Evaluation of Glycerylphytate Crosslinked Semi- and Interpenetrated Polymer Membranes of Hyaluronic Acid and Chitosan for Tissue Engineering

Abstract

In the present study, semi- and interpenetrated polymer network (IPN) systems based on hyaluronic acid (HA) and chitosan using ionic crosslinking of chitosan with a bioactive crosslinker, glycerylphytate (G₁Phy), and UV irradiation of methacrylate were developed, characterized and evaluated as potential supports for tissue engineering. Semi- and IPN systems showed significant differences between them regarding composition, morphology, and mechanical properties after physicochemical characterization. Dual crosslinking process of IPN systems enhanced HA retention and mechanical properties, providing also flatter and denser surfaces in comparison to semi-IPN membranes. The biological performance was evaluated on primary human mesenchymal stem cells (hMSCs) and the systems revealed no cytotoxic effect. The excellent biocompatibility of the systems was demonstrated by large spreading areas of hMSCs on hydrogel membrane surfaces. Cell proliferation increased over time for all the systems, being significantly enhanced in the semi-IPN, which suggested that these polymeric membranes could be proposed as an effective promoter system of tissue repair. In this sense, the developed crosslinked biomimetic and biodegradable membranes can provide a stable and amenable environment for hMSCs support and growth with potential applications in the biomedical field.

Keywords: chitosan; glycerylphytate; interpenetrated polymer network; mesenchymal stem cell; methacrylated hyaluronic acid; semi-IPN.

Figure 1

Polymer and crosslinker concentrations (wt.%) used for the fabrication of Ch membranes, semi-, and IPNs developed in this work (A); Digital images of the developed systems (B).

Figure 2

SEM micrographs (left) and AFM 3D perspective images with their respective calculated roughness parameters (right) for Ch (A), Ch/HA (B), and Ch/HAMA (C) polymeric membranes.

Figure 3

Evolution of elastic (G’, filled) and viscous (G’’, unfilled) moduli, and loss tangent (tan δ, half-filled) as a function of applied frequency at constant strain of 0.1% of Ch, Ch/HA, and Ch/HAMA polymeric membranes (A). The average mesh size values ξ of Ch, Ch/HA and Ch/HAMA polymeric membranes determined at a frequency of 1 Hz (B).

Figure 4

Effect of hydrogel membrane composition on swelling after incubation in PBS 7.4 at 37 °C for different periods of time (A). Weight loss of Ch, Ch/HA and Ch/HAMA membranes at different time points after soaking in PBS 7.4 at 37 °C under static conditions (B). Data represented the mean ± SD.

Figure 5

Release profiles of G₁Phy from the Ch, Ch/HA and Ch/HAMA membranes in 0.1 M Tris buffer (pH 7.4) at 37 °C. Data represented the mean ± SD.

Figure 6

ESEM images of Ch, Ch/HA and Ch/HAMA hydrogel membranes. Representative ESEM images of hydrogel membranes before hMSCs culture (A). Representative ESEM images of hMSCs growing on hydrogel membranes after 21 days (B).

Figure 7

Cytocompatibility of Ch, Ch/HA and Ch/HAMA hydrogel membranes with hMSCs. Representative confocal images of hMSCs stained with Calcein AM (living cells in green) and ethidium homodimer (dead cell in red) at days 1, 7 and 21 using the Live/Dead^®® assay (A). Cell proliferation at the hydrogel membranes after 1, 5, 7, 14 and 21 days (B). Values are represented as mean ± SD (n = 3) and normalized respect to day 1 values. Two-tailed Student T test analysis were performed for Ch/HA and Ch/HAMA samples with respect to Ch samples at each time at significance level of ** p < 0.01, and for Ch/HA samples with respect to Ch/HAMA samples at each time point at significance level of (## p < 0.01).