2.8.1. Animals

Wistar male rats (340 ± 38 g) were purchased from Vivarium of Academy of Medical and Technical Sciences (Russia). Animal care was performed according to European regulations on the protection of experimental animals (Directive 2010/63/UE) and Russian regulations (No. 742 from 13.11.1984, Ministry of Education and Science). The rats were divided into four groups and were kept in plastic cages under controlled conditions (at a temperature of 20 ± 3 ◦C and a humidity of 65 ± 10%) with running water and complete feed. The in vivo study was approved by the Institutional Ethical Review Board of the Kazan Federal University.

#### 2.8.2. Subcutaneous Implantation Model

The animals were anesthetized using tiletamine-zolazepam-xylazine (30/20/10 mg/kg, respectively) administered via IP injection. The upper back skin was shaved and disinfected with 70% ethanol solution. Two symmetrical full-thickness skin incisions were made horizontally with a width of 1.5 cm using fine scalpel. A subcutaneous pocket with about 1.5 cm long from the lower incision border was created on each side by detaching the skin from the underlaying tissues employing anatomical forceps (Figure 7). The incision was disinfected with 0.05% chlorhexidine solution and washed with sterile isotonic solution. The cryogel sheets were cut into 1 × 1 cm square pieces, additionally decontaminated with penicillin (5 kU/mL)/streptomycin (5 mg/mL) solution and equilibrated with excess of sterile isotonic solution. The studied materials were aseptically implanted into subcutaneous pockets so that the control (non-doped) and metal-doped cryogels (TM concentration = 0.2 mM) were alternately placed at the right and left sides of an animal. The procedure allowed us to decrease the number of animals per group to *n* = 6. No manifestations of pain, infection or any worsening of animal behavior were observed during the experiment.

#### *2.9. Histological Evaluation*

On days 5 and 10 post-implantation, the animals were sacrificed using tiletaminezolazepam-xylazine anesthesia and by applying incremental concentration of CO2. The treated skin with implanted material was surgically excised, then subsequently fixed in 4% neutral buffered formalin solution in PBS at room temperature for 48 h, washed with distilled water, dehydrated in a graded series of ethanol solutions (50, 70, 90, 96, 99.8%) and cleared in xylene. The explants were embedded in paraffin blocks and cut on a microtome HM 355S (Thermo Fisher Scientific) into 10–14 µm sections. The tissue sections were stained with Giemsa, hematoxylin and eosin (H&E) or Picrosirius red and analyzed by bright-field and polarized light microscopy on an Axio Observer Z1 microscope (Carl Zeiss).

For immunohistochemical analysis, glass slide-adhered tissue sections were permeabilized using 0.1% Triton X-100 in PBS for 30 min, thoroughly washed with PBS, and blocked with 1.5% BSA. The sections were subsequently incubated with primary antibodies (diluted 1:300 in 1.5% BSA/PBS) against CD-31 (PECAM-1), HIF-1α, MMP-2 or MMP-3 overnight at 4 ◦C followed by incubation with proper Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 647-conjugated donkey anti-mouse secondary antibodies (1:350 in 1.5% BSA/PBS), for 45 min at room temperature. DAPI was used to stain cell nuclei. LSCM images were acquired on an LSM 780 microscope.

#### *2.10. Statistical Analysis*

Data were presented as mean ± SD. Statistical significance was determined by oneway analysis of variance (ANOVA) followed by Tukey's Multiple Comparison post-test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

#### **3. Results**

#### *3.1. Characterization of Cryogels*

Cryogels were made by the cryotropic gelation of bovine gelatin cross-linked with glutaraldehyde. Co2+, Cu2+ and Zn2+ (further designated as Co, Cu and Zn) in the form of water-soluble salts were incorporated into the cryogel material during gelation. The addition of pectin to the gelatin cryogel has been used to facilitate the capture of metal ions by introducing additional anionic groups, such as galacturonic acid, into the polymer network. The metal content in the cryogels linearly depended on the concentration of TMs in the gel-forming solution from 0.04 to 5 mM; the TM-doped cryogels were designated by these concentrations. For the upper 1 mM concentration used in this study, the TM content in cryogels was previously shown to amount to ca. 3 <sup>×</sup> <sup>10</sup><sup>3</sup> ppm (Zn, Cu) and 1 <sup>×</sup> <sup>10</sup><sup>3</sup> ppm (Co) [19].

According to LSCM analysis, the TM-doped cryogels preserved an interconnected porous structure (Figure 1A), typical of cryogel scaffolds, with somewhat larger pores at the upper surface, commonly used for cell culture [34]. Non-doped cryogels possessed macropores with a calculated mean pore size of 80 ± 13 µm, which moderately decreased by 1.2–1.4 times in the TM-doped materials. According to rheological data, all cryogels displayed a linear viscoelastic region for a shear strain amplitude of γ ≤ 6% (Figure 1B). Both storage (G0 ) and loss (G") modulus relatively weakly depended on angular frequency, whereas G0 greatly prevailed over G" (Figure 1B), indicating a well-structured hydrogel network with dominant elastic behavior. The incorporated TMs increased the G0/G" ratio by 1.3–1.9-fold, with a relatively lower effect of Co, thus demonstrating that the metal component contributes to the elasticity and mechanical strength of the materials. The modulation of pore size and viscoelastic behavior of cryogels by the introduced TMs is explained by additional cross-linking of macromers (gelatin and pectin molecules) by the metal ions presumably involving not only ionic but also coordination bonding with biopolymer ligand groups.

#### *3.2. Behavior of Fibroblasts in Metal-Doped Cryogels*

#### 3.2.1. Effect of Cryogel Composition on Cell Proliferation

Mouse embryonic fibroblasts (3T3 cells) were seeded on the top surface of cryogels and allowed to grow for 72 h followed by cell detection using the MTS proliferation assay [34]. At a concentration of 0.04 and 0.2 mM, the introduced TMs did not inhibit cell proliferation, whereas 1 mM Co and Zn, unlike 1 mM Cu, caused a moderate inhibitory effect of up to 24% (Figure 2A). When supplemented into the culture medium, soluble TMs had IC<sup>50</sup> values of 170 ± 9 µM (Zn), 250 ± 17 µM (Co), and 407 ± 20 µM (Cu) (72 h). Assuming most of the amount of TMs in gelling solution to be attached to the cryogel [19], the above data together suggest that the entrapped metals are not readily released into the medium remaining less available and less cytotoxic toward the cells than dissolved metals. In total, 0.2 mM of Cu and 0.2 mM of Zn were found to noticeably stimulate cell proliferation, respectively, by 30 and 10% (Figure 2A); this intermediate concentration was, therefore, selected for further study and comparison of regenerative effects of the TM-doped cryogels.

Similar to 3T3 cells, human skin fibroblasts (HSFs) proliferated more rapidly in the cryogels with 0.2 mM Zn (by 29%) or Cu (by 40%) (*p* < 0.05), unlike Co (Figure 2B). Furthermore, binary TM compositions exhibited quite different effects on HSF behavior. In particular, Zn/Cu did provide additive stimulation of cell growth compared to the individual metals, whereas the stimulating effect was partially preserved for Zn/Co and disappeared for Cu/Co system (Figure 2B). Considering that such a mitogenic activity could be associated with Fenton-like reactions of TMs [29], the ability of cryogels to gen-

erate ROS in the reaction with hydrogen peroxide (H2O2) was analyzed with the aid of a DCFDA probe (Figure 3). The results show that the cryogel-formulated metal dopants are capable of reacting with H2O2, where Co and Cu, unlike Zn, effectively generate ROS (Co > Cu) in agreement with earlier observation for these TMs' behavior in solution [35]. When introduced together, Co and Cu showed additive prooxidant activity, whereas the individual activity of Co or Cu was profoundly inhibited in the presence of Zn co-dopant, reflecting its antioxidant/anticorrosive effect toward the metals with variable valency [28]. frequency, whereas G′ greatly prevailed over G″ (Figure 1B), indicating a well-structured hydrogel network with dominant elastic behavior. The incorporated TMs increased the G′/G″ ratio by 1.3–1.9-fold, with a relatively lower effect of Co, thus demonstrating that the metal component contributes to the elasticity and mechanical strength of the materials. The modulation of pore size and viscoelastic behavior of cryogels by the introduced TMs is explained by additional cross-linking of macromers (gelatin and pectin molecules) by the metal ions presumably involving not only ionic but also coordination bonding with biopolymer ligand groups.

*Gels* **2022**, *8*, x FOR PEER REVIEW 6 of 29

*2.10. Statistical Analysis*

**3. Results**

10<sup>3</sup> ppm (Co) [19].

(\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

*3.1. Characterization of Cryogels*

MMP-3 overnight at 4 °C followed by incubation with proper Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 647-conjugated donkey anti-mouse secondary antibodies (1:350 in 1.5% BSA/PBS), for 45 min at room temperature. DAPI was used to

Data were presented as mean ± SD. Statistical significance was determined by oneway analysis of variance (ANOVA) followed by Tukey's Multiple Comparison post-test

Cryogels were made by the cryotropic gelation of bovine gelatin cross-linked with glutaraldehyde. Co2+, Cu2+ and Zn2+ (further designated as Co, Cu and Zn) in the form of water-soluble salts were incorporated into the cryogel material during gelation. The addition of pectin to the gelatin cryogel has been used to facilitate the capture of metal ions by introducing additional anionic groups, such as galacturonic acid, into the polymer network. The metal content in the cryogels linearly depended on the concentration of TMs in the gel-forming solution from 0.04 to 5 mM; the TM-doped cryogels were designated by these concentrations. For the upper 1 mM concentration used in this study, the TM content in cryogels was previously shown to amount to ca. 3 × 10<sup>3</sup> ppm (Zn, Cu) and 1 ×

According to LSCM analysis, the TM-doped cryogels preserved an interconnected porous structure (Figure 1A), typical of cryogel scaffolds, with somewhat larger pores at the upper surface, commonly used for cell culture [34]. Non-doped cryogels possessed macropores with a calculated mean pore size of 80 ± 13 μm, which moderately decreased by 1.2–1.4 times in the TM-doped materials. According to rheological data, all cryogels displayed a linear viscoelastic region for a shear strain amplitude of γ ≤ 6% (Figure 1B). Both storage (G′) and loss (G″) modulus relatively weakly depended on angular

stain cell nuclei. LSCM images were acquired on an LSM 780 microscope.

**Figure 1.** (**A**) LSCM images of non-doped (Ctrl) and TM-doped cryogel sheets (top surface) visualized by autofluorescence upon argon laser excitation (488 nm). (**B**) Strain amplitude sweep test (angular frequency ω = 10 rad⸱s −1 ) and frequency sweep test (strain deformation δ = 1%) data for the cryogels. TM-doped cryogels (1 mM) were analyzed. **Figure 1.** (**A**) LSCM images of non-doped (Ctrl) and TM-doped cryogel sheets (top surface) visualized by autofluorescence upon argon laser excitation (488 nm). (**B**) Strain amplitude sweep test (angular frequency ω = 10 rad s−<sup>1</sup> ) and frequency sweep test (strain deformation δ = 1%) data for the cryogels. TM-doped cryogels (1 mM) were analyzed.

#### *3.2. Behavior of Fibroblasts in Metal-Doped Cryogels* 3.2.2. Cytokine and Growth Factor Profile

TM-doped cryogels.

3.2.1. Effect of Cryogel Composition on Cell Proliferation Mouse embryonic fibroblasts (3T3 cells) were seeded on the top surface of cryogels and allowed to grow for 72 h followed by cell detection using the MTS proliferation assay [34]. At a concentration of 0.04 and 0.2 mM, the introduced TMs did not inhibit cell proliferation, whereas 1 mM Co and Zn, unlike 1 mM Cu, caused a moderate inhibitory effect of up to 24% (Figure 2A). When supplemented into the culture medium, soluble TMs had IC<sup>50</sup> values of 170 ± 9 µM (Zn), 250 ± 17 µM (Co), and 407 ± 20 µM (Cu) (72 h). Assuming most of the amount of TMs in gelling solution to be attached to the cryogel [19], the above data together suggest that the entrapped metals are not readily released into the medium remaining less available and less cytotoxic toward the cells than dissolved metals. In total, 0.2 mM of Cu and 0.2 mM of Zn were found to noticeably stimulate cell proliferation, respectively, by 30 and 10% (Figure 2A); this intermediate concentration The effect of metal dopants on the production of cytokines and growth factors by HSFs in the cryogels was assessed (Figure 4A). The secretion of FGF-2, VEGF, IL-6, and IL-8 tightly involved in paracrine stimulation of angiogenesis [26] was profoundly stimulated (by 1.2–5.6 times relative to the non-doped cryogel) generally as follows: Zn < Co < Cu (Figure 4B). Monocyte chemoattractant proteins (MCP-1, MIP-1b) were also overproduced in the presence of TMs. In comparison with Cu and Co, Zn weakly affected the level of pro-inflammatory cytokines IL-6/IL-8, whereas it greatly increased the level of MCPs comparably with Cu and Co. The TMs induced the overproduction of EGF and PDGF-AA, similar to other pleiotropic growth factors, namely, FGF-2 and VEGF (by 1.9–3.2 times). Exceptions were that Co and Zn exhibited a lack of effect, respectively, in the case of EGF and VEGF (Figure 4B). The results demonstrate a strong ability of the cryogel-formulated TMs to increase key signaling molecules involved in regeneration-related processes.

was, therefore, selected for further study and comparison of regenerative effects of the

**A**

TM-doped cryogels.

**B**

for the cryogels. TM-doped cryogels (1 mM) were analyzed.

3.2.1. Effect of Cryogel Composition on Cell Proliferation

*3.2. Behavior of Fibroblasts in Metal-Doped Cryogels*

−1

test (angular frequency ω = 10 rad⸱s

**Figure 1.** (**A**) LSCM images of non-doped (Ctrl) and TM-doped cryogel sheets (top surface) visualized by autofluorescence upon argon laser excitation (488 nm). (**B**) Strain amplitude sweep

Mouse embryonic fibroblasts (3T3 cells) were seeded on the top surface of cryogels and allowed to grow for 72 h followed by cell detection using the MTS proliferation assay [34]. At a concentration of 0.04 and 0.2 mM, the introduced TMs did not inhibit cell proliferation, whereas 1 mM Co and Zn, unlike 1 mM Cu, caused a moderate inhibitory effect of up to 24% (Figure 2A). When supplemented into the culture medium, soluble TMs had IC<sup>50</sup> values of 170 ± 9 µM (Zn), 250 ± 17 µM (Co), and 407 ± 20 µM (Cu) (72 h). Assuming most of the amount of TMs in gelling solution to be attached to the cryogel [19], the above data together suggest that the entrapped metals are not readily released into the medium remaining less available and less cytotoxic toward the cells than dissolved metals. In total, 0.2 mM of Cu and 0.2 mM of Zn were found to noticeably stimulate cell proliferation, respectively, by 30 and 10% (Figure 2A); this intermediate concentration

) and frequency sweep test (strain deformation δ = 1%) data

**Figure 2.** (**A**) Proliferation of 3T3 fibroblasts in TM-doped cryogels at different metal concentrations (MTS assay, 72 h). (**B**) Effect of metal dopants (0.2 mM) on HSF proliferation in the cryogels (MTS assay, 72 h). The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01). **Figure 2.** (**A**) Proliferation of 3T3 fibroblasts in TM-doped cryogels at different metal concentrations (MTS assay, 72 h). (**B**) Effect of metal dopants (0.2 mM) on HSF proliferation in the cryogels (MTS assay, 72 h). The data are presented as mean ±SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01). When introduced together, Co and Cu showed additive prooxidant activity, whereas the individual activity of Co or Cu was profoundly inhibited in the presence of Zn co-dopant, reflecting its antioxidant/anticorrosive effect toward the metals with variable valency [28].

When introduced together, Co and Cu showed additive prooxidant activity, whereas the individual activity of Co or Cu was profoundly inhibited in the presence of Zn co-dopant, **Figure 3.** Generation of ROS in reaction of TM-doped cryogels with H2O<sup>2</sup> according to DCFDA fluorescence (λex = 490 nm, λem = 526 nm). **Figure 3.** Generation of ROS in reaction of TM-doped cryogels with H2O<sup>2</sup> according to DCFDA fluorescence (λex = 490 nm, λem = 526 nm).

The effect of metal dopants on the production of cytokines and growth factors by HSFs in the cryogels was assessed (Figure 4A). The secretion of FGF-2, VEGF, IL-6, and IL-8 tightly involved in paracrine stimulation of angiogenesis [26] was profoundly

**Figure 3.** Generation of ROS in reaction of TM-doped cryogels with H2O<sup>2</sup> according to DCFDA

The effect of metal dopants on the production of cytokines and growth factors by HSFs in the cryogels was assessed (Figure 4A). The secretion of FGF-2, VEGF, IL-6, and IL-8 tightly involved in paracrine stimulation of angiogenesis [26] was profoundly

fluorescence (λex = 490 nm, λem = 526 nm).

3.2.2. Cytokine and Growth Factor Profile

reflecting its antioxidant/anticorrosive effect toward the metals with variable valency [28].

3.2.2. Cytokine and Growth Factor Profile

processes.

stimulated (by 1.2–5.6 times relative to the non-doped cryogel) generally as follows: Zn < Co < Cu (Figure 4B). Monocyte chemoattractant proteins (MCP-1, MIP-1b) were also overproduced in the presence of TMs. In comparison with Cu and Co, Zn weakly affected the level of pro-inflammatory cytokines IL-6/IL-8, whereas it greatly increased the level of MCPs comparably with Cu and Co. The TMs induced the overproduction of EGF and PDGF-AA, similar to other pleiotropic growth factors, namely, FGF-2 and VEGF (by 1.9– 3.2 times). Exceptions were that Co and Zn exhibited a lack of effect, respectively, in the case of EGF and VEGF (Figure 4B). The results demonstrate a strong ability of the cryogelformulated TMs to increase key signaling molecules involved in regeneration-related

**Figure 4.** (**A**) Radar plot representation of secreted cytokine/growth factor profile for HSFs cultured in TM-doped cryogels (0.2 mM) for 24 h (pg/mL, log scale). (**B**) The corresponding levels of selected cytokines/growth factors. The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). **Figure 4.** (**A**) Radar plot representation of secreted cytokine/growth factor profile for HSFs cultured in TM-doped cryogels (0.2 mM) for 24 h (pg/mL, log scale). (**B**) The corresponding levels of selected cytokines/growth factors. The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

Human umbilical vein endothelial cells (HUVECs) were used as reference endothelial cells (EC) to compare angiogenic properties of the TM-doped cryogels. The metal dopants (0.2 mM) did not cause any inhibition of HUVEC proliferation, similar to that observed for HSFs, confirming the cytocompatibility of the materials for primary

factor of ca. 1.3 (Zn), 1.5 (Co) and 1.9 (Cu) compared to the control cryogel (Figure 5A). In comparison with HSFs, HUVECs were characterized by higher sensitivity to Cu and Co, whereas Zn almost abolished the stimulating effect of Cu and Co in binary compositions (Figure 5A), presumably in relation to the ROS-modulating activity of the TMs (Figure 3). In addition, HUVECs were visualized within the semi-transparent cryogels after cell staining with cresyl violet (Figure 5B). The calculated cell number per 1 mm<sup>2</sup> of the analyzed surface was as follows: 47 ± 8 (Ctrl), 53 ± 6 (Zn), 102 ± 16 (Cu) and 73 ± 11 (Co), supporting the fact that the corresponding MTS signals (Figure 4A) reflected the cell density in the matrix rather than the change in metabolic activity. Furthermore, HUVECs were well adhered and spread on the surface of all cryogels and their morphology was altered to spindle-shaped cells with developed extensions. Specifically, in the Cu-doped cryogel, up to 41% of the cells adopted more elongated morphology with at least three sprouts; the adjacent cells showed a tendency to migrate and connect to each other, which

*3.3. Behavior of HUVECs in Metal-Doped Cryogels*

is attributed to the induction of tubulogenesis.

**A**

3.3.1. Proliferation and Spreading

#### *3.3. Behavior of HUVECs in Metal-Doped Cryogels* (Figure 5A), presumably in relation to the ROS-modulating activity of the TMs (Figure 3).

3.3.1. Proliferation and Spreading

#### 3.3.1. Proliferation and Spreading In addition, HUVECs were visualized within the semi-transparent cryogels after cell

0.001).

Human umbilical vein endothelial cells (HUVECs) were used as reference endothelial cells (EC) to compare angiogenic properties of the TM-doped cryogels. The metal dopants (0.2 mM) did not cause any inhibition of HUVEC proliferation, similar to that observed for HSFs, confirming the cytocompatibility of the materials for primary human cells. Furthermore, HUVECs proliferated much faster in the presence of TMs by a factor of ca. 1.3 (Zn), 1.5 (Co) and 1.9 (Cu) compared to the control cryogel (Figure 5A). In comparison with HSFs, HUVECs were characterized by higher sensitivity to Cu and Co, whereas Zn almost abolished the stimulating effect of Cu and Co in binary compositions (Figure 5A), presumably in relation to the ROS-modulating activity of the TMs (Figure 3). staining with cresyl violet (Figure 5B). The calculated cell number per 1 mm<sup>2</sup> of the analyzed surface was as follows: 47 ± 8 (Ctrl), 53 ± 6 (Zn), 102 ± 16 (Cu) and 73 ± 11 (Co), supporting the fact that the corresponding MTS signals (Figure 4A) reflected the cell density in the matrix rather than the change in metabolic activity. Furthermore, HUVECs were well adhered and spread on the surface of all cryogels and their morphology was altered to spindle-shaped cells with developed extensions. Specifically, in the Cu-doped cryogel, up to 41% of the cells adopted more elongated morphology with at least three sprouts; the adjacent cells showed a tendency to migrate and connect to each other, which is attributed to the induction of tubulogenesis.

*Gels* **2022**, *8*, x FOR PEER REVIEW 10 of 29

**B**

*3.3. Behavior of HUVECs in Metal-Doped Cryogels*

**Figure 4.** (**A**) Radar plot representation of secreted cytokine/growth factor profile for HSFs cultured in TM-doped cryogels (0.2 mM) for 24 h (pg/mL, log scale). (**B**) The corresponding levels of selected cytokines/growth factors. The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* <

Human umbilical vein endothelial cells (HUVECs) were used as reference endothelial cells (EC) to compare angiogenic properties of the TM-doped cryogels. The metal dopants (0.2 mM) did not cause any inhibition of HUVEC proliferation, similar to that observed for HSFs, confirming the cytocompatibility of the materials for primary human cells. Furthermore, HUVECs proliferated much faster in the presence of TMs by a factor of ca. 1.3 (Zn), 1.5 (Co) and 1.9 (Cu) compared to the control cryogel (Figure 5A). In comparison with HSFs, HUVECs were characterized by higher sensitivity to Cu and Co, whereas Zn almost abolished the stimulating effect of Cu and Co in binary compositions

**Figure 5.** (**A**) Effect of metal dopants (0.2 mM) on proliferation of HUVECs cultured in TM-doped cryogels (MTS assay, 72 h). The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\*\* *p* < 0.001). (**B**) Corresponding bright-field microscopy images of HUVECs stained with cresyl violet in metal-free (Ctrl) and TM-doped cryogels. **Figure 5.** (**A**) Effect of metal dopants (0.2 mM) on proliferation of HUVECs cultured in TM-doped cryogels (MTS assay, 72 h). The data are presented as mean ± SD (*n* = 3, \* *p* < 0.05, \*\*\* *p* < 0.001). (**B**) Corresponding bright-field microscopy images of HUVECs stained with cresyl violet in metal-free (Ctrl) and TM-doped cryogels.

3.3.2. Angiogenic Differentiation The markers of HUVEC differentiation in the TM-doped cryogels were detected by In addition, HUVECs were visualized within the semi-transparent cryogels after cell staining with cresyl violet (Figure 5B). The calculated cell number per 1 mm<sup>2</sup> of the

**A**

**B**

morphological changes with reorganization of actin cytoskeleton (Figure 6A).

LSCM. In the presence of TMs, phalloidin CruzFluor™ 647-stained cells showed profound

analyzed surface was as follows: 47 ± 8 (Ctrl), 53 ± 6 (Zn), 102 ± 16 (Cu) and 73 ± 11 (Co), supporting the fact that the corresponding MTS signals (Figure 4A) reflected the cell density in the matrix rather than the change in metabolic activity. Furthermore, HUVECs were well adhered and spread on the surface of all cryogels and their morphology was altered to spindle-shaped cells with developed extensions. Specifically, in the Cu-doped cryogel, up to 41% of the cells adopted more elongated morphology with at least three sprouts; the adjacent cells showed a tendency to migrate and connect to each other, which is attributed to the induction of tubulogenesis.

#### 3.3.2. Angiogenic Differentiation

The markers of HUVEC differentiation in the TM-doped cryogels were detected by LSCM. In the presence of TMs, phalloidin CruzFluor™ 647-stained cells showed profound morphological changes with reorganization of actin cytoskeleton (Figure 6A).

In the Cu-doped cryogel, the cells were characterized by increased spreading with ca. 2.2 fold bigger area than control cells (mean cell area detected was <sup>5807</sup> <sup>±</sup> 506 and 2595 <sup>±</sup> <sup>468</sup> <sup>µ</sup>m<sup>2</sup> , respectively). According to immunofluorescence analysis, the expression of VEGF and ICAM-2 factors, which regulate EC viability, migration and microvasculature formation [36], was increased by the metal dopants as follows: Zn < Co < Cu, approximately 1.6–2.9-fold for VEGF and 2.6–5-fold for ICAM-2 (Figure 6B,C). This further suggests profound angiogenic activity of the TMs, especially Cu, under experimental conditions.
