Mineralization Strategy And Parameter Optimization Of Biomimetic Mineralized Collagen Promoting Interface Osseointegration In Titanium Alloy Micro-pores

Background:The ideal scaffold for bone tissue engineering must possess bone inductivity,bone conductivity,and bone integration functions to facilitate effective bone tissue repair.Bone integration at the micro-porous interface primarily relies on the mechanical interlocking formed by bone ingrowth into the micropores,directly impacting the stability and long-term success of the implant.Titanium and titanium alloys are the most commonly used materials for orthopedic implants due to their excellent biocompatibility and mechanical properties.While the titanium alloy micro-porous interface prepared by 3D printing exhibits certain bone conductivity,the high biological inertness of titanium and titanium alloy materials leads to a lack of bone inductivity in the micro-porous interface.This results in limited bone ingrowth rate and depth,ultimately leading to poor bone integration effects.Among the various factors affecting bone integration,the surface characteristics of implants play a crucial role,necessitating surface modification of titanium alloy micro-porous interfaces.Currently,common surface modification strategies include chemical modification,physical modification,and biological modification.Chemical modification involve complex chemical reactions,with implants typically exposed to chemical solutions or gases.Physical modification primarily alters the surface morphology and microstructure of titanium alloy materials through methods like laser treatment,high-energy particles,ultrasound,and magnetic fields.Biological surface modification mainly involves attaching organic bioactive substances,such as proteins,to the surface of implants through mechanisms like electrostatic attraction and hydrogen bonding.Unlike the other two methods,biological modification does not involve complex chemical reactions or high-temperature,high-pressure conditions,thereby preventing deactivation of the bioactive substances.Biomimetic mineralized collagen（BMC）possesses excellent biocompatibility,biodegradability,and osteoinductive properties.Some researchers have explored combining BMC with titanium alloy implants to enhance cell adhesion,strengthen the bone-conducting effect at the interface,increase osteoinductivity,and promote bone integration.There are various preparation strategies for BMC,with the most common being the classical ion-induced mineralization strategy（CIMS）and the polymer-induced liquid precursor pathway（PILP）.These strategies are capable of faithfully replicating the diverse nanostructures of mineralized collagen found in natural bone in vitro.Collagen,minerals,and non-collagenous proteins（NCP）are essential components for BMC preparation,and their variations can impact the in vitro mineralization process of collagen.Among the many factors influencing in vitro mineralization,minerals and NCP play pivotal roles,with the introduction of NCP leading to alterations in mineralization strategies,thereby directly affecting the type of mineralized collagen produced.Additionally,the concentration of mineralization solution and mineralization time parameters are crucial factors influencing the degree of BMC mineralization and surface morphology.However,current research predominantly focuses on BMC under specific strategies or parameters,lacking comprehensive and systematic comparative analysis of mineralization strategies and parameters for BMC modification on titanium alloy micro-porous interfaces.Therefore,this study,based on titanium alloy micro-porous interfaces,constructs a series of biomimetic bone interfaces with variations in structure,composition,and morphology through BMC modification.Comparative studies are conducted on physicochemical properties,bone inductivity,and bone integration,analyzing the impacts of CIMS and PILP strategies as well as mineralization time and mineralization solution concentration on BMC interfaces to identify the optimal strategies and parameters.This study primarily comprises the following two parts:ⅠThe Influence of Biomimetic Mineralized Collagen Modification with Various Mineralization Strategies on the Osseointegration of 3D Printed Titanium Alloy Micro-porous InterfacesObjective:Through comparative analysis of the differences in physicochemical properties,osteoinductivity,immune modulation,and bone integration of BMC interfaces prepared using CIMS and PILP strategies,this study aims to investigate their effects on BMC interfaces and determine the optimal preparation strategy.Method:This study utilized Ti₆Al₄V powder as the raw material and employed 3D printing technology to fabricate titanium alloy porous interfaces（PTi）.Subsequently,dopamine coating was prepared on the porous interface,followed by covalent grafting of collagen to prepare COL/PTi interface.Next,the EMC/PTi interface was prepared using the CIMS strategy,and the I-EMC/PTi interface was prepared using the PILP strategy.The physicochemical properties of these interfaces were characterized using SEM,EDS,confocal laser scanning microscopy,water contact angle measurements,and protein adsorption experiments.Following this,bone marrow mesenchymal stem cells（BMSCs）were extracted from rabbit femurs and underwent cell identification.Co-culturing BMSCs with each micro-porous interface,we evaluated biocompatibility through live/dead cell staining,CCK-8 assay,FITC-conjugated phalloidin/DAPI staining,and immunofluorescence staining.Subsequently,we assessed the osteogenic activity of each interface using ALP staining,Alizarin Red staining,RT-q PCR experiments,and immunofluorescence staining.Furthermore,we investigated the influence of each interface on macrophage polarization through immunofluorescence staining and RT-q PCR experiments.Establishing a co-culture model with conditioned medium using the rat-derived monocytic cell line（RAW 264.7）and BMSCs,we evaluated the effects of the immunomodulatory microenvironment involving macrophages on BMSC osteogenic differentiation through ALP staining,Alizarin Red staining,and RT-q PCR experiments.Finally,we implanted the micro-porous scaffolds into the distal femur of rabbits and observed the rate of new bone formation at the micro-porous interfaces using calcein/tetracycline fluorescence double labeling.The osseointegration effect at the bone-implant interface was evaluated through micro-CT,histological sections,immunohistochemistry staining,and mechanical push-out tests.Results:（1）The titanium alloy porous scaffold with a pore size of 800μm and a porosity of 70%has been successfully prepared,which possesses a biomimetic structure.Subsequently,three types of interfaces,namely COL/PTi,EMC/PTi,and I-EMC/PTi,were prepared based on PTi.Compared to PTi and COL/PTi,EMC/PTi and I-EMC/PTi exhibit higher surface roughness,stronger hydrophilicity,and protein adsorption capacity.Among them,EMC/PTi has the highest surface roughness.（2）BMSCs were successfully isolated,and it was demonstrated that all micro-porous interfaces did not compromise the viability of BMSCs,ensuring their proliferation.Compared to PTi,the other interfaces all facilitated the early adhesion of BMSCs.Among them,EMC/PTi and I-EMC/PTi exhibited the most effective cell adhesion,with higher expression of focal adhesion proteins in both the cell cytoplasm and periphery.Additionally,EMC/PTi and I-EMC/PTi demonstrated higher ALP activity,calcium nodule deposition,and expression of osteogenic genes（ALP,BMP-2,Runx-2,Col I）and osteogenic proteins（OCN,Runx-2）compared to PTi,indicating that both BMC interfaces possess stronger bone inductivity.（3）Compared to I-EMC/PTi,EMC/PTi significantly promoted M1 polarization of macrophages,inhibited M2 polarization,and concurrently upregulated inflammatory genes（TNF-αand i NOS）.Furthermore,compared to BMSCs co-cultured directly with EMC/PTi,the conditioned medium of EMC/PTi interface RAW 264.7 inhibited the ALP activity,calcium deposition,and the expression of osteogenic genes（ALP,BMP-2,Runx-2,Col I）in BMSCs.（4）Each micro-porous interface demonstrated non-toxicity to major organs in vivo.Compared to other groups,I-EMC/PTi exhibited the most rapid bone generation rate,highest volume of newly formed bone,deepest bone penetration,highest expression of surrounding osteogenic proteins ALP,BMP-2,and Runx-2,maximal peak push-out force,and manifested the optimal osseointegration effect.Conclusion:The BMC interfaces fabricated through both CIMS and PILP strategies significantly enhanced the surface roughness,hydrophilicity,and protein adsorption capacity of the interfaces,while also demonstrating excellent biocompatibility and osteogenic activity.Specifically,the BMC interfaces prepared using the PILP strategy were capable of promoting M2 polarization of macrophages and increasing the expression of anti-inflammatory genes,thus creating an immunomodulatory microenvironment conducive to BMSCs’osteogenic differentiation.In vivo experimental results revealed that this interface exhibited superior osseointegration.Consequently,the PILP strategy is deemed the optimal approach for BMC modification of micro-porous interfaces.ⅡThe Effects of Biomimetic Mineralized Collagen Modification with Different Mineralization Parameters on the Osseointegration of 3D Printed Titanium Alloy Micro-Pore InterfacesObjective:By comparing the effects of mineralization solution concentration and mineralization time parameters on the physicochemical properties,osteoinductivity,and osteointegration of the BMC interface,this study investigates their influence on the BMC interface and selects the optimal parameters.Method:Utilizing PTi interfaces as substrates and employing the PILP strategy,we prepared four interface groups（1X-2D/PTi,1X-4D/PTi,2X-2D/PTi,and 2X-4D/PTi）by modifying the PTi surface with BMC.This modification involved adjusting the mineralization solution concentration（1X,2X）and mineralization time（2 days,4 days）.Subsequently,these interfaces underwent comprehensive physicochemical characterization and evaluation for biocompatibility,osteoinductivity,and osseointegration effectiveness.Results:（1）Compared with PTi,each BMC interface exhibited a relatively rough surface morphology,significantly increased hydrophilicity,and protein adsorption capacity.Among them,the surfaces of 2X-2D/PTi and 2X-4D/PTi were the roughest,with the best hydrophilicity and strongest protein adsorption capacity.（2）Compared to the other groups,fewer viable cells adhere to the surface of 2X-4D/PTi,with cells exhibiting a more rounded morphology,poorer adhesion,and a significantly slower proliferation rate.This group was excluded from subsequent experiments.Additionally,1X-4D/PTi and 2X-2D/PTi exhibited higher ALP activity,calcium nodule deposition,and expression of osteogenic genes compared to PTi.（3）Compared to PTi,each BMC interface demonstrated faster bone generation speed and greater bone formation.Among them,the 2X-2D/PTi exhibited the fastest rate of new bone generation,the highest volume of new bone,the deepest bone ingrowth,the maximum peak push-out force,and showed the best bone integration effect.Conclusion:Using the PILP strategy and adjusting mineralization parameters,a series of BMC interfaces were constructed by introducing BMC onto the PTi surface.The study found that mineralization parameters have a significant impact on the physicochemical properties and biocompatibility of BMC interfaces.As the concentration of mineralization solution increases and the mineralization time extends,BMC interfaces become rougher,and their hydrophilicity and protein adsorption capability also increase.However,the 2X-4D/PTi interface exhibited unfavorable conditions for cell adhesion and proliferation,while other BMC interfaces demonstrated excellent biocompatibility,osteoinductivity,and osseointegration performance.Among them,the 2X-2D/PTi exhibited the best osteoinductive and osseointegration effects.Therefore,"double mineralization solution,mineralization for 2 days"was identified as the optimal parameter combination for BMC modification of micro-porous interfaces using the PILP strategy.Through the aforementioned studies,we identified the optimal strategy and parameters for BMC modification of micro-porous interfaces,establishing a relationship between strategy,parameters,performance,and effects.This research X provides a theoretical basis for the design of 3D printed titanium alloy micro-porous interfaces with excellent biological activity.