*Article* **RGD-Functionalized Hydrogel Supports the Chondrogenic Commitment of Adipose Mesenchymal Stromal Cells**

**Cristina Manferdini 1,† , Diego Trucco 1,2,3,† , Yasmin Saleh <sup>1</sup> , Elena Gabusi <sup>1</sup> , Paolo Dolzani <sup>1</sup> , Enrico Lenzi <sup>1</sup> , Lorenzo Vannozzi 2,3 , Leonardo Ricotti 2,3 and Gina Lisignoli 1,\***


**Abstract:** Articular cartilage is known to have limited intrinsic self-healing capacity when a defect or a degeneration process occurs. Hydrogels represent promising biomaterials for cell encapsulation and injection in cartilage defects by creating an environment that mimics the cartilage extracellular matrix. The aim of this study is the analysis of two different concentrations (1:1 and 1:2) of VitroGel® (VG) hydrogels without (VG-3D) and with arginine-glycine-aspartic acid (RGD) motifs, (VG-RGD), verifying their ability to support chondrogenic differentiation of encapsulated human adipose mesenchymal stromal cells (hASCs). We analyzed the hydrogel properties in terms of rheometric measurements, cell viability, cytotoxicity, and the expression of chondrogenic markers using gene expression, histology, and immunohistochemical tests. We highlighted a shear-thinning behavior of both hydrogels, which showed good injectability. We demonstrated a good morphology and high viability of hASCs in both hydrogels. VG-RGD 1:2 hydrogels were the most effective, both at the gene and protein levels, to support the expression of the typical chondrogenic markers, including collagen type 2, SOX9, aggrecan, glycosaminoglycan, and cartilage oligomeric matrix protein and to decrease the proliferation marker MKI67 and the fibrotic marker collagen type 1. This study demonstrated that both hydrogels, at different concentrations, and the presence of RGD motifs, significantly contributed to the chondrogenic commitment of the laden hASCs.

**Keywords:** adipose mesenchymal stromal cells; hydrogels; chondrogenic differentiation; RGD motif; hydrogel characterization; cartilage regeneration

### **1. Introduction**

The degeneration of articular cartilage due to trauma, osteoarthritis, or aging is a common joint disorder, with a high incidence worldwide [1,2]. It has been reported that the articular cartilage has limited intrinsic self-healing capacity, mainly due to its avascular and aneural nature [3]. Different clinical treatments, such as autologous chondrocyte implantation, mosaicplasty, and microfracture, have been used for inducing articular cartilage regeneration [4]. However, these techniques show limitations since they do not permit effective long-term cartilage regeneration and do not assure the formation of a fully differentiated articular cartilage structure, thus requiring the development of alternative strategies [5].

Tissue engineering approaches represent a promising alternative for cartilage regeneration and repair [6–8]. Hydrogels are among the most versatile kinds of materials used for various tissue engineering applications, since they can be engineered into almost any shape and size [9,10]. They can also be functionalized or enriched with micro/nanofillers

**Citation:** Manferdini, C.; Trucco, D.; Saleh, Y.; Gabusi, E.; Dolzani, P.; Lenzi, E.; Vannozzi, L.; Ricotti, L.; Lisignoli, G. RGD-Functionalized Hydrogel Supports the Chondrogenic Commitment of Adipose Mesenchymal Stromal Cells. *Gels* **2022**, *8*, 382. https://doi.org/ 10.3390/gels8060382

Academic Editors: Yanen Wang and Qinghua Wei

Received: 23 May 2022 Accepted: 13 June 2022 Published: 15 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for building composite hydrogels, giving them improved properties tailored to specific applications [11,12]. In general, hydrogels represent hydrophilic 3D networks composed of water-soluble natural (e.g., polysaccharides and proteins) and/or synthetic polymers crosslinked by chemical or physical methods to form a water-insoluble matrix [13,14]. Different from hydrogels, aerogels are porous materials obtained when the liquid phase of a hydrogel is replaced by a gas, while preserving the internal structure and the surface area of the initial hydrogel [15–17]. They have been proven to provide highly desirable 3D environments for the regeneration of the cartilaginous tissue, as shown by in vitro and in vivo studies [18–21]. Hydrogels are viscoelastic materials, and their mechanical properties are an important requirement for engineering cell functions, since their tuning can improve mechanosensitive signaling. In fact, matrix stiffness, permeability, swelling ability, and degradation provide a peculiar biomimetic environment able to create a niche suitable to drive cell migration, adhesion, proliferation, and chondrogenic differentiation [22]. These properties, associated with other factors (i.e., seeding conditions, hypoxia), modulate the differentiation processes [23–25].

The use of injectable and in situ-forming hydrogels enables the treatment of irregular cartilage defects and a proper alignment with the surrounding tissues, characteristics that make them superior to 3D-structured scaffold-based approaches [26,27]. Meanwhile, from the clinical viewpoint, implantation surgery can be avoided and replaced by a simple, minimally invasive injection [26,28]. Moreover, bioactive molecules or cultured cells can simply be incorporated into the hydrogel precursors before they are ready for injection, or 3D bioprinted [29–31].

Mesenchymal stromal cells (MSCs) have shown interesting results in the field of cartilage regeneration, mainly due to their accessibility, immunomodulatory and proregenerative capabilities, and chondrogenic differentiation potential [32–35]. It has been shown that MSCs from various origins, combined with scaffold materials, have great potential in the regeneration of cartilage, in both animal models and in humans, as suggested by recent clinical trials [27,28,30,36]. It has been recognized that MSCs, laden in natural or synthetic hydrogels, create a suitable environment for inducing their cellular differentiation [37].

Recently, it has been shown that VitroGel® (VG) hydrogels have the potential to mimic the cartilage extracellular matrix (ECM) [38]. This hydrogel can also provide binding sites for cell adhesion, thanks to the functionalization with arginine-glycine-aspartic acid (RGD) motifs, which is a well-known tri-peptide able to promote cell attachment and, at the same time, favor cell-matrix interactions by enhancing cellular function, like cell proliferation, migration, and differentiation [39,40]. It has been shown that the RGD motif is a crucial component of adhesive proteins in the ECM, working through integrin transmembrane receptors by transmitting the cell survival signaling within the cells [41]. However, it has been shown that this tri-peptide exerts controversial issues in cartilage tissue engineering [42]. It has been demonstrated that the hydrogel modified with RGD peptide (VG-RGD) can be easily injected for the treatment of the intervertebral disc in rats, promoting the proliferation and differentiation of nucleus pulposus (NP)-derived MSCs (NPMSCs), and also promoting the NPMSC's long-term retention and survival in the degenerated intervertebral disc, with the formation of a neo-ECM [38].

The chondrogenic commitment of human adipose-derived mesenchymal stromal cells (hASCs) in VG hydrogels has been never investigated before. The novelty of the study is to gain new insight into the microenvironment fostered by natural hydrogels, by investigating the 3D hydrogel environment created by VG without (VG-3D) and with RGD (VG-RGD) motif on the chondrogenic differentiation of encapsulated hASCs for the potential treatment of cartilage defects.

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

#### *2.1. Materials 2.1. Materials*

VG-3D and VG-RGD hydrogels were both purchased from Well Bioscience (North Brunswick, NJ, USA) and prepared following the manufacturer's protocol. Briefly, the VG-3D and VG-RGD solutions were directly mixed at room temperature (RT) with the Dilution Solution Type 1® (The Well Bioscience, North Brunswick, NJ, USA) at the ratio of 1:1 and 1:2 to obtain two different hydrogel formulation. Then, Dulbecco Modified Eagle Medium (DMEM) (Life Technologies, Bleiswijk, The Netherlands) was added to each pre-crosslinked solution at the ratio of 4:1 (pre-crosslinked solution: DMEM) at RT. Four different combinations (VG-3D 1:1, VG-3D 1:2, VG-RGD 1:1, VG-RGD 1:2) were analyzed. Sodium alginate powder (Sigma-Aldrich Merck, Saint Louis, MO, USA) was used as the control hydrogel. VG-3D and VG-RGD hydrogels were both purchased from Well Bioscience (North Brunswick, NJ, USA) and prepared following the manufacturer's protocol. Briefly, the VG-3D and VG-RGD solutions were directly mixed at room temperature (RT) with the Dilution Solution Type 1® (The Well Bioscience, North Brunswick, NJ, USA) at the ratio of 1:1 and 1:2 to obtain two different hydrogel formulation. Then, Dulbecco Modified Eagle Medium (DMEM) (Life Technologies, Bleiswijk, The Netherlands) was added to each precrosslinked solution at the ratio of 4:1 (pre-crosslinked solution: DMEM) at RT. Four different combinations (VG-3D 1:1, VG-3D 1:2, VG-RGD 1:1, VG-RGD 1:2) were analyzed. Sodium alginate powder (Sigma-Aldrich Merck, Saint Louis, MO, USA) was used as the control hydrogel.

#### *2.2. Experimental Plan 2.2. Experimental Plan*

The experimental workflow for this study is shown in Figure 1. Briefly, the VG hydrogels raw materials (Figure 1a) were characterized through FTIR analysis. Then, the rheological and mechanical properties of hydrogels (Figure 1b) were evaluated and the protein diffusivity was tested. hASCs were analyzed in terms of antigenic profile and differentiation capabilities (Figure 1c). Then, hASC-laden hydrogels (Figure 1d) were analyzed at two different time points to assess cell distribution, viability, cytotoxicity, and metabolic activity. Finally, the chondrogenic differentiation of hASCs encapsulated in hydrogels (Figure 1d) was evaluated through molecular biology assays and immunohistochemical staining. The experimental workflow for this study is shown in Figure 1. Briefly, the VG hydrogels raw materials (Figure 1a) were characterized through FTIR analysis. Then, the rheological and mechanical properties of hydrogels (Figure 1b) were evaluated and the protein diffusivity was tested. hASCs were analyzed in terms of antigenic profile and differentiation capabilities (Figure 1c). Then, hASC-laden hydrogels (Figure 1d) were analyzed at two different time points to assess cell distribution, viability, cytotoxicity, and metabolic activity. Finally, the chondrogenic differentiation of hASCs encapsulated in hydrogels (Figure 1d) was evaluated through molecular biology assays and immunohistochemical staining.

**Figure 1.** Depiction of the experimental plan: (**a**) characterization of raw materials through FTIR analysis; (**b**) evaluation of the rheological and mechanical properties of hydrogels and protein **Figure 1.** Depiction of the experimental plan: (**a**) characterization of raw materials through FTIR analysis; (**b**) evaluation of the rheological and mechanical properties of hydrogels and protein diffusivity

evaluation; (**c**) characterization of the hASCs in terms of antigenic profile and differentiation capabilities; (**d**) biological analysis to assess cell distribution, viability, cytotoxicity and metabolic activity, and chondrogenic assessment evaluated through molecular biology assays and immunohistochemical staining.
