Amyloid-like Oligomeric Nanospheres Modify Type Ⅰ Collagen To Promote Intrafibrillar Mineralization

Objectives There is a key challenge to obtain remineralized collagen with mineral contents and mechanical properties comparable to natural hard tissues.Here,we applied the reaction product of lysozyme and TECP,namely amyloid-like oligomeric nanospheres（ALONs）to promote intrafibrillar mineralization innovatively,to shorten mineralization time and to enhance the degree of intrafibrillar mineralization.Methods ALONs preparation:Lysozyme and TCEP has to be mixed in 2 minutes before using to ensure the presence of ALONs.Explore the factors that affect the size of ALONs:lysozyme was mixed with TCEP thoroughly（with a series of reaction time）,the products were detected and analyzed by dynamic light scattering（DLS）.Explore the surface modification effect of ALONs on typeⅠcollagen fibrils:The optimized ALONs in the above experiments was co-cultured with acid-soluble type I collagen solution.The modification effect was determined by SEM,FTIR and DMA.Explore the promotion effect of ALONs on collagen intrafibrillar mineralization:The thermodynamic properties of ALONs and calcium interaction were roughly characterized using Micro Cal PEAQ-ITC.The mineralization experiments were divided into two groups,the collagen was modified by ALONs before incubating in m-SBF in experimental group and the collagen without ALONs was used in control group.The mineralized collagen was examined by TEM,the mechanical properties were determined by DMA,and TGA was used to evaluate the mineral contents of mineralized collagen.Results DLS results showed the size of ALONs was closely correlated with reaction time,the optimized reaction time was 2 min,the presence of ALONs was determined by CD and congo red staining,the ALONs with a diameter of 100-200nm could be found by TEM,and it was the aggregated products of nanospheres with diameter of 20-50nm.FTIR,SEM and TEM results showed the modification of collagen by ALONs,DMA results showed collagen with ALONs modification possessed higher tensile strength.ITC results illustrated that ALONs spontaneously attracted Ca²⁺via hydrogen bonds and electrostatic interactions,the binding interaction was spontaneous and thermodynamically favorable.The mineralized collagen with ALONs modification achieved intrafibrillar mineralization in a shorter time and with higher mineral content,what’s more,the mechanical properties were stronger than control group as showed by DMA results.Conclusion Amyloid-like oligomeric nanospheres can promote intrafibrillar mineralization by modifying type I collagen,this finding may have implications for using supplements to promote intrafibrillar mineralization and serve as a reference for understanding the mechanism of intrafibrillar mineralization.