Biomimetic Synthetic Tissue Scaffolds for Bone Regeneration: A Dissertation

Injury to bone is one of the most prevalent and costly medical conditions. Clinical treatment of volumetric bone loss or hard-to-heal bony lesions often requires the use of proper bone grafting materials, with or without adjuvant anabolic therapeutics. Despite significant problems associated with autografting (donor site morbidity, limited supplies) and allografting (disease transmissions, high graft failure rates) procedures, synthetic bone grafts remain the least utilized clinically. Existing synthetic orthopaedic biomaterials rarely possess a combination of bone-like structural and biochemical properties required for robust osteointegration, scalable and user-friendly characteristics indispensable for successful clinical translations. This thesis tests the hypothesis that by recapitulating key structural elements and biochemical components of bone in 3- and 2-dimensional biomaterials, scalable synthetic bone grafts can be designed to enable expedited healing of hard-to-heal volumetric bone loss. Specifically, FlexBone, a 3-dimensional hydrogel scaffold encapsulating 50 wt% of structurally well integrated nanocrylstalline hydroxyapatite, the main inorganic component of bone, was developed. The large surface area of nanocrystalline hydroxyapatite combined with its intrinsic affinity to proteins and its excellent structural integration with the hydrogel matrix enabled FlexBone to both sequester endogenous protein signals upon press-fitting into an area of skeletal defect and to deliver exogenous protein therapeutics in a localized and sustained manner. We demonstrated that FlexBone enabled the functional healing of critical-size long bone defects in rats in 8 – 12 weeks with the addition of a very low dose of osteogenic growth factor BMP-2/7. This promising synthetic bone graft is now being explored for the delivery of multiple growth factors to expedite the healing of diabetic bony lesions. In addition, a 2-dimensional electrospun cellulose fibrous mesh was chemically modified with sulfate residues to mimic sulfated polysaccharide ECM components of skeletal tissues to enabled progenitor cell attachment and differentiation as well as controlled retention and localized/sustained delivery of protein therapeutics. This sulfated fibrous mesh is currently explored as synthetic periosteum to augment the osteointegration of devitalized structural allografts. Finally, a rat subcutaneous implantation model developed to examine the biocompatibility of newly developed biodegradable shape memory polymer bone substitutes is also presented

Vancomycin-bearing Synthetic Bone Graft Delivers rhBMP-2 and Promotes Healing of Critical Rat Femoral Segmental Defects

For graft-assisted repair of large volumetric bone loss resulting from traumatic orthopedic injuries, strategies that simultaneously promote osteointegration/graft healing and mitigate risks for infections are highly desired. Previously, we developed a poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) composite as a synthetic bone graft. The composite, when loaded with a single dose of 400-ng rhBMP-2/7 and press-fit into a 5-mm rat femoral segmental defect, led to bony callus fully bridging over the defect and substantial restoration of the torsional rigidity by 12 weeks. More recently, we showed that 4.8 wt% vancomycin can be encapsulated within the composite without compromising the structural and mechanical integrity. Additionally, FDA-approved rhBMP-2 can be absorbed onto the graft and both the vancomycin and rhBMP-2 can be released in a localized and sustained manner. Here we examine the efficacy of pHEMA-nHA-vancomycin grafts pre-absorbed with rhBMP-2 in repairing 5-mm rat femoral segmental defects, and determine if vancomycin hinders the repair. pHEMA-nHA-vancomycin or pHEMA-nHA with/without 3-µg rhBMP-2 were press-fit in 5-mm femoral defects in male rats. Histology, microcomputed tomography, and torsion testing were performed on 12-week explants to evaluate the extent and quality of repair. Partial bridging of the defect with bony callus by 12 weeks was observed with pHEMA-nHA-vancomycin without rhBMP-2 while full bridging with substantially mineralized callus and partial restoration of torsional strength was achieved with 3-µg rhBMP-2. The presence of vancomycin did not significantly compromise graft healing. The pHEMA-nHA-vancomycin graft, with the ability to deliver safe doses of osteogenic recombinant proteins and to simultaneously release the encapsulated antibiotics in a sustained manner holds promise in improving the clinical outcome of graft-assisted repair of traumatic bone injuries

Modulating Mechanical and Shape-Memory Properties while Mitigating Degradation-Induced Inflammation of Polylactides by Pendant Aspirin Incorporation

Synergistically modulating mechanical properties and improving shape-memory performance while mitigating degradation-induced chronic inflammation of polylactide (PLA)-based implants for biomedical applications remain elusive. We test the hypothesis that copolymerizing aspirin-functionalized glycolide with d,l-lactide could enhance the thermal processing, toughness, and shape-memory efficiency of the copolymer while mitigating local inflammatory responses upon its degradation. The content of pendant aspirin was readily modulated by monomer feeds during ring-opening polymerization, and the copolymers with approximately 10% or less aspirin pendants exhibited gigapascal-tensile moduli at body temperature and significantly improved fracture toughness and energy dissipation that positively correlated with the aspirin pendant content. The copolymers also exhibited excellent thermal-healing and shape-memory efficacy, achieving a \u3e 97% temporary shape fixing ratio at room temperature and facile shape recovery at 50-65 degrees C. These drastic improvements were attributed to the dynamic hydrophobic aggregations among aspirin pendants that strengthen glassy-state physical entanglement of PLA while readily dissociating under stress/thermal activation. When subcutaneously implanted, the copolymers mitigated degradation-induced inflammation due to concomitant hydrolytic release of aspirin without suppressing early acute inflammatory responses. The incorporation of aspirin pendants in PLA represents a straightforward and innovative strategy to enhance the toughness, shape-memory performance, and in vivo safety of this important class of thermoplastics for biomedical applications

pHEMA-nHA Encapsulation and Delivery of Vancomycin and rhBMP-2 Enhances its Role as a Bone Graft Substitute

BACKGROUND: Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7). QUESTIONS/PURPOSES: To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin. METHODS: We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively. RESULTS: Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture. CONCLUSIONS: pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses. CLINICAL RELEVANCE: The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk