Design and development of a novel 3D bioprinted bone tissue engineered drug delivery scaffold for the treatment of Bone Fractures

Abstract

Reconstruction of complicated bone defects remain a significant challenge, especially in patients with insufficient horizontal and vertical bone dimensions. Injury, age-related bone defects, and pathological disorders are a few of the most common impairments related to bone fractures. This generally results in an extensive healing time, and in some cases, relapses occur due to the therapy not reaching the specific site of fracture. Although autogenous bone grafts are predominantly deemed the gold standard for bone repair, their use is restricted due to increased donor site morbidity and graft resorption, as well as limited bone availability. Therefore, bone tissue engineered 3D constructs customized for patient-specific needs are being manifested as pseudo-bone scaffolds to improve bone repair and regeneration, as well as to increase bone cell and tissue differentiation. In this study, a 3D bioprinted pseudo-bone drug delivery scaffold was fabricated to display matrix strength, matrix resilience, as well as porous morphology, of healthy human bone. Polymers employed for formulating the 3D scaffold comprised of polypropylene fumarate (PPF), free radical polymerized polyethylene glycol- polycaprolactone (PEG-PCL-PEG) and pluronic (PF127). Simvastatin was incorporated into the 3D bioprinted scaffolds, to further promote bone healing and repair properties. The 3D bioprinted scaffold was characterized for its chemical, morphological, mechanical, in vitro and in vivo properties, for evaluation of its behavior for application as an implantable technology. Computer Aided Design (CAD) software was employed, for developing the 3D bioprinted scaffold. Further optimization of the bioink formulation was undertaken using MATLAB® software employing artificial neural networks (ANN). The 3D bioprinted scaffolds, evaluated as 39 design formulations using MATLAB® software, comprised of variables of PPF (8%w/v – 20%w/v) and PF127 (14%w/v – 16%w/v). These design scaffold formulations were studied in response to duration of release of simvastatin and the degree of thermo-gelation of the bioink formulation. It was observed that with incremental increases in the concentration of PPF at constant PF127 levels, a comparatively greater concentration of simvastatin was released for the formulations. This can be attributed to the increasing incompatibility created by the hydrophobic chain regions of PPF encapsulating simvastatin, a biopharmaceutics classification system (BCS) class 2 drug, at higher concentrations. The ANN-optimized 3D bioprinted scaffold displayed significant properties as a controlled release platform, demonstrating drug release over 20 days. The 3D bioprinted scaffold further displayed formation of a pseudo-bone matrix using a human clavicle bone model, induced with a butterfly fracture. The strength of the pseudo-bone matrix, evaluated for its Matrix Hardness (MH) and Matrix Resilience (MR), was determined to be as strong as healthy human clavicle bones, having a MH of 99% and a MR of 98%. Due to the porous nature of the 3D bioprinted scaffold, the strain distribution experienced by the scaffold ranged up to 13.2% over 60 sec, with a young’s modulus of 26,5 Mpa. Ex-vivo cytology studies determined distinct MG-63 cells with greater density within the 3D scaffold matrix, observing filopodias growth around the entire scaffold architecture. At day-30, it was clearly noticed that cells employed the larger surface area of the scaffold, reaching confluence and covering almost the entire surface of the scaffold. MTT assay for determining cell viability over 30 days, demonstrated a positive cell density after cell seeding occurred. Biodegradation analysis of the 3D bioprinted scaffolds reflected a gradual loss in polymer mass, with an initial 18% loss with 10 days of evaluation. It was also reflected that the scaffold maintained its circular morphology throughout the degradation process, with at least over half of the mass degraded within 20 days. In vivo studies using New Zealand White Rabbits (NZW) confirmed that the nasal bones, after being induced with a nasal fracture and treated with the 3D bioprinted scaffold, demonstrated greater evidence of bone healing and new bone formation. Treatment with simvastatin displayed considerable bone growth and proliferation, compared to unloaded simvastatin 3D bioprinted scaffolds. In comparison to the control defect where no treatment was administered, it was observed that an increased rate of bone healing and regeneration occurred by administration of the 3D bioprinted scaffold. This result was supported by both by X-ray and histological analysis. Evaluation of the nasal bone induced fracture observed after treatment of the statin-loaded 3D bioprinted scaffold after 4 weeks, resulted in sample area of focal nasal bone regeneration, with mild periosteal reaction typical of new bone formation and prominent osteoblastic activity. Furthermore, evaluation of the nasal bone induced fracture observed after treatment of the non-statin-loaded 3D bioprinted scaffold after 4 weeks, reflected focal defects visible in the nasal bone with granulation tissue observed. It can thus be proved that the 3D bioprinted drug delivery scaffold has significant potential for application in bone fracture treatment, with substantial benefits for new growth and repair.