In vitro and in vivo biocompatibility and inflammation response of methacrylated and maleated hyaluronic acid for wound healing

Abstract

Over the past few years, different in vitro and in vivo studies have been highlighting the great potentiality of hyaluronic acid (HA) as a biomaterial in wound healing treatment thanks to its good capability to induce mesenchymal and epithelial cell growth and differentiation, angiogenesis, and collagen deposition. However, the need to improve its mechanical properties as well as its residence time has led scientists to study new functionalization strategies. In this work, chemically modified HA-based hydrogels were obtained by methacrylic and maleic functionalization. Methacrylated (MEHA) and maleated HA (MAHA) hydrogels have shown important physico-chemical properties. The present study provides a deeper insight into the biocompatibility of both synthesized materials and their effects on tissue inflammation using in vitro and in vivo models. To this aim, different cell lines involved in wound healing, human dermal fibroblasts, human adipose-derived stem cells and human umbilical vein endothelial cells, were seeded on MEHA and MAHA hydrogels. Furthermore, an inflammation study was carried out on a murine macrophage cell line to assess the effects of both hydrogels on inflammatory and anti-inflammatory interleukin production. The results showed that both MAHA and MEHA supported cell proliferation with anti-inflammation ability as highlighted by the increased levels of IL-10 (57.92 ± 9.87 pg mL^-1 and 68.08 ± 13.94 pg mL^-1, for MEHA and MAHA, respectively). To investigate the inflammatory response at tissue/implant interfaces, an in vivo study was also performed by subcutaneous implantation of the materials in BALB/c mice for up to 28 days. In these analyses, no significant chronic inflammation reaction was demonstrated in either MEHA or MAHA in the long-term implantation.

Fig. 1. From synthesis to MAHA and MEHA photocrosslinked patches: (a) synthesis of MEHA and MAHA; (b) ¹H NMR of synthesized materials; (c) photocrosslinking procedure by UV light; (d), SEM images of photocrosslinked and dried samples (scale bars: left – 1 mm, right – 300 μm).

Fig. 2. Swelling and degradation behavior of MEHA and MAHA at different time points until 14 days in physiological conditions. “Q” is expressed as mean value ± standard deviation (n = 3).

Fig. 3. Cell proliferation investigation of HUVEC (a), HDF (b) and HAD-MSC (c) seeded on MEHA and MAHA at long time (1, 3 and 7 days). Cell proliferation was performed by CCK8 assay using manufacturer's protocol. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, no significant difference.

Fig. 4. Effect of MEHA and MAHA hydrogels on RAW 264.7 in terms of: (a) and (b) cell proliferation after 1, 3 and 7 days of cell culture. Macrophage proliferation was performed by CCK8 assay using manufacturer's protocol; (c) and (d) interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10) levels in basal condition. For (c) and (d), measurements were performed after 3 days of cell–material interaction and results (expressed as picograms per mL) are reported as mean ± standard deviation of 4 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, no significant difference.

Fig. 5. Appearance of the skin in the implantation site. No inflammatory reactions including erythema, swollen, infiltration and blister were observed during the 28 days of implantation. Images were obtained by means of Canon camera at different time points. Black arrows indicate MAHA or MEHA implantation site. Scale bar: 8 mm.

Fig. 6. H&E staining of tissues surrounding different implants at day 1, 3, 7, 14, 28 after implantation. MAHA and MEHA were observed in the implantation site, indicated by black arrows. Scale bar: 200 μm.

Fig. 7. Immunohistochemical staining of tissues surrounding different implants at day 1, 3, 7, 14, 28 after implantation. (a) Qualitative evaluation, scale bar: 200 μm; (b) magnification of the IHC image, scale bar: 40 μm; (c) quantitative assessment of CD11b+ cell density around the implants. “&” indicates the implantation area. “#” indicates cell number. *, p < 0.05; **, p < 0.01; ns, no significant difference.

Fig. 8. Blood vessel quantification in tissues surrounding different implants at day 1, 3, 7, 14, 28 after implantation. (a) Qualitative evaluation; (b) and (c) quantitative assessment of blood vessels around the implants. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, no significant difference. Black arrows indicate blood vessel. Scale bar: 100 μm.