The Application Of Lactoferrin Peptide Functionalized With Hydroxyapatite In Bone Defect Repaire

Bone tissue is always in the process of continuous reconstruction and has strong self-healing ability.However,bone defects caused by various diseases such as bone trauma and tumor are far beyond the scope of self-healing.In this case,the study on bone repair materials is of great significance.Lactoferrin（LF）is a multifunctional glycoprotein that can promote the proliferation and differentiation of osteoblasts.As a new bone growth factor,LF has great potential application in the field of bone tissue engineering.Therefore,the LF adsorption on the surface of hydroxyapatite（scaffold material）was investigated in this study,and its adsorption model and interaction mechanism were also analyzed.Also,the peptides with osteogenic activity from LF hydrolysate were screened,and the effect of peptide on proliferation and differentiation of osteoblasts and its mechanism were also investigated.At the same time,natural hydroxyapatite materials with good biological activity were prepared from fishbone.The natural hydroxyapatite functionalized with lactoferrin peptide and the effects of compound on the biocompatibility of osteoblasts and skull defects repair of rats were also analysized.The aim is to develop new bone growth factors and scaffold materials with good biocompatibility for the field of bone tissue engineering.The adsorption behavior of LF as bone growth factor on the surface of hydroxyapatite and its effect on the biological activity of hydroxyapatite were investigated.Two different forms of HAP were selected to analyze the adsorption behavior of LF on the surfaces of the two kinds of HAP.The results indicated that there existed an equilibrium adsorption of LF on the surfaces of nano-HAP and micro-HAP,the maximum adsorption capacity of LF on nano-HAP surface was 91.10 g/mg,and LF on micro-HAP surface was 50.76 g/mg.The adsorption curves both fit well with the Langmuir model which suggested that LF formed monomolecular layer on the two kinds of HAP surfaces,and the dominant force of the interaction is electrostatic.In addition,the HA-LF was characterized by TGA and FTIR,and the results showed that LF could stablely adsorbed on the HAP surface and LF could be released slowly from HAP-LF.What’s more,LF adsorption on HAP surface could significantly improve the proliferation of osteoblasts,strength the interaction between materials and cells,and significantly improve the biological activity of HAP.UPLC-QTOF and molecular docking technology were used to identify and screen LF enzymatic hydrolysis products in this study.LFP-C with osteogenic activity were obtained from LF pepsin hydrosate.The molecular docking results indicated that LFP-C had affinity osteoblast surface receptor EGFR,and could interact with it through hydrogen bonds,hydrophobic interaction and Van der Waals force.Besides,after solid phase synthesis of LFP-C,the effect of LFP-C on proliferation and differentiation of osteoblasts and its mechanism were investigated by CCK-8,flow cytometry,ALP detection,alizarin red staining and q RT-PCR.The results showed that LFP-C could significantly enhance the proliferative activity of osteoblasts MC3T3-E1 and promote the transformation of osteoblasts from G₀/G₁ phase to G₂/M phase.In addition,LFP-C could promote the expression of ALP and calcium deposition in osteoblasts,and promote the expression of differentiation related genes COL1α1,Runx2,OPN and OCN to promoting further differentiation of osteoblasts.LFP-C could also regulate the expression level of ERK1/2 gene and induce the phosphorylation of ERK1/2protein in MAPK signaling pathway,but it had no significant regulatory effect on the expression of JNK1/2 and P38 genes.The results showed that LFP-C could stimulate the activation of downstream MAPK signaling pathway by binding to osteoblasts surface receptors,thus inducing the proliferation and differentiation of osteoblasts.In addition,in this study,molecular dynamics was used to analyze the interaction between LFP-C polypeptide and hydroxyapatite（0,0,1）crystal surface,the interaction energy between LFP-C and hydroxyapatite（0,0,1）crystal surface is-7.06×10⁴ kcal/mol and electrostatic energy is-9.26×10⁵ kcal/mol.The results showed that LFP-C could stably bind to the hydroxyapatite（0,0,1）crystal surface through electrostatic interaction.The natural HAP materials were prepared by using calcining method from fish bone which is the waste product from food processing.Natural HAP was characterized by SEM,XRD,FTIR and EDS,and its effect on proliferation and differentiation activity of osteoblasts was investigated by MTT and ALP detection.The results showed that natural HAP derived from 650°C calcined salmon bone could retain Mg²⁺and CO₃^2-ions,which were naturally found in fish bones,and promoted the cell activity of osteoblasts and the expression of ALP indicating that the natural HAP had good biological activity.Also,natural HAP porous scaffold was prepared by organic template method using natural HAP powder material.The results show that the scaffold has a connected pore structure,good mechanical properties andsuitable degradability indicationg that it can work as scaffold material and may also have good performanc in bone tissue engineering field.Also,the interaction between bone growth factor LFP-C and HAP was analyzed to construct HAP-LFP-C composite material with good biocompatibility.The biocompatibility of composite scaffold materials was investigated by in vitro and in vivo experiments.HAP-LFP-C porous scaffold material can significantly promote cell adhesion and cell viability on the surface of the porous scaffold.The HAP-LFP-C scaffold can significantly promote the new bone growth in the skull defect of SD rats after 12 weeks of implantation.Therefore,the natural HAP porous scaffold combined with bone growth factor LFP-C had good biocompatibility and exhibited a good application prospect in the field of bone tissue engineering.In this thesis,HAP-LFP-C porous scaffold with good biocompatibility was constructed,which has a good application prospect in the field of bone tissue engineering and provide a theoretical foundation for the application of LF in the field of bone defect.