**1. Introduction**

Treatment of severe organ/tissue injuries generally requires the replacement of a post-traumatic defect with a scaffold such as an autologous/decellularized graft or preferably biomimetic biodegradable material capable of supporting cell growth and functional activity while overcoming the limitations of donor grafts mostly related to their scarcity and host immunogenic responses [1]. Although a number of biomimetic scaffolds composed of synthetic or/and naturally occurring biopolymers have been proposed, these materials by themselves are not able to provide sufficient regenerative responses without special bioactivation [2].

Transplanted cells such as mesenchymal stem cells from different sources [3], skin fibroblasts [4], neural cells [5], as well as related products (e.g., platelets [6] and extracellular vesicles [7]), were proved to enhance tissue regeneration activity of biomaterials. However, these biological products unavoidably feature typical limitations of donor tissues, primarily, low availability, variability of characteristics, and health risks. Given that the regenerative

**Citation:** Yergeshov, A.A.; Zoughaib, M.; Ishkaeva, R.A.; Savina, I.N.; Abdullin, T.I. Regenerative Activities of ROS-Modulating Trace Metals in Subcutaneously Implanted Biodegradable Cryogel. *Gels* **2022**, *8*, 118. https://doi.org/10.3390/ gels8020118

Academic Editors: Arish Dasan, Ashokraja Chandrasekar and Nupur Kohli

Received: 16 January 2022 Accepted: 10 February 2022 Published: 14 February 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/).

potential of the transplanted cells is mainly attributed to secreted signaling molecules, recombinant growth factors can be used instead to improve tissue-replacing scaffolds [8], although this is complicated by the increased manufacturing cost of pure growth factors and their deactivation upon immobilization and storage. Therefore, more stable, reproducible and available active components of biomaterials are still demanded in tissue engineering and regeneration applications.

Trace metals (TM) are essential bioactive microelements involved in the maintenance and regulation of cell metabolism, the functioning of the immune system, turnover and the regeneration of soft and hard tissues. Since TM deficiency accompanies many degenerative and traumatic diseases, their local administration in combination with biomaterials represents a promising therapeutic strategy [9,10]. The regenerative activities of such TMs (primarily, divalent ions of Co, Cu, Zn, Mn, and Fe metals) were established mainly for solid osteoinductive materials based on inorganic scaffolds doped with metal ions or nanoparticles. For instance, collagen scaffolds functionalized with Cu-eluting bioactive glass particles possessed profound in vitro angiogenic activity toward rMSCs and HUVECs as well as antibacterial and osteogenic effects in vivo [11]. Implanted Co-containing borosilicate glassbased scaffolds remarkably enhanced bone regeneration and the vascularized network of the calvarial defective site in rats [12]. Likewise, Zn incorporation into Ca-silicate-based cements increased the osteostimulative activity of the composite material in a maxillofacial bone defect model in rabbits [13].

Hydrogels have been considered among the most promising materials for tissue repair, showing successful results in pre-clinical trials owing to their appropriate physicochemical and hydration properties similar to those of soft body tissues [14,15]. Earlier, gelatin methacrylate hydrogels embedded with Cu nanoparticles supported the attachment and proliferation of 3T3 fibroblasts and inhibited bacterial growth in vitro, in addition to promoting effective wound closure in mice without inflammatory response [16]. Zn crosslinked alginate-polyacrylamide hydrogel supported increased vascular growth, collagen deposition, granulation tissue formation and wound healing along with reduced inflammation [17]. The co-encapsulation of Co and Ca ions within gauze-alginate composite hydrogel resulted in enhanced local VEGF and TGF-β1 protein expression and accelerated wound healing in a mouse bacteria-infected wound model [18]. We have shown recently that macroporous hydrogels prepared by the cryogelation technique, namely, cryogels composed both of bio- and synthetic polymers, represent a promising type of scaffold for bulk bioactivation with TMs in a controllable and stable manner [19,20]. The advanced porous structure of cryogels ensures enhanced mammalian cell infiltration and activity of the incorporated metal dopant within the scaffold [19–21]. The increased healing of an excisional skin defect treated with the Zn-doped gelatin cryogel [19] as well as enhanced in vitro angiogenic responses of poly(2-hydroxyethyl methacrylate) cryogels modified with Cu2+ (via complexation with GHK peptide) [20] were demonstrated.

Further clarification of mechanisms of local regenerative and adverse effects of TMcontaining hydrogels is demanded. These effects are often controversial, depending on biomaterial formulations, which may, for instance, show both prooxidant [22–24] and antioxidant [25,26] activities for the same TMs. Our previous studies prove cryogels as a relevant platform both for the examination of therapeutic effects of the TM compounds and for the development of advanced bioinductive materials [19,20].

In this work, we conducted a comparative investigation of regenerative activities of biodegradable gelatin cryogel doped with Zn, Cu or Co divalent metals as one of the most therapeutically relevant TMs [10]. Considering that TMs can participate in nonenzymatic redox-reactions, such as the Fenton-type generation of reactive oxygen species (ROS) involved in cell signaling [27–29], the TM-doped cryogels were assessed in relation to redox-modulating and cytokine-regulating in vitro activities of the metal dopants. To charactrize in vivo regenerative activities of the TM-doped cryogels, a subcutaneous implantation model with a comprehensive histological evaluation was optimized, considering the relevance of this model for understanding fundamental effects of biomaterials on host

tissue responses related to cellularization, angiogenesis, and inflammation [30–32]. Thus, specific localized effects of the cryogel-formulated TMs on different skin structures and underlying tissues were studied and compared.

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

#### *2.1. Materials*

Bovine skin gelatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent), 40 ,6-diamidino-2-phenylindole (DAPI), phenazine methosulfate (PMS), and Triton X-100 and 20 ,70 -dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich. Monochlorobimane (MCB) was purchased from ThermoFisher Scientific. CuSO4·5H2O, ZnCl2, CoCl2·6H2O, glutaric dialdehyde (GDA), and cresyl violet acetate were obtained from Acros Organics. Citrus pectin (classic CM 201) was obtained from Herbstreith&Fox.

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS reagent) was purchased from Promega. Phalloidin CruzFluor™ 647 conjugate, anti-VEGF (C-1) mouse monoclonal, anti-ICAM-2 (S-16) goat polyclonal, anti-MMP-3 goat monoclonal and anti MMP-3 goat monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti-HIF-1a mouse monoclonal antibody, donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647, and donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 555, were obtained from ThermoFisher Scientific. Anti-CD31 (PECAM-1) rabbit monoclonal antibody was obtained from Abcam. Hematoxylin and Eosin, and Giemsa staining were purchased from BioVitrum (Russia). Cell culture media and reagents were purchased from Paneco (Russia).

#### *2.2. Preparation and Characterization of Cryogels*

Cryogels were prepared from bovine skin gelatin using cryotropic gelation method as previously described [19] with some modifications. Briefly, the reaction mixture contained gelatin (2.5 wt%), pectin (ca. 0.1 wt%) and TM (0.04–1 mM) in aqueous solution. The gelation was initiated by adding 0.25 wt% GDA to the solution upon stirring followed by its pouring into a glass Petri dish and cooling at a temperature of −12 ◦C for 4 h in a thermostat and then at −18 ◦C for additional 24 h in a freezer. The resultant ~3 mm thick cryogel sheet was thawed at room temperature, washed and stored in 25% ethanol solution in the fridge.

Rheological properties of the cryogels were analyzed using MCR 302 rotational rheometer (Anton Paar) at 25 ◦C. The strain sweep and frequency sweep tests were performed by applying 0.01–100% strain amplitude (ω = 10 rad s−<sup>1</sup> ) and 0.01–100 rad s−<sup>1</sup> angular frequencies (γ = 1%), respectively. The storage (G0 ) and loss (G00) modulus of the materials were presented as a function of strain and frequency. Frequency dependences of G0 and G00 were detected within linear viscoelastic region (LVR). Porous structure of the cryogels was analyzed using laser scanning confocal microscopy (LSCM) using LSM 780 microscope (Carl Zeiss) equipped with argon laser excitation (488 nm). Zeiss ZEN black software was used for acquisition. Pore size of the cryogels was evaluated using ImageJ software (NIH, USA).

#### *2.3. Cell Maintenance and Seeding*

NIH 3T3 mouse embryonic fibroblasts (ATCC) and primary human skin fibroblasts (HSFs) isolated as described earlier [33] were grown in α-MEM supplemented with 10% FBS, penicillin (100 U/mL)/streptomycin (100 µg/mL) and L-glutamine (2 mM). Freshly isolated human umbilical vein endothelial cells (HUVECs) were kindly provided by Dr. Ilnur Salafutdinov (Kazan Federal University). HUVECs were grown in RPMI 1640 supplemented with 20% FBS, penicillin (100 U/mL)/streptomycin (100 µg/mL), L-glutamine (2 mM), sodium pyruvate (2 mM), heparin (100 µg/mL), and 30 µg/mL endothelial cell growth supplements (ECGS). The cells were cultured in a temperature- and humiditycontrolled incubator at 37 ◦C. The culture medium was refreshed every 2 days. Primary cells (HSFs and HUVECs) were studied between passages 3 and 6.

Prior to cell seeding, round cryogel sheets (14 mm in diameter) were incubated in penicillin (2.5 kU/mL)/streptomycin (2.5 mg/mL) antibiotic mixture for 1 h, rinsed with HBSS and equilibrated in the culture medium. Cells were seeded onto the cryogel surface using top seeding method in 24-well plate at a density of 4.88 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/cm<sup>2</sup> of cryogel area and incubated for 1.5 h under standard culture conditions to allow for cell attachment.

#### *2.4. Cell Detection in Cryogels*

The cryogels with cultured cells were collected at day 3, transferred into new wells containing 0.5 mL MTS/PMS reagents in fresh culture medium to assess cell metabolic activity [34]. After incubation for 1.5 h under standard culture conditions (37 ◦C, 5% CO2), the absorbance was determined at 490 nm on an Infinite M200 PRO microplate analyzer (Tecan).

For bright-field microscopy analysis, the cryogels with cultured HUVECs were fixed with 4% *p*-formaldehyde for 2.5 h and gently washed with PBS. The fixed cells were subsequently stained with cresyl violet (0.1% *w*/*v* in ultrapure water) for 5 min and visualized using AxioObserver Z1 microscope (Carl Zeiss).

#### *2.5. Immunocytochemistry*

The fixed cryogel matrices were incubated in 0.1% Triton X-100 in PBS for 15 min for cell membrane permeabilization and washed three times with PBS. Non-specific binding sites in the materials were blocked using 1.5% bovine serum albumin (BSA) for 30 min at room temperature. The matrices were subsequently incubated with primary antibodies (1:500 in 1.5% BSA/PBS) against VEGF or ICAM-2 overnight at 4 ◦C followed by incubation with Alexa Fluor 647-conjugated donkey anti-mouse or Alexa Fluor 555-conjugated donkey anti-goat secondary antibodies (1:350 in 1.5% BSA/PBS) for 45 min at room temperature. Following washing with PBS, the cell nuclei were stained with 40 ,6-diamidino-2 phenylindole (DAPI). For cytoskeleton visualization, F-actin was labeled using phalloidin CruzFluor™ 647 conjugate in 1% BSA for 30 min. LSCM images were acquired on an LSM 780 microscope.

#### *2.6. Detection of ROS and Glutathione*

To assess extracellular ROS-generating ability of the TM-doped cryogels, the materials in a 24-well plate were incubated with H2O<sup>2</sup> (21.5 mM) in PBS for 60 min in the presence of 5 µM DCFDA. The probe fluorescence (λex/λem = 490/526) in the solution was monitored during the reaction using an Infinite M200 PRO microplate analyzer.

Intracellular effects of dissolved TMs on both ROS and reduced glutathione levels were additionally studied. 3T3 cells were seeded in 96-well plate at a density of <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well and grown overnight. The cells were exposed to dissolved CuSO4, ZnCl<sup>2</sup> or CoCl<sup>2</sup> at a concentration of 1 or 10 µM in HBSS for 60 min in CO<sup>2</sup> incubator. Subsequently, the treated cells were stained with 20 µM DCFDA fluorescent probe for 40 min or with 5 µM monochlorobimane probe for 60 min to assess intracellular ROS and reduced glutathione, respectively. The cellular fluorescence of DCFDA (λex/λem = 490/526) and MCB (λex/λem = 380/480) was detected. The data are presented as mean ± SD.

#### *2.7. Multiplexed Fluorescent Bead-Based Immunoassay*

Top seeded HSFs were grown within non-doped and metal-doped cryogels as mentioned above in 2.3. At 24 h post-seeding, the conditioned culture medium containing cell secretion was collected and immediately frozen at –80 ◦C. The analysis of secreted levels of cytokines was performed using xMAP Luminex technology on a Bio-Plex MAGPIX analyzer (BioRad, USA) according to the manufacturer's recommendations. A commercially available MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (HCYTMAG-60K-PX41) was used to quantitatively measure cytokine/chemokine levels.

Standard reference curve was used to determine the concentration of analytes in each sample according to their fluorescence intensities. Background levels of analytes in cell-free culture medium were subtracted. Bio-Plex Manager 4.1 software (Bio-Rad Laboratories) was used to analyze the data.

#### *2.8. In Vivo Study*
