Elsevier

Acta Biomaterialia

Volume 9, Issue 11, November 2013, Pages 9012-9026
Acta Biomaterialia

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: In vitro and in vivo studies

https://doi.org/10.1016/j.actbio.2013.06.046Get rights and content

Abstract

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (∼200 μm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress–strain behavior and hysteresis loops during loading–unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.

Introduction

Soft tissue deficiency is a common problem after traumatic injury, tumor ablation, congenital underdevelopment and the natural changes of the aging process. An autologous fat graft is currently considered the gold standard in soft tissue augmentation procedures. The advantages are that the graft is autogenous, eliminating the risk of immunogenic reactions, and the harvest of the fat graft is simple and safe, with minimal complications. However, autologous fat grafts have been found to have a 40–60% resorption rate [1], [2]. Therefore, multiple procedures are required to achieve satisfactory results. Sometimes central necrosis results in microcalcifications that can be palpated and are difficult to differentiate from tumors in the patients. In addition, the donor source is limited in slim patients when a large amount of fat graft is needed. Adipose tissue engineering is a promising method to solve these problems [3]. In tissue engineering, cells can be seeded onto or into an artificial structure that is referred to as the scaffold, which mainly gives mechanical support in the formation of a three-dimensional (3-D) tissue engineered cells/scaffold construct that is subsequently implanted into the host tissue.

Scaffolds have an important role in tissue engineering. For successful tissue engineering, the scaffold must be biodegradable and biocompatible, with a 3-D porous architecture and inter-pore connections and the appropriate mechanical properties similar to those of the reconstructed tissue [4]. Scaffolds must also be produced to allow cell distribution and to direct their growth into the 3-D structure. Several techniques have been developed to produce 3-D porous scaffolds. These techniques include solvent-casting/particulate-leaching, gas foaming, phase separation, electrospinning, melt molding, rapid prototyping and freeze-drying [5]. However, most of those traditional production methods are complex, require specific equipment, use high temperatures or involve hazardous organic solvents.

Cryogelation is a simple approach for producing macroporous scaffolds [6]. This method allows effective control over the pore size by using ice crystals as templates and produces a porous structure without the involvement of organic solvents or any additives during the production process. The method involves the cryogenic treatment of polymeric gel precursors in the moderate freezing, storage (in the frozen state) and subsequent thawing steps. The cryogel matrices are cross-linked at sub-zero temperatures, whereas part of the solution remains unfrozen in a liquid microphase, in which the phases are separated after undergoing chemical reactions [7]. The reactions that occur in the liquid microphase lead to gel formation, with the ice crystal acting as porogen substrates. In thawing the ice crystals, a system of large interconnected pores is formed within the gel. The compulsory displacement of the polymeric precursors into a non-frozen microphase results in the polymer gel being concentrated in the pore walls, which augments the mechanical strength of the cryogel. Wide, interconnected pores form in the cryogel as each ice crystal grows until it contacts adjacent crystals during the freezing of the initial aqueous solution, providing a labyrinthine system of interconnected channels after the frozen sample is thawed. Freezing at moderate temperatures that do not compromise the scaffold’s mechanical properties could offer a more cost-efficient process than freeze-drying [8]. Hence, cryogels have some important general characteristics, including an interconnected, highly porous structure, mechanical stability, elasticity, reversible and very rapid size changes induced by external forces and good swelling in aqueous media [9]. They are very tough and can withstand high levels of deformation, including tensile, compressive or flexural strains [10]. However, all of these properties depend on the materials used.

Gelatin, a partially degraded product of collagen, is believed to have a lower antigenicity than collagen. Moreover, gelatin is not only more economical than collagen, but it also contains amino acid sequences, such as the RGD of collagen, which can enhance cell attachment. Therefore, gelatin has been blended with other natural or synthetic biomaterials to fabricate 3-D scaffolds using various methods for different tissue engineering applications. Gelatin microspheres containing basic fibroblast growth factor were shown to induce preadipocytes to form adipose tissue at the implant site [11]. Macroporous gelatin spheres were reported to be suitable transplantation vehicles and biodegradable scaffolds for the guided regeneration of soft tissues [12]. Gelatin sponges have been used for adipose tissue engineering employing human marrow stromal cells [13], [14].

Hyaluronic acid (HA) is a naturally polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by alternating (1  4) and (1  3) linkages. As an important component of the extracellular matrix (ECM), HA can increase cell attachment and cell migration into the scaffold due to its high water retention and intrinsic swelling property. Therefore, HA has been used alone or cross-linked with other biomaterials for applications in tissue engineering, such as skin, cartilage, bone and fat. Borzacchiello et al. reported using an ester derivative of HA, HYAFF II, as a potential 3-D scaffold for adipose tissue engineering, both in vitro and in vivo [15]. An aminated hyaluronic acid-g-poly(N-isopropylacrylamide) thermosensitive hydrogel was shown to have a 3-D porous structure able to encapsulate adipose-derived stem cells (ADSCs) to form adipose tissue in vivo [16]. Recent studies also demonstrated the utility of HA-based scaffolds in enhancing adipose tissue development in vitro [17], [18] and in vivo [19], [20], [21].

In view of the successful use of gelatin and HA-based scaffolds in adipose tissue engineering, we hypothesized that a cryogel scaffold fabricated from those precursors would be suitable for in vitro and in vivo adipogenesis using ADSCs. To the best of our knowledge, there are no reports discussing the optimal scaffold for adipose tissue engineering from the aspect of its mechanical properties. For adipose tissue engineering, the designed scaffold should not be as hard as bone tissue but must be elastic and capable of withstanding cyclic mechanical strains. We conjectured that gelatin/HA cryogels could withstand static and dynamic deformational loading without cracking or permanent deformation. The characteristics of the gelatin/HA cryogels were analyzed in terms of pore size, porosity, swelling kinetics and mechanical properties using static and dynamic compressive testing. To test the biocompatibility and potential for applications in adipose tissue engineering, the cryogels were seeded with porcine ADSCs to study the cell proliferation, adipogenic gene expression and protein synthesis both in vitro and in vivo using biochemical analyses, quantitative real-time polymerase chain reactions (qRT-PCR) and histology.

Section snippets

Materials

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was obtained from Acros. Hyaluronic acid (sodium salt from Streptococcus equi, molecular weight 1500–1750 kDa), gelatin (type A from porcine skin, 300 bloom), 2-morpholinoethane sulfonic acid (MES) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) were all purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM, Sigma) and fetal bovine serum (FBS, HyClone) were used for cell culture. Rhodamine-phalloidin and the 4′,

Synthesis and characterization of the gelatin/hyaluronic acid cryogels

Initially, different gelatin to HA mass ratios ranging from 1:0.25 to 15:0.25 were used to prepare cryogels with a fixed mass of HA. The selection of the optimum ratio of gelatin to HA was based on the cryogel properties, which should fulfill the minimal criteria set for scaffolds intended for soft tissue engineering. The final gelatin to HA ratio was determined to be between 4:0.25 (GH4) and 10:0.25 (GH10), whereas other combinations did not yield good mechanical or morphological properties.

Discussion

GH cryogels were synthesized using EDC as the cross-linking agent at −20 °C. EDC and glutaraldehyde are two of the most commonly used chemical agents, but they work in distinctly different manners. EDC is limited to cross-link gelatin/HA molecules that are directly adjacent to each other (1 nm), whereas glutaraldehyde can cross-link molecules that are more separated. However, the incorporation of glutaraldehyde into scaffolds can have implications for biocompatibility. EDC, conversely, is known

Conclusion

We demonstrated that macroporous elastic cryogels could be fabricated from gelatin and hyaluronic acid, two biopolymers suitable as adipose tissue engineering scaffolding materials. The GH cryogel is endowed with unique mechanical properties, such as high extensibility, moderate toughness and total recovery from large strains. Furthermore, the superior physico-chemical properties, such as high porosity, large pore size, fast swelling kinetics and high swelling ratio, combined with mechanical

Acknowledgements

This work was supported by Chang Gung Memorial Hospital (CMRPD2C0081) and National Science Council, Taiwan, ROC.

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    These authors contributed equally to this work.

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