR confirmed that the microspheres contained Mg P and Sil MA.Scanning electron microscopy and thermogravimetric analysis showed that Mg P on the surface of Sil MA microspheres increased gradually with the prolongation of reaction time,and the increase was very small and close to saturation after 24 hours of reaction.Ion release experiments showed that Mg²⁺released from microspheres was relatively stable in the first two weeks,which is consistent with the actual time of magnesium ion acting on bone regeneration reported in the current literature.Degradation experiments showed that the microspheres could degrade effectively.Section 3:In vitro biological study of magnesium phosphate mineralized microspheresMethods:First,the effects of magnesium phosphate mineralized Sil MA microsphereson the toxicity,proliferation,migration and adhesion of bone marrow mesenchymal stem cells（BMSCs）were investigated by live/dead cell staining,CCK8 assay and Transwell cell migration assay,respectively.BMSCs on co-cultured microspheres were stained by phalloidin,and cell adhesion and expansion on the microsphere surface were observed by laser scanning confocal microscopy（CLSM）.The angiogenic ability of Sil MA@Mg P microspheres in vitro was detected by tube formation assay.In terms of osteogenic performance experiments,the effects of Sil MA@Mg P microspheres on osteogenic differentiation of BMSCs were mainly studied by biological assays such as alkaline phosphatase staining,alizarin red staining,RT-q PCR and Western-blot.Finally,the potential mechanism of Sil MA@Mg P microspheres in promoting osteogenic differentiation of BSMCs was analyzed by transcriptome sequencing.Results:Live/dead cell staining showed that Sil MA@Mg P and Sil MA microspheres had no significant toxicity on BMSCs.CCK-8 cell proliferation assay showed that Sil MA@Mg P microspheres showed good cell proliferation promoting effect on days 7and 10.Transwell migration assay confirmed that Sil MA@Mg P microspheres could significantly promote the migration of BMSCs to microspheres at 24 hours of co-culture.CLSM observation showed that BMSCs could adhere and expand well on Sil MA@Mg P microspheres.Tube formation experiments showed that Sil MA@Mg P microspheres had good angiogenic ability.BMSCs showed significantly increased alkaline phosphatase activity after 7 days of osteogenic induction with Sil MA@Mg P microspheres;after 21days of co-culture,they showed more calcium nodule formation.RT-q PCR results showed that Sil MA@Mg P microspheres could significantly increase the expression of related m RNAs,including Col-1,ALP,OCN and OPN;western blot results showed that Sil MA@Mg P microspheres could significantly increase the expression of osteogenesis-related proteins Col-1,OCN and OPN.The results of transcriptome sequencing showed that the molecular mechanism by which Sil MA@Mg P microspheres promoted osteogenic differentiation of BMSCs may be related to the PI3K-Akt pathway,ECM-receptor interaction and focal adhesion.Section 4:Animal study on repairing bone defect with magnesium-based mineralized microspheresMethods:BMSCS-laden Sil MA@Mg P microspheres（Bone regenerationenhancement Units,BREUs）were obtained by co-culturing Sil MA@Mg P microspheres with BMSCs.Further,the osteogenesis-promoting effect of BREUs was verified by animal experiments.We constructed a critical bone defect model of the distal femur in rats by drilling,and then implanted BMSCS-laden Sil MA microspheres,Sil MA@Mg P microspheres,and BREUs,respectively.At different time points after surgery,Micro-CT imaging was performed to evaluate the repair effect from bone morphology and bone micro-architecture.Then HE and Masson staining were further performed on the samples to compare the repair effect of each group from a histological point of view.Results:Micro-CT results showed that there were more new bone tissues in theBREUs group,and quantitative analysis showed that the bone volume fraction,trabecular thickness,and trabecular separation in the BREUs group were significantly better than those in the other three groups.In the 8th week after surgery,the bone defects in this group basically achieved full-thickness and full-area repair.The results of HE and Masson staining of femoral specimens suggested that BREUs could effectively promote the formation of new bone,and the new bone in this group was more mature and dense,and the results were consistent with the results of Micro-CT.ConclusionsIn summary,inspired by the natural biomineralization of hard tissue,we reported a new enzymatic reaction-based strategy to prepare magnesium-based mineralized microspheres of Sil MA@Mg P.Firstly,Sil MA microspheres with uniform size have been prepared via a combined technique of microfluidics and photo-crosslinking.Then,the in-situ mineralization strategy was successfully used to prepare magnesium-based mineralized microspheres,by inducing the formation of flower-like spherical Mg P on Sil MA microsphere.The mineralization was originated by the reaction of ALP-catalyzed hydrolysis of adenosine triphosphate（ATP）.The minerals of Mg P formed in this enzymatic reaction have presented a flower-like spherical structure composed of random Mg P nanosheets with a crystal phase of magnesium phosphate hydrate（JCPDS 49-0752）.The as-prepared Sil MA@Mg P microspheres with uniform size and porous structure have displayed a facile degradability and the ability for sustained release of Mg²⁺.Furthermore,the in vitro studies have demonstrated that the Sil MA@Mg P microspheres have high bioactivities in promoting the proliferation,migration,and osteogenic differentiation of BMSCs.Transcriptomic analysis showed that the osteoinductivity of Sil MA@Mg P microspheres may be related to the activation of PI3K/Akt signaling pathway.Finally,the BREUs were constructed by seeding BMSCs on the Sil MA@Mg P microspheres.The in vivo bone repair performance of BREUs have been studied in a rat distal femur bone defect model.The results of micro-CT assay and histological staining have clearly presented the remarkable efficient of the BREUs in promoting the bone regeneration.This study not only provides a biomaterial of cell-microsphere complex with superior bioactivity for bone regeneration,but also demonstrates a biomimetic strategy for designing composite materials with defined structure and combination functions.