Morphology and structure of Ch-PVA and silk fiber
Polymer solutions were continuously extruded from the tip of the needles in co-electrospinning process while drawing the polymer solutions towards the collector continues under the influence of the electric field. During process crystalline cone created and the jet thrown toward the collector observed [1, 2, 5, 8, 11]. Smooth and uniform fibers with porous structure was fabricated from neat PVA. The fiber morphology did not change with addition of Ch and incorporation of silk solution in co-electrospinning process (Fig. 2B and C). The produced Ch-PVA + Silk fibrous mat was bead-free and smooth without any branching in regular shape during the process and observation showed that blended fibers get greater fiber flexibility. The fibrous mats were tightly packed and highly entangled without node formation (Fig. 2A-C). While, the mean diameter of fiber mat produced from neat PVA was 842 ± 205 nm (Fig. 2A). The diameters of composite Ch-PVA and Ch-PVA + Silk increased at 1070 ± 340 and 1200 ± 321 nm respectively (Fig. 2B and C). However, statistically there was no significant difference in size distribution or average diameter of fibrous mats in different compositions (Fig. 2E, F and G). These proved that suitable concentration and ratio of polymers as well as applied voltage in agreement with previous studies [2, 5, 8, 11]. In order to achieve a good electrospun fiber scaffold, suitable concentration of polymer and solubility in solvent are vital parameters to obtain homogenous, continuous and uniform size specimen which could result lack of phase separation traces [1, 2, 5, 8, 11]. Meanwhile, the average pore sizes of PVA and Ch-PVA were 1.33 ± 0.49 and 0.9 ± 0.31 µm. The mean pore size of Ch-PVA + Silk fibrous mat was 2.2 ± 0.9 µm which indicated better pore size for permeation of oxygen and metabolites (Table 1). The Ch-PVA + Silk fiber had a porosity of 68 ± 4.4% of the total scaffold volume in comparison with 74 ± 3.9% and 52 ± 4.7% for neat PVA and hybrid Ch-PVA fibrous mats (Table 1). The high porosity of the PVA fiber is desired due to ease transfer of nutrients, oxygen and other metabolites as well as support moisture and hydrophilicity of structure. Moreover, the pore size and interconnectivity of pore network would be facilitated cellular migration and in following angiogenesis which is important in wound healing and tissue engineering. The smallest pore size and porosity of fiber mat found for Ch-PVA fiber which could be due to water solubility of polymers as well as proper selection of processing parameters [5, 8, 14, 21, 26].
Fig. 3 showed the FTIR spectrums of the fabricated PVA fiber. The peaks of PVA were revealed by the bands 829, 1110, 1731, 2925 and 3185 cm-1 for vibration stretching of C–C in alkyl chain back bone, C-O stretching, C=O stretching, C-H of alkyl stretching mode and OH vibrations respectively. The FTIR spectrum of Ch-PVA fiber mat showed a broad peak at 3338 cm-1, attributed to the O-H and N-H stretching vibrations which
shifted to the higher wave number with addition of Ch to PVA materials (Fig. 3). The resonance band at 1645 and 1512 cm-1 related to amide I band (C=O stretching) and amide II (N-H bending and C-H stretching). Additionally, the pair peaks of 1129 and
921 cm-1 were arising from saccharide structure of Ch and also 1089 and 1027 cm-1 were related to the skeletal vibrations of C=O stretching of the glucosamine
residues. The main characteristic absorption bands at 1636 cm-1 and 1537 cm-1 corresponded to amide I, C=O stretching and for amide II, secondary N-H bonding due to b-sheet structure in silk structure. The peaks at 1341 cm-1 and 727 cm-1 assigned for amide III, C-N and N-H functionalities and for amide V respectively. The created fiber structure in Ch-PVA + Silk are due to intermolecular hydrogen bonds which occur between the hydroxyl group in PVA and the -N-H/C=O bonds in grafted Ch and silk. However, noted shifts were not detected in the FTIR as there was no functional group alternation in the composite fiber. The FTIR spectroscopy could detect presence of the chemical bonds of specific polymers utilized for composite Ch-PVA + Silk fibrous mat.
Swelling and hydrophilicity
Hydrophilic/hydrophobic property of hydrogel manipulates cellular behavior including cell attachment, proliferation and migration potential through permeation of nutrient, metabolites and oxygen [5, 8, 10, 14]. Moreover, wettability of dressing area is an important element to maintain tissue moisture during wound healing process [5, 8, 10, 14]. The PVA fiber showed trivial water absorption at 21% of initial weight of fiber (Fig. 4 A). The hybrid fibers of Ch-PVA and Ch-PVA + Silk absorbed water molecules up to 51% and 74% respectively which indicate addition of Ch and silk could improve swelling behavior of fibers (Fig. 4 A). High water absorbency of hydrogel fibers containing Ch and silk could be due to hydrogen bonding among polymeric chains and water molecules [1, 5, 6, 10, 14, 27]. The hydrophilic functional groups in polymeric structure of Ch-PVA + Silk such as amine (NH2), hydroxyl [-OH], amide (-CONH-, -CONH2) and sulphate (-SO3H) would be improved swelling degree of fibers [5, 10, 14, 27]. As seen in Fig. 4B, average contact angles of the fibers decreased by hybridization pf polymers from 112° ± 8.1° for neat PVA until 54° ± 6.4° for hybrid Ch-PVA + Silk fibers which means higher hydrophilic characteristics of resultant fibers. The utilization of Ch and silk enhanced hydrophilic characteristic of fibrous mats owing to the presence of hydrophilic groups of Ch and silk on their structures and also Wenzel wetting model that occurs on heterogeneous surface geometries [26, 28]. The Ch and silk could circumvent poor hydrophilicity of PVA fiber and provide desirable microenvironment for cell culture, tissue engineering as well as wound healing application [5, 8, 14, 21, 27, 28].
Mechanical property and degradability
Blending of PVA with Ch or incorporation of silk have an obvious impact on the mechanical properties of PVA fibrous mats (Fig. 5A-C). Mechanical strength and Young’s modulus of PVA based fibrous mat reduced by admixing Ch to the PVA solution (Fig. 5A and B). The Ch-PVA + Silk fibrous mat exhibited improvement in mechanical strength and Young’s modulus with incorporation of co-electrospinning silk and these values were more than two times of results of PVA or Ch-PVA fibrous mat (Fig. 5A and B). Although, porosity and average pore size of Ch-PVA fibrous mat was lower than PVA fiber which means tight fiber network. However, the results suggest that chemical interaction of co-electrospun silk and blended Ch with PVA could be reason of enhanced strengthening [5, 12, 20]. The PVA fiber mat was break at about 100% elongation. The breaking strain of fibers was found to increase from around 100% to 220% upon utilization of Ch and silk in composite fibrous mat (Fig. 5C). Degradation of fibrous mat is important parameter in applicability of tissue engineering scaffolds which allow cell proliferation, migration and extracellular matrix replacement during cellular growth [2, 12, 14, 19, 20, 28]. The neat PVA fiber mat showed lowest rate of weight loss compared to Ch-PVA and Ch-PVA + Silk mats (Fig. 5D). The weight loss of PVA specimen was lower than 20% until 14 days. The hybrid fibers comprising Ch-PVA and Ch-PVA + Silk mats showed an accelerated weight loss at about three and two times compared with PVA sample until 14 days (Fig. 5D). The Ch-PVA can be easily dissolved and hydrolyzed in aqueous media. Meanwhile, incorporation of silk in Ch-PVA+ Silk fiber due to highly fibroin b sheet in the silk structure resulted slower degradation compared with Ch-PVA fiber mat [10, 27]. These results would be confirmed by corresponding mechanical stiffness and hydrophilicity behavior of produced fibrous mats.
Stem cell characterization and differentiation
Isolated MSCs on culture dish could be adhered to dish culture and totally were spread and elongated in homogenous shape and morphology (Fig. 6A). Presence of MSC markers including CD73 and CD105 and the absence of endothelial cell marker CD31 as well as hematopoietic stem cell marker CD45 confirmed by flow cytometry analysis of the MSCs with specific antibodies Fig. 6B [3, 29-31]. Therefore, flow cytometry verified the bMSC characteristics for isolated cells. To induce keratinocyte differentiation of MSCs in long-term cultures, the isolated cells were exposed specific differentiating media [32, 33]. The MSC differentiation observed by phase contrast microscopy and immunocytochemical analysis compare with mice foreskin-derived keratinocytes as a positive control (Fig. 6A and C). The microphotograph shows evidences of polarization in the treated cells with differentiating media on day 18 and cells were in polygonal and cobblestone morphology with colonies likewise foreskin-derived keratinocytes which is specification of keratinocyte (Fig. 6A). As shown in Fig. 6C the differentiated MSCs into keratinocytes were positively expressed keratinocyte proteins including cytokeratin-19 (CK-19), involucrin (IVL) and vimentin (Vim) markers after 18 days in conditioned culture media similar results with foreskin-derived keratinocytes. It can be seen MSCs were negative to express CK19, IVL and Vim in undifferentiated media and did not expressed essential keratinocyte proteins. The utilization of calcium stimulus and EGF which bind to EGF receptor triggering signals was reliable approach for isolated MSC differentiation into keratinocytes. These data support the possibility of wider applications of MSCs for tissue engineering of skin through evidence for epidermal differentiation induction.
Cell viability and proliferation
To evaluate the biocompatibility of fabricated fibrous mats, cell culture experiments were performed by seeding MSC-derived keratinocytes on samples. The cellular morphology and metabolic activity of cells detected by SEM, DAPI staining and MTT assays for seeded cells on fibrous mats as well as polystyrene tissue culture plate (PS; control). Electronic microscopy photographs showed that cells attached, grew and spread extensively with cytoskeleton extension on the Ch-PVA and Ch-PVA + Silk fibrous mats (Fig. 7A and B) which proves Ch and silk might be appropriate structure for cell culture systems [5, 34]. Whereas, the MSC-derived keratinocytes on PVA fibrous mat did not attached and were in spheroid shape (Fig. 7C). As can be seen, there were no significant differences between the cell viability of PVA and Ch-PVA fibrous mats in initial time of culture (Fig. 7D). The Ch-PVA + Silk and polystyrene tissue culture plate (PS) conditions showed higher number of attached cells in initial time of culture at 12 h. Interestingly, viability of MSC-derived keratinocytes enhanced during extend time of incubation for Ch-PVA + Silk fibrous mat and cellular growth was reached 3 times higher than neat PVA fibrous mat group (Fig. 7D). The cells could maintain their viability on PVA and Ch-PVA during extended time of incubation and lower cellular growth could be due to lack of sufficient cell adhesive ligands in these fibrous mats. However, cells seeded on PS showed higher value compared with other cell seeded fibrous mat conditions (Fig. 7D). Besides, as seen in Fig. 7E-G, composite Ch-PVA + Silk mats showed higher number of viable cells and their accumulation using dapi staining which confirmed proliferation of MSC-derived keratinocytes. Results in desired cell proliferation could be due to existence of cell recognition signals as well as hydrophilic surface tendency of Ch-PVA + Silk fibers (Fig. 4A and B) [3, 7, 16, 27, 28, 32, 35]. In summary, these data substantiated that Ch-PVA + Silk fibrous mat possessed proper cytocompatibility and incorporation of Ch as well as silk resulted increase in cell adhesion and proliferation. Hence, hybrid Ch-PVA + Silk fiber revealed high biocompatibility and potential applicability for wound healing and tissue engineering applications (Fig. 7).
Wound closure evaluation and histological analysis
Due to proper in vitro results of Ch-PVA+ Silk fiber mat (Fig. 7), this fiber construct was utilized for in vivo wound treating experiments. We performed in vivo wound treating experiments using paraffin sterile gauze (as a negative control; Ctrl), and Ch-PVA + Silk fiber with and without MSC-derived keratinocytes for 14 days. The wound sizes show treatment-dependent and time-dependent differences in rate of wound contractions as indicated in Fig. 8A. The wound closure measurements indicated significant wound healing after 7 days treating by Ch-PVA + Silk fibrous mat with and without MSC-derived keratinocytes compared to paraffin sterile gauze condition (Fig. 8A). The decrease in size of the wound area in comparison to its original size was 87.4 3.4 % for Ch-PVA + Silk fibrous mat with MSC-derived keratinocytes, 79.1 4.1 % for Ch-PVA + Silk fibrous mat while it was only 57.2 5.4 % for the paraffin sterile gauze condition at the 14th day. High degrees of wound healing suggest that the Ch-PVA + Silk fibrous mat due to worthy moisture condition and distinctive hemostatic properties could actively promote wound healing process with higher degrees of re-epithelialization [16, 19, 20, 27, 28, 32].
Histological analysis of the wounds in the paraffin sterile gauze group displayed evident leukocyte infiltration, granulation tissue formation, and a crusty scab, however, epidermal layer has not been formed (Fig. 8B). Histopathological evaluation of Ch-PVA + Silk group showed moderate infiltration of inflammatory cells into the wound area (Fig. 8B). Although, the epithelialization process was initiated, this process was incomplete in all samples in this group and the epithelial layer was thinner than normal skin. The inflammatory cells were significantly reduced at Ch-PVA + Silk group in comparison to paraffin sterile gauze group. Besides, we did not observed wound infection for treated conditions. It is expected that the antimicrobial effect of Ch due to destabilization of the outer membrane of gram-negative bacteria and permeabilization of the microbial plasma membrane be the reson for obtained results [3, 29, 36-39]. However the antibacterial effect and possible mechanism of the combination therapy is required to study through agar plate method and bacteriostatic ring test [38, 40].
Histopathological evaluation of the wounds treated by Ch-PVA + Silk fiber with MSC-derived keratinocyte group showed a considerable reduction of inflammatory cells in comparison to the other groups (Fig. 8B). A complete epithelial layer with the presence of rete ridges was formed in Ch-PVA + Silk fibrous mat containing cells. This group showed more resemblance to normal skin, with a thin epidermis (normal thickness of skin layers), presence of normal rete ridges, rejuvenation of the hair follicles (skin appendages). It seems that this treatment showed the best results while compared to other experimental groups. It is reported that keratinocytes play a vital role in epidermal restoration during wound healing through proliferation, migration and re-epithelialization. The differentiated MSC to keratinocyte would implement its impact through provision of structural support, protection of epithelial cells from mechanical and non-mechanical stress and the regulation of apoptosis and protein synthesis [23, 40]. The wound healing process is heavily dependent on collagen synthesis. Therefore, to further investigate the effect of different treatments on wound healing, sections of animal skin tissues were stained with MT staining. This staining was used to recognize the progress of collagen synthesis during granulation tissue (GT) formation and matrix remodeling. The collagen fibers were stained blue-green in MT staining method which the intensity of this color corresponds to the relative amount of deposited total collagen and reflects the advancement of collagen synthesis and remodeling [13, 14, 16, 33, 36]. The results indicated that among the experimental groups, the Ch-PVA + Silk with MSC-derived keratinocyte group had the greatest collagen synthesis. On the other hand, the rate of collagen fiber synthesis and deposition in wound were the lowest in paraffin sterile gauze group.
The histomorphometric analysis was done and the results have been presented in Table 2 and 3. Amongst all groups, re-epithelialization in control groups was minimum and it was mostly filled with immature GT formation. The best re-epithelialization was seen in the Ch-PVA + Silk + MSC-derived keratinocyte treatment group. Moreover, the total number of inflammatory cells in Ch-PVA + Silk + MSC-derived keratinocyte treatment group was significantly reduced in comparison with others (P-value < 0.01). Overall, the healing results of Ch-PVA + Silk + MSC-derived keratinocyte treatment group was more similar to that of the normal skin with normal thickness of epidermal layer and rejuvenation of the hair follicles and other skin appendages.