Preparation of an adipogenic hydrogel from subcutaneous adipose tissue

Abstract

The ability to generate controlled amounts of adipose tissue would greatly ease the burden on hospitals for reconstructive surgery. We have previously shown that a tissue engineering chamber containing a vascular pedicle was capable of forming new fat; however, further refinements are required to enhance fat formation. The development and maintenance of engineered adipose tissue requires a suitable source of growth factors and a suitable scaffold. A hydrogel derived from adipose tissue may fulfil this need. Subcutaneous fat was processed into a thermosensitive hydrogel we refer to as adipose-derived matrix (ADM). Protein analysis revealed high levels of basement membrane proteins, collagens and detectable levels of growth factors. Adipose-derived stem cells exposed to this hydrogel differentiated into adipocytes with > 90% efficiency and in vivo testing in rats showed significant signs of adipogenesis after 8 weeks. ADM’s adipogenic properties combined with its simple gelation, relatively long shelf life and its tolerance to multiple freeze–thaw cycles, makes it a promising candidate for adipose engineering applications.

Introduction

Congenital, traumatic or post-surgical deformities such as mastectomy often require restoration of contour, usually involving adipose tissue. Not only does adipose tissue act as a reservoir for lipids, it also provides insulation and physical protection to the underlying tissue. Being vascularized, it makes an excellent graft bed for other tissues and can be used in complex reconstructive scenarios for which no appropriate donor tissue exists. Adipose tissue engineering has recently received much attention as it promises enhanced efficacy, reproducibility and predictability, compared with the contemporary methods used to treat disfiguring contour imperfections. Autologous free fat grafting with processed lipoaspirate has unpredictable results due to post-graft resorption with sometimes as little as 10% of the original fat volume retained [1], [2], [3]. While the use of vascularized fat flaps generally has more favourable results, complications such as flap failure, infections and pulmonary embolisms exist, along with morbidity relating to the donor site [4].

Once removed from their native environments, stem cells differentiate less efficiently [5], [6]. Therefore, when designing a suitable replacement three-dimensional (3-D) scaffold for thick tissues such as fat, it is important to incorporate characteristics of the native cellular environment to maintain optimal tissue development. For adipose tissue engineering, the ideal scaffold would be a self-gelling injectable material capable of inducing adipogenesis. Such a scaffold should contain the necessary extracellular matrix (ECM) components to initiate angiogenesis and subsequently induce resident stem cells to undergo adipogenesis. Of particular importance is the regulatory and structural role of the native extracellular matrix and associated factors [7]. The mechanotransduction between the ECM and cells plays a critical role in the regulation of angiogenesis [8] and in directing cells towards specific differentiation pathways [9]. Not only does the extracellular matrix provide structural support for the cells, it is also a reservoir for tissue-specific growth factors and signalling molecules that an entirely synthetic scaffold lacks.

Chemical crosslinking agents are successful in creating scaffolds from soluble proteins; however, the introduction of artificial linkages risks converting an otherwise native protein into something that may hinder cell infiltration and maturation [10].

Naturally derived scaffolds and hydrogels have been used for some years [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] and have shown great potential in supporting cell growth whilst maintaining their volume. Sharma et al. demonstrated that an adipocyte-derived ECM extract supported hepatocytes with a higher metabolic activity than with Matrigel, a commercially available ECM hydrogel [14], whereas Flynn et al. showed that matrices prepared from acellular placental ECM and hyaluronic acid support the differentiation of adipose-derived stem cells (ADSCs) [18], [19], [20], [21], [22]. In another study, freeze-dried, injectable powders were prepared from human lipoaspirate [11]. This scaffolding material combined with ADSCs resulted in well-vascularized adipose tissue after implantation into nude mice. When prepared as a hydrogel, adipose-derived extracts have been shown to not only support the growth of seeded ADSCs [17], but also showed signs of inducing neoadipogenesis when implanted along the rat epigastric artery vascular pedicle [15]. However, many of the current formulations of adipose-derived hydrogels either require additional steps to initiate gelation or have inconsistent gelation. Many collagen-based extracts require acid solubilization, which may adversely affect some proteins in the extract.

From what can be gathered in the literature, it is clear that adipose-derived products are capable of promoting cellular infiltration and have the potential to help form new vascularized adipose tissue once implanted in animals. In this study our aims were twofold: first, to produce a hydrogel from adipose tissue containing intact, bioactive proteins, displaying consistent gelation under physiological conditions; and second, to determine the adipogenic potential of this product both in vitro and in vivo.