Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro
Introduction
Large numbers of plastic and reconstructive surgical procedures are performed every year to repair soft tissue defects that result from deep burns, tumor resections and hereditary and congenital defects such as Romberg's disease and Poland syndrome [1]. Despite the increasing clinical demand, the optimal strategy for the reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery. Adipose tissue engineering strategies have commonly involved the use of seeding preadipocytes on appropriate polymeric scaffolds; however, they are already committed to the adipogenic pathway and have reduced proliferation ability, unpredictable variability based on anatomical sites, and limited availability [2], [3], [4], [5], [6].
Recently, a number of attempts have been made in vitro and in vivo to engineer adipose tissue using mesenchymal stem cells [7], [8], [9], [10]. Stem cells may be ideal for tissue engineering applications due to their ability to divide and renew themselves over long periods of time, to differentiate into various cell types, and their relatively easy isolation and expansion [11], [12]. The capacity of stem cells to differentiate into endothelial cells and adipocytes upon receiving proper stimuli may be promising for developing vascularised fat graft for reconstructive purposes [13]. In particular, endogenous adipose stem cells which can populate a construct and be induced into adipose tissues would have the added advantages of not requiring in vitro expansion and lacking immunological complications. We have developed such a system using vascularised three-dimensional chambers filled with adipo-inductive matrices. Matrigel supplemented with FGF-2 potently induces adipose tissue in a mouse model [14], and that this is dependent on inductive influences of pre-existing fat [15], [16]. We have recently found that when these are seeded with fat xenografts, the new adipose tissue is of host origin [17]. Similarly, Matrigel/FGF-2 has been found to be proadipogenic when injected subcutaneously alone [18] or in conjunction with a gelatin microspheres [19]. In the rat, we have found that devitalised muscle tissue, or a basement membrane rich extract derived from skeletal muscle (Myogel) both cause adipose induction without added cells [20]. We have also found that FGF-2-supplemented collagen supported adipogenesis in the mouse chamber [21], however the resultant construct was smaller than that achieved with Matrigel, presumably due to rapid degradation of the collagen. Since Matrigel is not suitable for human applications, we continue to seek viable alternatives.
In plastic and reconstructive surgery, injectable materials are favoured as they leave no scars from insertion. Pluronic F-127 is a synthetic hydrogel, which is non-toxic, biocompatible, bioabsorbable and is FDA approved for use in humans [22], [23], [24]. Pluronic F-127 has been widely used in drug delivery and controlled release applications [25], [26]. There have been several studies on use of Pluronic F-127 for in vivo tissue engineering (cartilage and lung) [24], [27], [28], [29]. Given our previous success in engineering adipose tissue in vivo using type I collagen gel [21], we proposed to add type I collagen to Pluronic F-127 as a favoured adhesive substrate to make the composite scaffold more cell-interactive. The objective of this study was to demonstrate adipogenic permissiveness of BM-MSC in a 3-D in vitro environment using Pluronic F-127 and the Pluronic F-127/type I collagen blend as hydrogel scaffolds. The adipogenic differentiation was demonstrated by cellular morphology and Oil Red O staining.
Section snippets
Materials
α-MEM, penicillin G and streptomycin sulphate were purchased from Invitrogen (Carlsbad, California, USA). Sodium pyruvate, l-glutamine, dexamethasone, indomethacin, IBMX (3-isobutyl-1-methylxanthine) and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS), pH 7.4 was obtained from Oxoid Ltd. (Hampshire, England). Fetal calf serum (FCS) was obtained from CSL Ltd. (Melbourne, Victoria, Australia). Cell culture-grade Pluronic F-127 and sodium bicarbonate
Results
The bone marrow-derived mesenchymal stem cells (BM-MSC) were homogeneously distributed in the hydrogels [Fig. 1(A) and (C)]. After 20 h, multiple cell aggregates were observed in wells receiving pluronic F-127 treatments [Fig. 1(B)]. This suggested that BM-MSC were able to interact with each other in a three-dimensional (3-D) environment and move throughout the Pluronic F-127 gel. In the blend, a single cluster of aggregated cells entangled within collagen fibrils was observed 20 h after cell
Discussion
Preadipocytes are the most commonly utilised cells for adipose tissue engineering applications, however these cells lose their capacity to differentiate after extensive passaging [30]. BM-MSC can be easily obtained and purified, and provide an unlimited source of cells for tissue engineering applications [13]. Furthermore, the capacity of these cells to differentiate and proliferate is similar for most donors, which makes autologous transplantation possible in most patients [31]. In this study,
Acknowledgement
This study was supported by the Australian National Health and Medical Research Council Grant 299872.
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