3.4.2. Cell Viability and Proliferation Assays

Proliferation of hMSCs cultured in Ch, Ch/HA and Ch/HAMA hydrogel membranes was evaluated with AlamarBlue®® assay at 1, 5, 7, 14 and 21 days of cell culture (Figure 7B). Results demonstrated that cell proliferation increased from day 1 until 21 days in all the systems. No In order to evaluate the feasibility of Ch, Ch/HA and Ch/HAMA hydrogel membranes as an adequate support for cell survival, the viability of the seeded hMSCs on the top of the membranes was

significant differences were found between Ch/HAMA and Ch membranes regarding cell

attachment and proliferation coming from entangled HA and a higher content of G1Phy in the semi-IPN system leading to a higher released concentration as it was observed in the G1Phy release profile (Figure 5), which after being released at short times of incubation could be assimilated by hMSCs, exerting a positive effect on cell adhesion and proliferation as it has been previously observed for similar polymeric systems [25,53]. Correira et al. [46] demonstrated that the biological performance of polysaccharides based systems was affected by their physicochemical factors, which could be tuned by the incorporation of HA to Ch at different proportions. In fact, they found that the addition of HA to Ch scaffolds up to 5% improved both physicochemical and biological properties of Ch scaffolds [46]. Additionally, previous works of the authors demonstrated the benefits of the glycerylphytate crosslinker on cell adhesion and proliferation of 3D scaffolds [25]. In overall, the better biological performance of the semi-IPN system can be attributed on the one hand, to its physicochemical features regarding surface roughness, mechanical properties, approaching those of

evaluated. The live/dead assay was employed to visualize the presence of living and dead cells after 1, 7 and 21 days in the hydrogel membranes (Figure 7A). Confocal images showed hMSCs growing on all the membrane surfaces at days 1 and 7. The number of living cells was much higher at day 21 and cells appeared covering the hydrogel membranes with few dead cells. These results indicated that Ch, Ch/HA and Ch/HAMA hydrogel membranes can provide an amenable environment that supports hMSCs growth and confirmed the cell viability with no cytotoxic effects. systems containing HA for tissue regeneration due to its similarities to ECM composition [27,54]. For example, Pescosolido et al. [54] combined HA with photocrosslinkable dextran to overcome instability problems of HA derived from its high hydrophilicity. The presence of the bioactive HA provided excellent biological properties to their systems. For its part, Skaalure et al. [27] developed a semi-IPN consisting on poly(ethylene glycol) and entrapped HA, whose incorporation clearly led to an enhanced cell adhesion and proliferation.

best candidate to mimic the native tissue ECM. Some authors have claimed the benefits of semi-IPN

*Polymers* **2020**, *12*, x FOR PEER REVIEW 14 of 18

cartilage, and wettability. On the other hand, the enhancement of cell viability and proliferation of Ch/HA sample in comparison to Ch/HAMA system can derived from the presence of linear HA embedded in the semi-IPN and its higher ability to be interchanged with the medium respect to

**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 **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).

Proliferation of hMSCs cultured in Ch, Ch/HA and Ch/HAMA hydrogel membranes was evaluated with AlamarBlue®® assay at 1, 5, 7, 14 and 21 days of cell culture (Figure 7B). Results demonstrated that cell proliferation increased from day 1 until 21 days in all the systems. No significant differences were found between Ch/HAMA and Ch membranes regarding cell proliferation at any time. For its part, Ch/HA demonstrated a significantly enhanced cell proliferation in comparison to Ch at 5, 7, 14 and 21 days, and in comparison to Ch/HAMA system at 7, 14 and 21 days. This result may be due to the supportive microenvironment of semi-IPNs system for cell attachment and proliferation coming from entangled HA and a higher content of G1Phy in the semi-IPN system leading to a higher released concentration as it was observed in the G1Phy release profile (Figure 5), which after being released at short times of incubation could be assimilated by hMSCs, exerting a positive effect on cell adhesion and proliferation as it has been previously observed for similar polymeric systems [25,53]. Correira et al. [46] demonstrated that the biological performance of polysaccharides based systems was affected by their physicochemical factors, which could be tuned by the incorporation of HA to Ch at different proportions. In fact, they found that the addition of HA to Ch scaffolds up to 5% improved both physicochemical and biological properties of Ch scaffolds [46]. Additionally, previous works of the authors demonstrated the benefits of the glycerylphytate crosslinker on cell adhesion and proliferation of 3D scaffolds [25]. In overall, the better biological performance of the semi-IPN system can be attributed on the one hand, to its physicochemical features regarding surface roughness, mechanical properties, approaching those of cartilage, and wettability. On the other hand, the enhancement of cell viability and proliferation of Ch/HA sample in comparison to Ch/HAMA system can derived from the presence of linear HA embedded in the semi-IPN and its higher ability to be interchanged with the medium respect to crosslinked HAMA, which is longer retained in the IPN membrane [36,46], along with the higher content of the bioactive G1Phy for this membrane. Thus, in our study, the semi-IPN highlights as the best candidate to mimic the native tissue ECM. Some authors have claimed the benefits of semi-IPN systems containing HA for tissue regeneration due to its similarities to ECM composition [27,54]. For example, Pescosolido et al. [54] combined HA with photocrosslinkable dextran to overcome instability problems of HA derived from its high hydrophilicity. The presence of the bioactive HA provided excellent biological properties to their systems. For its part, Skaalure et al. [27] developed a semi-IPN consisting on poly(ethylene glycol) and entrapped HA, whose incorporation clearly led to an enhanced cell adhesion and proliferation.

### **4. Conclusions**

Semi-IPN and IPN systems based on HA and Ch crosslinked with G1Phy were developed as biomimetic and degradable membranes with potential application in TE. Significant differences between semi-IPNs and IPNs were observed in terms of surface topography, mechanical, swelling and degradability properties. IPNs demonstrated to enhance HA retention, as well as mechanical properties of the polymeric network thanks to covalent crosslinking mediated by UV-light irradiation. Dual crosslinking processes of IPNs, consisting of ionic crosslinking of Ch and photopolymerization of HAMA, provided membranes with long-term stability and increased swelling. Moreover, the IPN framework led to flatter surfaces in comparison to semi-IPN. All the studied systems demonstrated high biocompatibility, supporting hMSCs adhesion and proliferation on their surfaces. However, the semi-IPN significantly increased cell proliferation over time respect to IPN, arising as the best candidate of the studied systems. This behavior could be due to the surface features of the semi-IPN (i.e., hydrophilic nature, granular topography and mechanical properties mimicking those of native cartilage), the higher content of the bioactive crosslinker and the entangled HA what seems to be key properties to favor hMSCs performance. These finding suggest that Ch/HA semi-IPNs ionically crosslinked with G1Phy have potential to be proposed as an effective promoter system of tissue repair. Further studies will be carried out to evaluate both in vitro and in vivo differentiation abilities of hMSCs seeded on these biomimetic ECM membranes and their potential application for guided bone regeneration.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4360/12/11/2661/s1, Figure S1: (A) <sup>1</sup>H-NMR spectrum of HAMA with 4.5% methacrylation degree in D2O, and (B) ATR-FTIR spectra of HA and HAMA, Figure S2: ATR-FTIR spectra of Ch, Ch/HA, and Ch/HAMA membranes.

**Author Contributions:** Conceptualization and methodology, M.L.L.-D., A.M.-B., E.L.-R.; physicochemical analysis, M.L.L.-D., A.M.-B.; Biological characterization, E.L.-R., G.J.; resources, and data interpretation, M.L.L.-D., A.M.-B., E.L.-R.; writing—original draft preparation, M.L.L.-D., A.M.-B.; writing—review and editing, A.M.-B., M.L.L.-D., B.V.-L.; supervision, B.V.-L., P.G.-M.; funding acquisition, P.G.-M., J.S.R., J.A.M., J.L.P., M.R.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors thanks to "La Caixa" Foundation (ID 100010434), which supported Ana Mora-Boza (scholarship code LCF/BQ/ES16/11570018) and to the Spanish Ministry of Economy and Competitiveness for financial support (project RTC-2016-5451-1) and the Fundación Mutua Madrileña (project FMM-AP17196-2019). M. R. Aguilar and B. Vázquez-Lasa are members of the *SusPlast platform (Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy)* from the Spanish National Research Council (CSIC).

**Conflicts of Interest:** The authors declare no conflict of interest.
