Differentiation of human bone marrow mesenchymal stem cells grown in terpolyesters of 3-hydroxyalkanoates scaffolds into nerve cells

Abstract

Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to their suitable mechanical properties, blood and tissue tolerance and in vivo biodegradability. As a new member of PHA family, terpolyester of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate, abbreviated as PHBVHHx, was compared with polylactic acid (PLA), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) for their respective functions leading to differentiation of human bone marrow mesenchymal stem cell (hBMSC) into nerve cells. Results indicated that 3D scaffolds promoted the differentiation of hBMSC into nerve cells more intensively compared with 2D films. Smaller pore sizes of scaffolds increased differentiation of hBMSC into nerve cells, whereas decreased cell proliferation. PHBVHHx scaffolds with pore sizes of 30–60 μm could be used in nerve tissue engineering for treatment of nerve injury. The above results were supported by scanning electron microscope (SEM) and confocal microscopy observation on attachment and growth of hBMSCs on PLA, PHBHHx and PHBVHHx, and by CCK-8 evaluation of cell proliferation. In addition, expressions of nerve markers nestin, GFAP and β-III tubulin of nerve cells differentiated from hBMSC grown in PHBVHHx scaffolds were confirmed by real-time PCR.

Introduction

The gold standard for treatment of nerve defects is to bridge the peripheral nerve gap with an autograft [1]. However, limitations still exist in these repairs of nerve injuries, partly because of the limited availability of donor tissues and partly due to the severity of the local pain suffered at the donor operative site [2]. Despite the good surgical advances, functional recovery was often poor [3], [4]. The development of biomaterials for stem cell carriers has been regarded as a useful source of alternative tissue equivalents that provide a favorable microenvironment for tissue regeneration [5]. Tissue engineering techniques which enhance the beneficial endogenous responses to nerve injury could provide an alternative repair strategy [3].

Human bone marrow stromal cells (hBMSC) can continuously self-renew and differentiate into nerve cells in vitro under certain conditions [6]. There are also many evidences showing that hBMSC may be non-immunogenic or hypo-immunogenic [7]. hBMSC transplanted at sites of nerve injury are thought to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons [8]. Many researchers have attempted to regenerate nerve tissue by combining suitable biomaterials with hBMSC [9]. Artificial nerve scaffolds with cell and tissue compatibility have been used as carriers of cells to improve regeneration for nerve injury repair [10], [11], [12], [13], [14], [15]. In the last few years, various biodegradable and non-biodegradable scaffolds have been tested using different experimental models of nerve injury [12], [13]. In rat hemi-section spinal cord injury model, PLGA scaffolds with neural stem cell were implanted into the injured site, resulted in a functional improvement. Tissue loss and glial scarring were reduced by transplantation [14].

PHA, with their biocompatibility, biodegradability and strong mechanical properties, have been widely investigated for tissue engineering applications. Previous studies showed that PHB and PHBHHx could be used as potential candidate materials for peripheral nerve tissue engineering [15], [16]. Novikova et al. demonstrated that a PHB scaffold promoted attachment, proliferation and survival of adult Schwann cells, and supported marked axonal regeneration within the graft [16]. An ideal scaffold should have a high affinity for cells to attach and proliferate, should be compatible to in vivo tissues and blood, should have durable strength. PHBHHx appears to be a material meeting these requirements [17]. Similar to PHBHHx, PHBVHHx is a new member of PHA family, our previous study showed that PHBVHHx had better biocompatibility compared with tissue cuture plates (TCP), PLA and PHBHHx [18]. Based on this finding, it had become interesting to investigate the possibility of PHBVHHx for hBMSC proliferation and differentiation.

Scaffold spatial structures have been shown to have effects on cell proliferation and differentiation in 3D directions [19]. In this study, porous PHBVHHx scaffolds intended for nerve tissue engineering were fabricated using thermally induced phase separation (TIPS), and these scaffolds with different pore sizes were studied for hBMSC proliferation and differentiation.