In situ conjugation-co-fabrication of bioarchetypes employing 3D print processing

Abstract

Bone tissue has the natural ability to self-regenerate or self-heal, however, large-size (critical-sized) bone defects do not have the ability to self-heal to completion without external intervention. Traditional repair treatments for critical-sized or non-union bone defects have been centered on orthopaedic implants, allografts, and autografts. However, these techniques suffer from a number of limitations such as lack of available donors and inadequate biomechanical properties. A constructive development of novel fabrication methods and the design of novel composite 3D printed biomaterial scaffolds will address the challenge of complete healing of critical-sized bone defects. Furthermore, 3D printing technology have emerged as an innovative tool, with great potential in designing and fabrication of novel composite 3D biomaterial scaffolds. Recently, there has been a huge interest in 3D printing biomaterial scaffolds for bone tissue engineering application. A number of reinforced multi-biomaterials 3D printed scaffolds have been developed and investigated as a key solution for the mechanical and biological properties. But there is still a huge gap for appropriate composite biomaterials that will yield composite 3D biomaterial scaffolds with acceptable mechanical and biological properties for bone tissue engineering application. Developing novel fabrication methods, which will increase the limited printable biomaterial, yielding 3D printed biomaterial scaffolds for bone tissue engineering application, will be of great advantage in bone tissue engineering research and be a solution in the healing of critical-sized bone defect. The use of single-component biomaterials independently poses limitations in their properties as scaffolds for bone tissue engineering application. In this research, a composite 3D printed biomaterial scaffold was fabricated with desirable mechanical and biological properties for bone tissue application. This 3D biomaterial scaffold was engineered by modifying sodium alginate (NaAlg) with silica gel (Si), through in situ conjugated with polyethylenimine (PEI) yielding a composite 3D printed biomaterial scaffold to regulate the regeneration of new bone tissue formation. The method of fabrication was based on the novel single step in situ conjugation-co-fabrication of the biomaterial scaffold employing 3D bioprinter technology as an innovative tool. The 3D printed biomaterial scaffold maintained its 3D architecture for the duration of the 28 day degradation investigation, while potentially permitting the infiltration of nutrients, growth factor, and cells, evident by the increased solvent penetration into the scaffold observed via Magnetic Resonance Imaging (MRI) studies. The scaffold porosity and pore size were found to be 60% and 210μm, respectively. Biomechanical evaluation revealed a Young’s modulus of 60MPa highlighting that the scaffold in its current form possesses the mechanical capabilities for certain bone tissue engineering applications. In vitro studies, which include: biomineralization (e.g. Ca-P) and biological parameters (such as cell viability, cell adhesion, and cell proliferation) were investigated and analysed. Osteoblast-like MG63 cells were used and cultured on the novel composite 3D printed biomaterial scaffolds to determine the various biological responses/parameters. Cells-scaffolds were cultured for seven days, thereafter, light microscopy and electron scanning microscopy (SEM) were used to qualitatively examine cell adhesion, cell proliferation and surface morphology; MTS assay was used to quantitatively determine cell viability. Therefore, the novel composite 3D printed biomaterial scaffold exhibited biomineralization (e.g. Ca-P) confirmed through the EDX and FTIR analysis. Light microscopy and scanning electron microscopy (SEM) revealed that the osteoblast-like MG63 cell cultured on the scaffold were clearly attached to its surface and proliferated within the scaffolds pores. Viability of cells cultured on the composite 3D printed biomaterial scaffold and on the control increased over time (P < 0.05), however, at respective time point, cell viability was not significantly different between the two groups (P > 0.05). Therefore, these results suggest that the novel composite 3D printed biomaterial scaffold is a biocompatible material. In vivo studies were performed using New Zealand White Rabbits model to assess new bone formation from the composite 3D printed biomaterial scaffold and control. Therefore, the final composite 3D printed biomaterial scaffold provided a pro-regenerative platform, rich in mechanical, topographical and biological cues and guides the cells towards functional regeneration. Histology results revealed that there was significant new bone formation especially at week 8 in all induced bone defects. It was also noted that the scaffold containing growth factors had complete bone formation (week 8 complete) compared to the scaffold without growth factors and the empty defect (the control). There was no sign of pain and advance tissue reaction, therefore; the composite 3D printed biomaterial scaffold proved to provide a pro-regenerative platform for bone tissue engineering application.