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

**Ana Mora-Boza 1,2,**† **, Elena López-Ruiz 3,4,5,6,**† **, María Luisa López-Donaire 1,2,\* , Gema Jiménez 3,4,5,6 , María Rosa Aguilar 1,2 , Juan Antonio Marchal 3,4,6,7 , José Luis Pedraz 2,8 , Blanca Vázquez-Lasa 1,2,\* , Julio San Román 1,2 and Patricia Gálvez-Martín 9**


Received: 19 October 2020; Accepted: 7 November 2020; Published: 11 November 2020

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

### **1. Introduction**

Hydrogels derived from natural polymers exhibit potential for tissue engineering (TE) applications as they closely mimic the extracellular matrix (ECM) of native tissues. They also provide a suitable environment for supporting cell adhesion and growth compared to other materials due to their biocompatibility, swelling ability, and possibility of diffusion for nutrients and waste exchange [1,2]. Thus, hydrogel membranes that present mechanical and physicochemical properties similar to those of native tissues have gained much attention in the latest years [3].

Polysaccharides-based hydrogels are promising candidates to fulfil the diversified demands in a variety of biomedical applications [4]. Chitosan (Ch) and hyaluronic acid (HA) are two polysaccharides that can form hydrogels and are widely exploited for its use as scaffolds for TE [2]. Ch is a linear polysaccharide widely applied in the biomedical field due to its structural similarity to the naturally occurring glycosaminoglycans and its susceptibility to degradation by enzymes in humans [2,5]. Ch shows also antimicrobial and hemostatic properties attributed to its cationic nature of amino groups [4–6]. HA is a glycosaminoglycan found in ECM that plays a key role as an environmental cue to regulate cell behavior during embryonic development, healing processes, inflammation [4,7,8]. HA participates in important cell signaling pathways due to the presence of cell surface receptors like CD44 and RHAMM, which is a receptor for hyaluronan-mediated motility [9]. Moreover, HA demonstrated to play powerful multifunctional activity in homeostasis and tissue remodeling processes [6,10]. Ch and HA have been combined to fabricate different matrices for several TE applications [1,2,4,11–17], such as polyelectrolyte complexes for dental pulp regeneration [12] or injectable hydrogels [1,2,14,17,18] for cartilage repair [14,18], peripheral nerve regeneration [17], and adipose tissue regeneration [19], among others. Ch and HA combination has been particularly attractive for osteochondral regeneration applications due to their physicochemical and compositional similarities with native cartilage [7,11,20–22]. For example, Mohan et al. [11] performed a profound study about the regeneration capacity of Ch/HA gels on critical osteochondral defects in knee joints of New Zealand white rabbits, claiming the potential regenerative capacities of their systems. In other work carried out by Erickson et al. [7], HA and Ch were used to fabricate a bilayer scaffold to repair osteochondral defects, showing excellent cellular proliferation results.

Among all available types of polymeric-based matrices, interpenetrating networks (IPNs) and semi-IPNs membranes provide highly tunable platforms regarding composition and physicochemical properties by the combination of different polymers and crosslinking processes. These systems have showed attractive features in terms of enhanced stability and mechanical properties, mainly due to the molecular reinforcement resulted from the network/s of different polymers [4,9,23]. As it is known, an IPN consists of a combination of two (or more) polymer networks which are physically or chemically crosslinked and entangled within each other. For its part, in a semi-IPN, only one of the polymers is crosslinked and the linear polymer is entangled within the network [9]. The present approach provides a promising candidate system for TE applications in the form of natural-occurring polysaccharides semi- and IPN systems using a novel recently developed crosslinker glycerylphytate (G1Phy). G1Phy is a natural derived crosslinker that possesses reduced cytotoxicity and antioxidant properties [24], and showed enhanced cellular adhesion and proliferation in comparison to other traditionally used phosphate-based crosslinkers like tripolyphosphate [25]. Specifically, we develop and evaluate semiand IPN systems formed by Ch/HA and Ch/methacrylated HA (HAMA), respectively, ionically crosslinked with G1Phy [25]. Although Ch [4] and HA [9,20,26,27] have been combined with other polymers for the preparation of semi- and IPN systems, the reported polymeric composition and applied crosslinking strategies in this work has not been explored before. The obtained materials were characterized by a set of techniques in terms of composition, physicochemical, morphological and mechanical properties as well as in vitro behavior, observing clear differences that are expected to influence its efficacy on their biological performance. Biological assays regarding viability, cell adhesion and proliferation were assessed on human mesenchymal stromal cells (hMSCs). The excellent biocompatibility of our systems was demonstrated by large spreading areas of hMSCs on the hydrogel

surfaces. Moreover, semi-IPN system showed a significantly enhancement of hMSCs proliferation over time in comparison to the other systems. Our findings suggested that surface properties and composition, can play a key role in the final application of semi- and IPN membranes as effective matrices for TE, tentatively to guided bone regeneration applications.

### **2. Materials and Methods**
