Introduction: Components of the extracellular matrix (ECM) may foster the development of bioinspired, functional biomaterials selectively controlling and promoting skin regeneration. Glycosaminoglycans (GAG) including sulfated hyaluronan (sHA) are attractive starting materials for the generation of skin-compatible scaffolds due to their cytocompatibility, binding capacity for mediator proteins and relatively quick biodegradation. However, manufacturing of stable HA hydrogel scaffolds with reproducible properties and interconnected pores is challenging. A promising approach is the preparation of porous hydrogels from crosslinkable GAG polymers. We developed and characterized photo-crosslinked hydrogels made of acrylated HA, acrylated and sulfated HA and collagen and demonstrated the biological compatibility for the culture of primary dermal fibroblasts (Fb) in vitro and in vivo with the perspective to develop a scaffold material for dermal wound treatment.
Materials and Methods: Low molecular weight HA, and sulfated HA (sHA) were acrylated as described by Becher et al[1]. using phase transfer conditions. For hydrogel formation acrylated GAGs, collagen type I and photo-initiator lithium phenyl-2,4,6-trimethyl-benzoylphosphinate solutions were mixed, applied to silicon molds and cross-linked by UV light followed by freeze drying and washing. The resulting hydrogels were characterized regarding their composition, release behavior, morphology, mechanical and swelling properties.
For biological testing the hydrogels were seeded with human foreskin and mouse skin Fb. Attachment, seeding efficiency and survival, gel penetration and matrix deposition were investigated in vitro. Furthermore, pre-seeded and cell-free hydrogels were applied to full thickness wounds of mouse skin. After 7 days the hydrogels and wound tissue were removed and analyzed with respect to cell and matrix content.
Results and Discussion: Hydrogels containing HA or HA combined with sHA as well as fibrillar collagen type I displayed a reproducible stable structure, high porosity after freeze drying with pores larger than 100 µm and good swelling properties adsorbing the maximum of water within 5 minutes. Desorption experiments over 8 days at 37°C proved a small release of sulfated GAGs, while the loss of collagen was higher.
An effective seeding procedure enabling the growth of human Fb for at least 12 days was established. Fb settled the hydrogels completely (up to 600 µm) and deposited their own matrix. The hydrogels enabled cellular survival for up to 7 days when applied to full thickness wounds and either pre-seeded with mouse dermal Fb or used as empty scaffolds, suggesting good biocompatibility in vivo as well.
Conclusions: Hydrogels prepared from acrylated HA, sulfated GAGs and fibrillar collagen are effective substrates for Fb in vitro and in vivo. Cells can growth within these gels, proliferate and deposit their own matrix within the pores. Taken together, HA-and hsHA-based hydrogels are promising biomaterials for the development of skin equivalents incorporating cells, bioactive mediators or modified GAG components.
The authors would like to thank the DFG (TRR 67, subprojects A2, A3, B4, Z3) for financial support.
References:
[1] Becher J, Möller S, Schnabelrauch M. Phase transfer-catalyzed synthesis of highly acrylated hyaluronan. Carbohydr Polym 2013;93:438–41.