Differentiation of Bone Marrow Derived Mesenchymal Stem Cells (BM-MSCs) Using Engineered Nanofiber Substrates

Abstract

Bone loss caused by trauma or disease often renders the use of bone graft materials to facilitate bone healing. Autografts are still the gold standard treatment option. However, there are several drawbacks that are associated with the use of autografts such as patient site morbidity and limited availability of healthy bone especially in younger patients and elderly patients who might suffer from osteoporosis. As such, the use of bone graft substitutes (such as coralline hydroxyapatite (HA), bioglass, calcium-based materials etc.) provides an attractive alternative. However, such materials usually act as passive scaffolding, leading to the lack of bone remodeling. A myriad of research has focused on developing tissue-engineered bone grafts with the aim to replace the use of autologous bone grafts and improving on the clinical performance of the current state of bone graft substitutes. In this project, it was hypothesized that biomimetic mineralized nanofibrous scaffolds (NFS) mimicking natural extracellular matrix (ECM) provided efficient cell attachment and enhanced osteogenic differentiation to promote bone regeneration.

Structurally, bone encompasses fiber bundles that are made up of collagenous nanofibrils laced with HA nanocrystals. Electrospinning was used to produce the NFS to mimic the structure of the bone nanofibrils. The choice polymer used to fabricate the NFS was poly-l-lactide acid (PLLA). A biomimetic approach of nano-hydroxyapatite (n-HA) mineralization was employed on the NFS to attempt to mimic the native ECM in bone. Co-blending type I collagen (Col) with PLLA had shown to enhance n-HA deposition, due to the presence of nucleation sites for n-HA mineralization. Such rapid nVII HA deposition was achieved at room temperature. It was demonstrated that mineralized NFS enhanced early cell capture of osteoblasts within 30 minutes. The osteogenic differentiation potential of bone marrow derived mesenchymal stem cells (BM-MSCs) was achieved by manipulating the physical, biochemical and environmental conditions. The nanoscale topography on the mineralized NFS was able to stimulate osteogenic BM-MSC differentiation without the use of any osteogenic supplements. Cell mineralization, a late osteogenic differentiation marker, which usually occurred on day 28 in culture, was seen after 14 days on mineralized NFS, where the BM-MSCs secreted bone nodules. The Ca/P ratio of the bone nodules was comparable to that of native HA in bone. Since cells are subjected to different nanotextures within a 3D ECM niche in vivo, 3D NFS (nanoyarns) can be an effective carrier for rapid cell capture, which can provide an in-situ therapeutic bone graft option for bone regeneration. Mineralized nanoyarns were enriched with bone marrow aspirate and the cell capture rate was 80%. Biomimetic mineralized nanoyarns could augment bone healing due to its high resemblance to the native bone fibrils as seen in a rabbit model. Speckles of bone was observed within the defect site, suggesting that the presence of n-HA alone could elicit an osteoinductive bone formation process. Therefore, this study suggested that there was great potential for NFS to become efficient bone grafts.