MVs-mimicking Micelles Loaded With PEG-Serine-ACP Nanoparticles To Achieve Biomimetic Intra/Extra Fibrillar Mineralization Of Collagen In Vitro

Objectives:Bone is composed of inorganic minerals(mainly hydroxyapatite,HAP)and organic matrix(mainly type I collagen and non-collagenous proteins,NCPs).In the formation of bone tissues in vivo,mature osteoblasts have been demonstrated to secret collagen fibrils and matrix vesicles(MVs).MVs can mediate extrafibrillar mineralization in vivo,but how MVs induce intrafibrillar mineralization of collagen in vivo has not been elaborated.Inspired by the polyelectrolytes nature of NCPs,researchers have used analogues of NCPs,inducing the mineralization of the collagen in vitro,due to their anionic polyelectrolytes nature to stabilize the amorphous calcium phosphate(ACP),which subsequently infiltrate into the collagen via the gap zone to achieve intrafibrillar mineralization of collagen.The primary purpose of the present study was to establish a MVs–mimicking model in vitro for studying the exact mechanism of MVs-mediated intrafibrillar mineralization of collagen in vivo.By adjusting the pH and surface tension of this model of MVs carrying ACP in vitro two forms of minerals(crystalline mineral nodules and ACP nanoparticles)were released to achieve the extrafibrillar and intrafibrillar mineralization,respectively.Also,this model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.Methods:1.Preparation and characterization of PEG-S-ACP loaded micelle(PEG-S-ACP/micelle).High-concentration serine was used to stabilize the amorphous calcium phosphate(S-ACP),which was subsequently mixed with polyethylene glycol(PEG)to form PEG-S-ACP nanoparticles.The nanoparticles were loaded in the polysorbate 80 micelle through micelle self-assembly process.This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model.DLS,Raman spectrum,TEM,Fluorescence spectrum,STEM–EDX mapping and ICP were used to analyze the micelles.2.Compare tow forms of the micelle loaded with different states of the minerals.By adjusting the pH and surface tension of the PEG-S-ACP/micelle,two forms ofminerals(crystalline mineral nodules and ACP nanoparticles)were characterized.For the one type,when the pH of the micelle system was increased,methods(XRD,TEM,and SAED)were used to confirm the minerals.For the other,surface tension,ICP,TEM,and DLS methods were used to characterize the micelle and the minerals.3.The self-assembled collagen was mixed with two forms of the mineralization medium(1 mL)in a 100% humidity chamber for 5 days to mineralize the collagen fibrils.TEM,STEM–EDX mapping and SAED methods were used to observe the mineralized collagen.Results:1.TEM,Raman spectroscopy and STEM–EDX mapping confirmed that polysorbate80-PEG6000 micelles at a dimension of 300 nm were prepared with ACP nanoparticles evenly distributed.In addition,the percentages of encapsulated calcium and phosphate were high.2.Two different contents,crystalline mineral nodules and ACP were released.By adjusting the pH of the system,an amorphous phase in the PEG-S-ACP/micelle transformed into thermodynamically stable HAP.In addition,the shape of the micelle eventually became irregular and mineralized nodules formed similar to those in vivo.The XRD pattern and SAED further confirmed the time dependent crystallite growth.For the other,IPA was managed to destroy the structure of the PEG-S-ACP/micelle at pH 6.0,with releasing the ACP nanoparticles.The significant decrease happened at IPA volume ratio of 0.2 with the high dissolving efficiency.3.Two different contents,crystalline mineral nodules and ACP were used to mineralize the self-assembled collagen to simulate the bone mineralization process in vivo.After mixing the PEG-S-ACP/micelle at with self-assembled collagen for 5 days,a needle-shaped apatite deposited on the surface of the collagen fibrils leading to extrafibrillar mineralization.When self-assembled collagen fibrils were exposed to the ACP releasing micelle for 5 days,the ACP nanoparticles infiltrated into the unstained fibrils and deposited mainly at the gap zone to accomplished the intrafibrillar mineralization.Conclusions:A MVs-mimicking model,PEG-S-ACP/micelle was synthesized and two forms ofminerals(crystalline mineral nodules and ACP nanoparticles)were released to achieve extrafibrillar and intrafibrillar mineralization,respectively.The PEG-S-ACP/micelle reproduced the mineral nodules mediating in vivo extrafibrillar mineralization.Also,this model implies that in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs.Accordingly,based on the MVs-mimicking model,scaffolds of mineralized collagen with the chemical composition,structure and mechanical properties being close to those of natural bone tissues can be developed to repair bone defects in bone tissue engineering.