A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue

Background

The main goal of tissue engineering is to generate new tissue by providing the body with a scaffold possessing similar properties to those of the target site. This allows for optimal remodeling and enables formation of de novo endogenous tissue. In the field of adipose tissue engineering, biomaterials derived from adipose tissue are of particular interest because adipose tissue is widely available and in theory provides the best possible environment for induction of adipogenesis (Flynn et al., 2007; Flynn, 2010; Uriel et al., 2008; Choi et al., 2009; Young et al., 2011). It has been established that adipocytes secrete adipogenic factors (Li et al., 1998; Shillabeer et al., 1989; Shillabeer et al., 1990) and that conditioned medium produced from either adipocytes or excised adipose tissue is able to induce adipogenesis in vitro (Sarkanen et al., 2012).

The development of an injectable hydrogel with properties closely matching those of healthy adipose tissue would potentially be of great use in regenerative medicine. The ideal hydrogel would be acellular, contain proteins that are representative of those found in natural adipose tissue, be structurally capable of maintaining a space after implantation, and be capable of inducing adipose tissue growth (Cheung et al., 2014; Drury and Mooney, 2003). We have previously reported on the production of a thermoresponsive hydrogel from excised adipose tissue which was shown to be adipogenic both in vitro and in vivo (Poon et al., 2013). The gel induced adipogenic differentiation of adipose-derived stem cells in vitro and was capable of producing adipose tissue from 8 weeks post-implantation in the subcutaneous layer of the rat back (Debels et al., 2015).

As originally reported, our adipose-derived hydrogel used dispase to decellularise the tissue prior to extraction. Although dispase is capable of efficiently decellularising tissue (Uriel et al., 2008; Prasertsung et al., 2008), we have since observed the degree of digestion varies greatly from batch to batch due to differences in tissue surface area. Additionally, the slight differences in decellularisation led to variations in lipid content between batches which altered protein extraction efficiency and clarity of the final product. The washing and delipidation steps were also of concern as they increased variation of the gel’s final physical properties. Since our original publication, we have developed a practical and efficient method to replace these early processes.

Here we provide a detailed protocol for producing a soft tissue-derived hydrogel which addresses many of these concerns and reduces batch-to-batch variability. In our new method, proteins are extracted from the tissue first in order to retain as much soluble protein as possible for subsequent re-addition. Decellularisation by dispase digestion has been replaced with cold homogenisation and nuclease treatment. Dispase is capable of efficiently decellularising tissue; it works by cleaving fibronectin and collagen IV, but there is a problem, these proteins may provide important functional groups which would otherwise be lost after dispase digestion (Gregoire et al., 1998; Khoshnoodi et al., 2008). Delipidation is no longer performed over the course of multiple salt washes and centrifugation steps, now it is done as part of the homogenisation and solubilisation steps. This method has been used to successfully produce thermoresponsive hydrogels from multiple tissues including skeletal muscle and organs such as the liver and heart. These hydrogels containing a collection of soluble proteins present in the native tissues may provide others in the field the basis for further developments in biomaterials research.