Citrate-Based Biphasic Scaffolds For The Repair Of Large Segmental Bone Defects

BackgroundCurrent challenges in the management of large segmental bone defects have driven research in the field of skeletal tissue engineering in the direction of creating biologically inspired substitutes to replicate the anisotropy, nonlinearity, and local mechanical properties of the native bone tissue[15-17].Bone is a relativelyrigid and lightweight organ optimized to withstand external loads through its unique tissue architecture, which is comprised of a bimodal distribution of highly porous cancellous bone surrounded by a dense layer of cancellous bone[18,19]. The elucidation of this natural structure has provided insight into the design of biomimetic alternatives focused on tissue functionality[17].For example, various multiphasic and gradient porosity scaffold design strategies have been introduced to mimic the stratified native architecture of bone and provide superior mechanical and biological properties over conventional scaffolds of uniform porosity[18,20]21].Thus, bysimultaneously replicating the respective porosities of dense cortical bone and open network of cancellous bonewithin a single construct, novel tissue engineered scaffoldscan be produced to not only provide long-term regenerative capabilities, but also immediate structural and load bearing support[17]. In addition to recreating the native tissue architectures, selecting the appropriate biomaterial for scaffold fabrication is equally important in the success of an orthopedic tissue-engineered device[22]. Huge efforts have been devoted to the hybridization of biodegradable polymers and inorganic bioceramics to improve the mechanical properties and bioactivity of each component for orthopedic applications[23]. Although promising, many of the previous composite materials are unable to match the native bone composition, provide adequate mechanical strength, minimize inflammatory responses, promote bone regeneration, and fully integrate with the surrounding tissue[24]. For example, many of the current materials are limitedby the total amount of bioceramic that can be incorporated into the composite before becoming too brittle, which ultimately limits their osteogenic potential in load bearing applications[25].Therefore, carefully selecting the candidate polymers to composite with bioceramics at the molecular level may constitute a significant aspect in bone biomaterial design.Designed to address the limitations of the previous materials,citrate-based hydroxyapatite composites are a new class of orthopedic biomaterial, which offer numerous advantages for orthopedic tissue engineering[25-29]. Citrate, a historically known metabolic byproduct of the Kreb’s cycle, is naturally abundant in bone tissue and has been recently found to be a bound and integral part of apatite nanocrystal structure playing important roles in bone formation, stability, strength, and maintenance[30-31]. When incorporated into biomaterial design, citrate provides valuable pendant carboxyl chemistry, which improveshydroxyapatite (HA) calcium chelation and allows for the ability to incorporate up to65wt.-%HAin the composite for improvedosteoconductivity and osteointegration[18].Previous citrate-based composites are able to inducerapid mineralization in vitro, up regulate alkaline phosphatase (ALP) and osterix(OSX) gene expression, accelerate osteoblast phenotype progression, and promote osteoconductivity and osteointegrationin vivofrom their ability to better replicate the native bone citrate and inorganic mineral content[25,27,29].Due to these multiple benefits, we believe that citrate should be considered in orthopedic biomaterial and scaffold design.We have recently developed a clickable biodegradable citrate-based elastomer, poly (octanediol citrate)-click (POC-Click), which employsazide-alkyne cycloaddition (click chemistry) as an additional crosslinking mechanism to improve the mechanical strength of the bulkmaterial without sacrificing valuable pendant citric acid carboxyl chemistry for hydroxyapatite (HA) calcium chelation [32]. In this study, biomimetic POC-Click-HA biphasic scaffolds were developedto simulate both the architectural and compositional properties of native bone tissue in order to provide the necessary mechanical properties and porosity. It is hypothesized that a citrate-based hydroxyapatite composite can provide an osteoconductive surface for bone regeneration and tissue integration, while the biphasic scaffold design can mimic the hierarchical organization of cancellous and cortical bone. A scaffold with this type of architecture can potentially provide the necessary porosity in the internal phase for tissue ingrowth with reduced porosity in the external phase to meet the immediate mechanical demands for the repair of large segmental bone defects[18] POC-Click-HA scaffolds were fabricated and characterized for their resulting geometries, mechanical properties, and efficacy in a10mm segmental rabbit radius long bone defect model[32,33].ObjectivesThe elucidation of native tissue architectures has lead to the design of biomimetic scaffolds with a specialfocus on functionality. In this study, biomimetic citrate-based poly (octanediol citrate)-click hydroxyapatite (POC-Click-HA) biphasic scaffolds were developed to simulate both the compositional and architectural properties of native bone tissue and provide immediate structural support for large segmental defects following implantation.MethodsBiphasic scaffolds were fabricated with70%internal phase porosity and various external phase porosities of5-50%to mimic the bimodal distribution of cancellous and cortical bone, respectively. POC-Click-HA biphasic scaffolds displayed compressive strengths up to37.45±3.83MPa, which could be controlled through the external phase porosity. The biphasic scaffolds were also evaluated in vivo for the repair of10mm segmental radius defects inrabbitsand compared to scaffolds of uniform porosity as well as autologous bone graftsafter5,10, and15weeks of implantation.Resultsall POC-Click-HA scaffolds exhibit good biocompatibility and extensive osteointegration with host bone. Biphasic scaffolds significantly enhanced the efficiency of new bone formation with higher bone densitiesin the initial stages after implantation.Biomechanical and histomorphometric analysis supported a similar outcome with biphasic scaffolds initially providing increased compression strength, interfacial bone ingrowth, and perio steal remodeling in early time points, but were comparable to all experimental groups by15weeks.ConclutionThese results confirm the ability ofbiphasic scaffold architecturesto restore bone tissue and physiological functions in the early stages of recovery, and the potential of citrate-based biomaterials in orthopedic applications.