| BackgroundTendon-bone interface(TBI)injuries,including anterior cruciate ligament(ACL)and rotator cuff(RC)injuries,represent prevalent sports-related pathologies.Restoring the physical integrity of the joint structure remains a primary treatment modality,typically achieved through tendon/ligament grafting wherein the transplanted tendon is affixed within bone tunnels to replace the native tissue’s function.Despite the common utilization of interface screws to enhance stability and load-bearing capacity at the tendon-bone junction,attaining complete restoration of patients’ functional mobility remains challenging due to suboptimal TBI healing outcomes.The primary reasons for the suboptimal healing effect in tendon-bone junctions are attributed to an unfavorable environment for bone-tendon repair,including excessive inflammation,poor vascularity,and abnormal mechanical conditions.The current research is predominantly centered on advancing bone and tendon regeneration,as well as targeting pivotal processes in tendon-bone healing such as inflammation modulation and vascular reconstruction to facilitate the restoration of bones and tendons.This encompasses the localized administration of bioactive agents,utilization of biologically enhanced screw materials,application of periosteum or ion-loaded stem cell membrane materials for tendon grafting,among other techniques.While the process of tendon-bone healing entails not only the regeneration of two distinct tissue interfaces but also the harmonization of soft and hard tissue interfaces,with careful consideration for the mechanical milieu conducive to both tissue types.Therefore,asymmetrical design of implants to concurrently enhance bone and tendon repair offers a promising strategy for providing an appropriate environment for tendon-bone healing,potentially yielding improved outcomes at the tendon-bone interface.PurposeThis study proposes an asymmetric hydrogel composite implant capable of sustained release of functional ions,serving as a platform for bone-tendon integration.The primary goal of the implant is to establish a mechanical environment conducive to the growth of bone and tendon,create a microenvironment conducive to bone and tendon formation through controlled ion release,and manifest anti-inflammatory and angiogenic properties.This endeavor aims to tackle challenges related to sluggish tissue repair and suboptimal results arising from excessive inflammation and inadequate vascularization,thereby fostering tendon-bone healing and augmenting the load-bearing capability of reconstructed tendons.This approach promotes tendon-bone healing and enhances the load-bearing capacity of reconstructed tendons.MethodsThe study comprised three parts:establishing an implant system to bolster bone tunnel interface regeneration,developing a hydrogel system to promote regeneration at bone tunnel and intra-soft tissue interfaces,and amalgamating the strengths of the preceding components to fabricate an asymmetrical hydrogel composite implant system geared towards facilitating tendon-bone interface healing post-anterior cruciate ligament reconstruction(ACLR).Part 1:Polyetherimide(PEI)-based implants with complex porous structures were prepared through 3D printing,followed by sulfonation and air-plasma treatment.The comparative analysis of sulfonation and air-plasma treatment was conducted to evaluate their respective advantages and disadvantages in enhancing the biological performance and bone tunnel repair of the implants,in order to develop a method that enables uniform and efficient surface modification of both internal and external surfaces of implants with complex porous structures.Scanning electron microscopy(SEM),white light interferometry,X-ray photoelectron spectroscopy(XPS),and water contact angle measurements were employed to examine the characteristics.Part 2:The CS/PVA hydrogel loaded with magnesium(Mg)and phosphorus(P)(CS/PVA/βGP-MgO)was prepared using a freeze-thaw method.Comprehensive material characterization encompassing surface topography,chemical composition,mechanical properties,degradation kinetics,pH dynamics,and ion liberation was performed to ascertain the successful fabrication of the material and its capacity for anticipated biological functionality.The biocompatibility,in vitro osteoinductive and tenogenic potential of the material were evaluated using murine bone marrow-derived mesenchymal stem cells(BMSCs)and TT-D6 cells through assessments of cell proliferation,morphology,alkaline phosphatase(ALP)activity,calcium nodules deposition,and gene expression profiling.Furthermore,the material’s in vitro angiogenic proficiency was appraised employing human umbilical vein endothelial cells(HUVEC)through analyses of tubulogenesis,scratch closure,cell migration,and gene expression studies.The material’s in vitro anti-inflammatory efficacy was gauged using RAW 264.7 cell line,involving scrutiny of cellular morphological alterations,phenotypic variations,immunofluorescence staining,and gene expression patterns.Part 3:The Mg and P-loaded CS/PVA hydrogel(CS/PVA/βGP-MgO)was synergistically integrated with sulfonated 3D-printed porous PEI implants(PEI-s)via freeze-thaw methodology.Subsequent to co-culturing with RAW 264.7 cells for a day,supernatant was harvested and diluted with standard culture medium at a 1:4 ratio to culture BMSC and TT-D6 cells for assessing the in vitro impact of the composite implant on inflammation-mediated osteogenic and tenogenic responses.Serum inflammatory factors were assayed three days post subcutaneous implantation in rodents,followed by histological analysis of subcutaneous tissues and vital organs to evaluate in vivo inflammatory modulation and biocompatibility of the implant.The composite implant was implanted in rabbit ACLR,with sample retrieval at 6 and 12 weeks post-surgery for magnetic resonance imaging(MRI),micro-computed tomography(Micro-CT),and biomechanical testing to evaluate the capacity of enhancing tendon-to-bone healing in vivo.ResultsPart 1:Both sulfonation and air-plasma treatment demonstrated consistent and uniform surface modification of the implants with complex porous structures,improving the hydrophilicity of the material and thus providing a foundation for enhancing biological properties such as cell adhesion and osteogenic differentiation.Sulfonation treatment was found to alter the micro/nano morphology,increase roughness,and introduce functional groups like-SO3H,while air-plasma treatment primarily introduced functional groups like-OH on the material surface,with minimal impact on morphology.Air-plasma treatment enhanced cell adhesion,whereas sulfonation treatment accelerated osteogenic differentiation,promoting bone regeneration and integration in vivo,indicating that roughness had a greater influence on osteogenesis.Furthermore,it was observed that combining sulfonation with air-plasma treatment did not synergistically utilize the advantages of each treatment;instead,it introduced free radicals such as peroxides,adversely affecting osteogenesis.In summary,sulfonated 3D printed PEI porous implants(PEI-s)are more suitable for promoting bone interface regeneration in tendon-bone healing implants.Part 2:The CS/PVA hydrogel loaded with Mg and P(CS/PVA/βGP-MgO)enables slow degradation and gradual release of functional ions,with approximately 64%Mg2+and 46%PO34-released over a period of 6 weeks.Additionally,the degradation of MgO creates a mildly alkaline microenvironment,providing a favorable basis for tissue repair.Specifically,the presence of Mg regulates inflammation by suppressing M1 polarization of macrophages and promoting the expression of anti-inflammatory genes such as IL-4 and IL-10.It also enhances the expression of angiogenesis-related genes like VEGF and PDGF,facilitating vascularization and creating an optimal environment for tendon-bone healing.Furthermore,Mg promotes Scx expression,inhibits the expression of genes MMP-2,MMP-13,and ColⅢ-α1,and thus promotes tendon formation through the TGF-β/Smad 2/3 pathway.The presence of P,in synergy with the weak alkaline microenvironment created by MgO degradation,promotes alkaline phosphatase(ALP)secretion,extracellular matrix(ECM)mineralization,and the expression of osteogenic-related genes(Runx-2,BMP-2,OPN,ColⅠ),thereby facilitating osteogenic differentiation.These findings highlight the potential of the sustained-release CS/PVA hydrogel loaded with Mg and P in tendon-bone healing and its suitability for application at the tendon interface of tendon-bone healing implants.Part 3:This section,which constitutes Chapter 4,combines the selected rigid and flexible systems identified in Chapters 2 and 3 to fabricate an asymmetrical tendon-bone repair implant capable of sustained release of dual-functional ions(CS/PVA/βGP-MgO@PEI-s).CS/PVA/βGP-MgO@PEI-s promotes tendon and bone healing by regulating macrophage polarization towards the M2 phenotype,leading to the release of anti-inflammatory factors and activation of the TGF-β/Smad 2/3 and Hif-1α pathways,thereby enhancing the expression of tendon-related and bone-related genes.In in vivo experiments,compared to the PEI-s group,the CS/PVA/βGP-MgO@PEI-s group significantly suppresses inflammation and fibrosis,exhibits improved tendon growth rates and faster repair,and demonstrates superior bone tunnel healing at 6 and 12 weeks post-ACLR based on gross observation,MRI,X and Micro-CT analysis.The mechanical testing reveals that the reconstructed tendon using the CS/PVA/βGP-MgO@PEI-s implant achieves higher maximum failure load and tensile strength,indicating better load-bearing capacity.Overall,the CS/PVA/βGP-MgO@PEI-s group serves as a long-term,stable nutrient supplier and microenvironment for tendon and bone repair,regulating inflammation and promoting tendon-bone healing.ConclusionThis study amalgamated a Mg and P-loaded CS/PVA hydrogel with sulfonated 3D printed porous PEI implants to engineer an asymmetrically designed implant aimed at fostering tendon-bone healing.The asymmetrical configuration provides tailored mechanical environments conducive to the growth of both soft and hard tissues,while the sustained release of bioactive ions such as Mg and P from the implant ensures a prolonged and stable nutritional supply for effective tendon-bone repair and integration.Moreover,the anti-inflammatory properties and vascular reconstruction effects attributed to Mg ions establish an optimal microenvironment for tissue regeneration.These factors collectively enhance the load-bearing capacity of the transplanted tendons.Overall,this research offers valuable insights into implant materials for tendon and ligament reconstruction,providing new avenues for clinical application and theoretical research in tendon-bone healing. |
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