A 3-dimensional biomimetic in-vitro bone model for testing small orthopaedic implants

摘要

Introduction: Bone's extraordinary properties include its ability to heal itself. While small bone defects heal spontaneously, critical size defects may exceed the body's regenerative capabilities. Such defects usually require surgical intervention involving the use of bone substitutes and implants. To date, in-vitro and in-vivo testing of implants remain the gold standard for rigorous mechanical stability and biological safety checks. However, current 2-D in-vitro testing is limited by a lack of dynamic environment and an inability to investigate mechanical strength of attachment between the bone-matrix and implant surface. Also, 3-D in-vivo tests are limited by differences in behaviour and structure of human and animal cells, high costs and difficulty of replicating human aging effects. Therefore, our aim is to develop a biocompatible and osteoconductive PU-HA scaffold with optimal mechanical properties and beneficial bioactivity that enhances in-vitro bone regeneration, to serve as a biomimetic in-vitro bone model for implant testing of small orthopaedic and dental implants. Methods: Using a novel physical mixing particulate leaching technique, scaffolds were fabricated with 15%wt PU dissolved in 70/30 DMF/THF solvent and NaCl particles(~250 urn). Composite scaffolds with micro-hydroxyapatite particles were fabricated in a ratio of 3PU:1 HA. Scaffold morphology, pore size and interconnectivity were accessed with SEM and Micro-CT. Scaffolds were biologically characterized in-vitro with hES-MP progenitor or MLO-A5 osteoblastic cells and assayed over a 56-day period for cell attachment and viability. To investigate bone matrix deposition onto different orthopaedic screws, 10 mm cortex or osteopenia titanium screws were inserted into cylindrical scaffolds on either DayO or Day28 of culture, and cultured for a further 28 days in-vitro. Micro-CT, confocal microscopy, Sinus-Red and Alizarin-Red stains were used to assess extracellular and mineralised matrix deposition at each time-point. For in-vivo studies, scaffolds were implanted in cortical defects created in 24 rabbits, and sacrificed on Day7 and Day45 after implantation. Results and Discussion: The presence of hydroxyl and phosphate bending peaks in PAS-FTIR characterization confirmed the presence of HA in all composite scaffolds. With an average pore size of 211±59μm, PU-only scaffolds had Young's Modulus(E) of 0.543±0.032MPa, in compression. Interestingly, these were significantly higher than mHA composites with E of 0.190± 0.028MPa and pore size of 171±55μm, which contrasts data from literature. Micro-CT, confocal and histological analysis demonstrated that all scaffolds supported cell attachment, proliferation and the deposition of calcified matrix in-vitro by D56 of culture. mHA composites had the highest cell viability and collagen-matrix deposition. This was significantly higher than that of PU-only scaffolds. Additionally, extracellular matrix deposition was observed on inserted metallic screws after D28 of in-vitro culture. This deposition was higher on cortex screws inserted on DO, as compared to those inserted on D28 of culture, and for both DO and D28 in the osteopenia screw-group. After D45 of in-vivo studies, osteoid bone formation was present in all scaffolds, which was only present in composite scaffolds by D7, emphasising the need for HA in bone-matrix formation. Conclusion: We have developed novel PU-HA scaffolds that serve as a biomimetic in-vitro bone model for testing small orthopaedic implants.