Osteogenic Differentiation Of IPSC-MSCs On The Biomimetic Nanofibrous Scaffolds Of HAp/Col/CTS

In the field of tissue engineering and regenerative medicine (TERM), a combination ofbiomimetic nanofibrous scaffolds with renewable stem cells has recently emerged as a newstrategy for the regenerative repair of damaged tissues or organs. In this context, the mainobjective of this thesis is to examine the osteogenic differentiation capacity of iPSC-MSCscultured on a biomimetic nanofibrous scaffold of hydroxyapatite/collagen/chitosan (HAp/Col/CTS)in vitro, and the efficacy of using the cell-scaffold constructs of iPSC-MSCs seeded HAp/Col/CTSto repair and regenerate bone defects in a rat skull model in vivo.As a kind of new stem cells, induced pluripotent stem cells (iPSCs) generated from somaticcells by reprogramming have received great attention in the TERM community because of theirself-renewal capacity and pluripotent differentiation potential. iPSCs are known to possess theadvantages of homology and circumventing the ethical issues with respect to embryonic stem cells(ESCs); and can be abundantly obtained with ease and less pain for patients compared withmesenchymal stem cells (MSCs). In terms of iPSCs-derived MSCs (i.e., iPSC-MSCs), they havethe merits of high proliferation ability, expressing the MSCs maker genes able to be directedtowards the typical three-lineage differentiations, and controllable differentiation directions withreduced tumourgenicity contrast to the iPSCs. Therefore, iPSC-MSCs have been considered to bea promising seed-cell source for bone tissue engineering (BTE). However, how to control thedifferentiation of iPSC-MSCs into functional osteoblasts is a critical issue to be addressed. In viewof the fact that naturally it is the reciprocal interactions between stem cells and their immediatevicinity (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 arational and promising approach to manipulate the differentiation of iPSC-MSCs.In this study, HAp/Col/CTS nanofibrous scaffolds are used to mimic the natural bone interms of composition and nanostructure. In this scaffold, HAp as the main natural inorganiccomponent of bone can promote bone tissue repair because of its osteoconductive andosteoinductive properties. Biologically compatible Col is the major organic constituent of bone matrix. It can be combined with HAp at the nano-dimension to form the basic building blocks ofHAp/Col composite nanofibers, from which hierarchically organized bone structure are assembled.CTS, a kind of polysaccharides with its chemical structure similar to that of theglycosaminoglycans (GAGs) found in the native extracellular matrix (ECM), has tunabledegradation rate by varying the acetyl content, which helps offset the uncontrollable property ofCol in degradation. Thus, formulating the components of HAp/Col/CTS into nanofiber form canachieve high level of biomimicking to the natural bone matrix, attractive for engineering bonetissue. We therefore hypothesize that the biomimetic scaffold of HAp/Col/CTS will promote theadhesion, growth and osteogenic differentiation of iPSC-MSCs.First of all, HAp/CTS nanocomposites were synthesized using a well-establishedco-precipitation method. HAp/Col/CTS nanofibers could be subsequently fabricated byelectrospinning a collagen (15%, relative to the CTS content) and PEO (15%, relative to the CTScontent) doped solution of the HAp/CTS hybrid dissolved in diluted acetic acid. Throughelectrospinning performed at ambient conditions of2530%humidity at3035C, HAp/Col/CTSnanofibers with a diameter of190-230nm and relatively uniform distribution of HApnanoparticles within the biopolymeric matrix of CTS were obtained. Due to the good interactionsbetween the constituents, both thermal stability and mechanical performance were enhanced. Itwas found that compared to collagen, the decomposition temperature of electrospunHAp/Col/CTS increased to235C from172C of the pure collagen, and tensile strengthincreased to2.54MPa. These properties generally suggest its suitability for bone tissuescaffolding.Secondly, iPSC-MSCs derived from the adherent culture of embryoid bodies were preparedand characterized. It was found that some representative pluripotent genes of iPSC-MSCs, e.g.,Nanog, Oct3/4, Sox2, were kept on a flat low level from passage4thonwards, which ensures safetyand high proliferation ability of the iPSC-MSCs. Meanwhile, the markers of MSCs, CD90, CD29,CD44reached up to high levels of99.62%,99.14%,86.12%, respectively. However, the marker ofhematopoietic stem cell, CD45, was as low as0.29%. These results demonstrated that iPSC-MSCspossessed not only the characteristics of iPSCs such as high homology, abundance in availability,potent differentiation in vitro, but also the attributes of MSCs in terms of safety, mesodermdifferentiation, and osteogenic differentiation. Thereby, iPSC-MSCs can be used as a kind ofexcellent seed cells in our current bone tissue engineering research.Then, iPSC-MSCs were cultured on nanofibrous scaffolds of CTS, HAp/CTS, andHAp/Col/CTS and the tissue culture plate (TCP) to examine their adhesion, growth, migration andosteogenic differentiation behaviours. The results showed that nanofibrous scaffolds ofHAp/Col/CTS had excellent cellular affinity and cytocompatibility by supporting the iPSC-MSCs to spread and grow well. The expressions of bone-associated genes including Runx2, Ocn, Alp andCol, were significantly upregulated. For instance, it is noted that the expression of Runx2onHAp/Col/CTS nanofibers was3.82,3.29, and2.67-fold higher than that on TCP, nanofibrous CTSand HAp/CTS, respectively. Meanwhile, high positive rate of marker proteins OCN, RUNX2andover expression of secretory proteins ALP, Col all confirmed that nanofibrous HAp/Col/CTSscaffold significantly promoted iPSC-MSCs differentiation toward osteogenic lineage.Lastly, mouse cranial defects were created to investigate the efficacy of using theiPSC-MSCs combined nanofibrous HAp/Col/CTS scaffold for regenerative repair in vivo.Examinations by dual-source computed tomography (CT) imaging, bone mineral density andhematoxylin and eosin (HE) staining demonstrated that the cell-scaffold construct ofiPSC-MSCs/HAp/Col/CTS could effectively promote bone tissue regeneration. After6weeks ofimplantation, the bone mineral density of iPSC-MSCs/HAp/Col/CTS group was found to be3.35,2.91,2.88, and2.14-fold higher than that of the blank, CTS, HAp/CTS, and HAp/Col/CTS groups,respectively. The animal test results obtained thus suggest the great potential of using theiPSC-MSCs/HAp/Col/CTS for achieving optimal outcome in bone defect repairs.Taken together, iPSCs have been speculated to be advantagous and of great potential forrealizing personalized treatment in future. In this study, for the first time we investigated theosteogenic differentiation of iPSC-MSCs on the biomimetic nanofiberous scaffolds ofHAp/Col/CTS. The results showed that HAp/Col/CTS nanofibers could significantly induce theiPSC-MSCs to differentiate into osteogenic lineage by promoting the functional expression in thecells and enhancing repairing efficacy in the mouse cranial defects. The newly developedcell-scaffold system could be used as a new candidate in the future BTE research and applications.