Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide

Abstract

BACKGROUND: Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties. OBJECTIVE: The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37). METHODS: Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis. RESULTS: We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days. CONCLUSIONS: Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.

Advancements and Challenges in Hydrogel Engineering for Regenerative Medicine

“…Extracellular matrix (ECM)-derived and decellularized hydrogels, utilizing materials from sources like the porcine urinary bladder (pUBM) [9,49] and decellularized tissues such as the small intestine submucosa (SISMA) [22], offer a biomimetic environment conducive to cell attachment, proliferation, and differentiation.…”

“…Hydrogels are increasingly tailored to modulate cellular behaviors and facilitate the regeneration of specific tissues. This customization is achieved by incorporating bioactive molecules such as bioligands [29], growth factors [42,54], and bioactive peptides [49,55], which trigger specific cellular responses conducive to processes like osteogenesis, angiogenesis [8], and cell differentiation [35,56,57]. Such strategic incorporations are designed to meet the regenerative requirements of targeted tissues.…”

“…Research into various crosslinking methods for chitosan/gelatin/egg-white composite hydrogels has identified TPP crosslinking as particularly effective in enhancing their physical and biological properties, fostering new vessel growth and angiogenesis [17]. The biocompatibility and angiogenic potential of extracellular matrix hydrogels from the porcine urinary bladder, biofunctionalized with the LL-37 peptide, have been evaluated, showing the capacity of these hydrogels to induce angiogenesis in vivo without adversely affecting cell proliferation, thus demonstrating their suitability for tissue regeneration therapies [49].…”