Osteogenic Differentiation Of MMSCs On The Biomimetic Nanofibrous Scaffolds Of Hydroxyapatite/Chitosan

In recent years, there has been increasing interest in the restoration of damaged bone tissue architecture by transplantation of cells combined with a suitable three-dimensional scaffold. Considering their many merits such as ease of isolation, high proliferation capability, multilineage differentiation potential, and lower immunogenicity, mesenchymal stem cells (MSCs) have gained immense interest and are generally considered to be a highly promising cell source for bone tissue engineering applications. Nevertheless, how to precisely regulate and control MSC differentiation toward specific cell type for desired tissue development both in vitro and in vivo remains an intensively investigated subject in the field of stem cells and tissue engineering. In view of the fact that naturally it is the reciprocal interactions between stem cells and their immediate vicinity (or microenvironment) that essentially direct and coordinate a variety of cellular processes, designing new artificial matrices or scaffolds that can mimic the stem cell living niche would be a rational and promising approach to manipulate the differentiation of stem cells.Natral bone, at nanoscale of its hierarchical organization, is made up of platelet-like HAp nanocrystal-incorporated collagen nanofibers. Thus, in the past few years, electrospun inorganic-organic biocomposite nanofibers, capable of compositionally and structurally emulating the basic building blocks of those natural mineralized collagen nanofibers, has received increasing attention in the past few years. Many studies have shown that electrospun biomimetic composite nanofibers can manipulate and promote the differentiation of stem cells. Chitosan (CTS), as a natural biopolymer with many merits, has been one of the most intensively investigated scaffolding matrices in bone tissue engineering. Although it was previously shown that electrospun chitosan-based hydroxyapatite composite nanofibrous scaffolds are capable of promoting proliferation and biological mineralization of the seeded human fetal osteoblasts, relatively little is known whether HAp/CTS nanofibrous scaffolds could accommodate continuous differentiation of MSCs into osteoblasts. The aim of the present work was therefore to design and fabricate biomimetic and bioactive nanofibrous scaffold of HAp/CTS and to study its effects on the differentiation of mMSCs into osteoblasts, in the absence and presence of osteogenic supplementation, respectively.We firstly prepared HAp/CTS nanocomposites containing different percents of HAp nanoparticles by a co-precipitation method. SEM observation showed homogeneous dispersion of nano HAp within the CTS matrice. FTIR analysis indicated there existed specific interactions between the organic and inorganic phases. XRD patterns present the crystalline characteristics of the constituents HAp and CTS, and the characteristic peak of HAp became attenuated with decreasing the HAp loading ratios, partially suggesting molecular interactions between HAp and CTS. TG analysis revealed that thermal stability of HAp/CTS nanocomposites rised by 30℃and loss of the sample weight increased with increasing CTS content.Next, we performed electrospinning to make HAp/CTS composites nanofibers containing different HAp contents. Through a great deal of try-and-error and optimization, continuous and geometrically fairly uniform HAp/CTS nanofibers loaded with 15% ultrahigh molecular weight polyethylene oxide (UHMWPEO) could be gracely electrospun if the ambient humidity is in a range of 22-25%. Both the FTIR and XRD analysis again indicated the interaction between HAp and CTS within the composite. Likewise, thermal analysis showed that thermal stability of HAp/CTS composite nanofibers rised by 30℃compared to the pure CTS.Lastly, an mMSC cell line, C3H10T1/2, was used to study influence of HAp/CTS nanofibers on the growth and osteogenic differentiation of the stem cells. SEM and proliferation results showed that cells on the HAp/CTS composite nanofibers exhibited consistently better attachment and growth than that of CTS nanofibrous scaffolds. In the absence of osteogenic supplementation, expression of all the tested series of bone-associated genes (i.e., ColⅠ, Runx2, ALP and OCN) and proteins confirmed that the HAp/CTS composite nanofibers significantly promoted mMSCs differentiation toward osteogenic lineage. Furthermore, we found that addition of biochemical cue of osteogenic factors could further enhance the ostegenic differentiation capability of mMSCs.In summary, we have demonstrated that electrospun biomimetic nanofibrous scaffolds of HAp/CTS significantly promoted growth and differentiation of the mMSCs toward osteogenic lineage even in the absence of osteogenic supplementation. The performance of biomimetic HAp/CTS nanofibrous scaffolds warrants future work aimed at in vivo level assessment of osteogenesis and osseointegration. Current results also provided further evidence of the potential in using the biomimetic nanofibrous HAp/CTS scaffolds for tissue engineering and regenerative therapies of bone.