*2.2. The Quantification of Aggregate Protein at the Wound Site*

An adequate supply with proteins is necessary for consistent wound healing. Therefore, protein content can be used to evaluate the conditions of wound healing [27]. Total protein expression at the wound site of all groups in the excision wound model is shown in Table 1. On day 3 and 7, protein content of the experimental groups was higher than the control and the woundplast groups (*p* < 0.05), and the protein content was high in BCE I and BCE II treated groups compared to other groups on day 7 (*p* < 0.05). On day 14, protein content of the experimental groups was significantly higher than control group (*p* < 0.05). Protein content of ACS treated group was significantly higher than BCS, BCE I, and BCE II treated groups (*p* < 0.05).


**Table 1.** The effect of each group on total protein content in wound area tissues (*x* ± *s*, *n* = 6).

Note: Different superscript alphabets in each column represent statistical significance (*p* < 0.05).

#### *2.3. Hydroxyproline (Hyp) Content at the Wound Site*

Hyp is a specific component of the protein collagen. Therefore, Hyp content might be used as an indicator to determine collagen deposition to measure the speed of wound healing [28]. The significant increase of Hyp in collagen treated group implied faster rate of wound healing process than control group (*p* < 0.05). Indeed, collagen is a major protein of the extracellular matrix and it ultimately contributes to wound healing [29]. Hyp content at the wound site of all groups in the excision wound model is shown in Table 2. Hyp content was higher in ACS and BCE II treated groups (5.87 ± 0.42 mg/g, 5.66 ± 0.12 mg/g tissue) on day 3 and in PCS, ACS, BCE II treated groups on day 7 (*p* < 0.05). On day 14, PCS and ACS treated groups had high Hyp content (7.32 ± 0.43 mg/g, 7.41± 0.42 mg/g tissue) than control and woundplast treated groups.


**Table 2.** The effect of each group on the Hydroxyproline content in wound area tissues (*x* ± *s*, *n* = 6).

Note: Different superscript alphabets in each column represent statistical significance (*p* < 0.05).

### *2.4. Histopathological Examination*

In control group, a large number of inflammatory cells appeared in the wound than collagen-treated groups on day 3 (Figure 2). Granulation tissue and fibroblasts activity were more pronounced in collagen-treated groups compared to control group. A large number of inflammatory cells, blood vessels, small amount of collagen fibers and fibroblasts were seen in woundplast and BCS groups. Fibroblast cells were important in the wound site and predominant collagen expression could be seen in PCS, ACS, BCE I, and BCE II treated groups. On day 7, healed regions of the wounds were covered by epithelial tissue with significant fibroblast proliferation, collagen deposition and granulation tissue formation. However, the granulation tissue organization and vascularization in unhealed regions of collagen-treated groups were notably different from the control group. Collagen-treated groups developed collagen deposition and vascularization than the control group. The H&E staining revealed that in collagen-treated wound on day 7, cutaneous appendages like hair, hair follicles and sebaceous glands began to appear, which signify the formation of epidermal layer. A large number of inflammatory cells and collagen fibers and fibroblasts were observed in control group. A large number of fibroblasts and collagen were seen in woundplast and BCS treated groups and skin appendages and skin tissues were gradually formed in collagen treated groups. More fibroblasts and collagen and less inflammatory cells were seen in PCS, ACS, BCE I, and BCE II treated groups than the control group. On day 14, a sufficient number of fully formed skin adnexal and epithelial tissues were present in collagen-treated groups. PCS, ACS, BCE I, and BCE II treated groups showed well-formed stratified epithelial layer, granulation tissue formation and collagen deposition in healed regions than the control, woundplast and BCS groups. As shown in Table 3, a scoring system was used to quantify the pathological results of the H&E stained samples. In this scoring system five criteria were scored. An increasing score denotes the better wound healing. The histological scoring result showed that the PCS, ACS, BCE I, and BCE II treated groups had better wound healing than the control group.

**Table 3.** Histological evaluation of wound tissue in all groups by H&E staining.


**Figure 2.** Histological analysis of H&E stained wounded tissues (Magnification, ×100) with histological scoring. The alphabetic letters e, d, gt and hf represent epidermis, dermis, granulation tissue, hair follicle, respectively. (Each bar represents the mean ± SD. \* *p* < 0.05: significantly different from the control group.).

#### *2.5. Collagen Promotes the Expression of EGF, FGF, and CD31 in the Wounds*

Immuno-histochemical analysis of the reconstituted tissue was shown in Figures 3–5. EGF stands for epidermal growth factor, which can promote proliferation and differentiation of keratinocyte. EGF can induce fibroblasts proliferation and collagen synthesis, resulting in epithelization. Fibroblast growth factor (FGF) can accelerate migration and proliferation of fibroblast, and vascularization. EGF and FGF play an important role in the wound healing process. Prolonging the treated time, the expression of EGF and FGF in control group showed an increasing trend, the expression of EGF and FGF in woundplast and BCS groups increased in the beginning and then decreased, and the expression of EGF and FGF in PCS, ACS, BCE I, and BCE II treated groups showed a decreasing trend. On day 3 and 7, the expression of EGF and FGF in PCS, ACS, BCE I, and BCE II treated groups was higher than control, woundplast and BCS groups. CD31 is a platelet endothelial cells adhesion molecule-1, stands for vessel proliferation. The relative quantity and distribution of CD31 in the construct-treated wound bed is important in wound healing. Brown granules were positive signal in Figure 3. The expression of CD31 showed similar trends same as EGF and FGF of collagen treated groups.

**Figure 3.** Immunohistochemical analysis of EGF expression in wounded tissues (Magnification, ×100); the histogram shows the total of positively stained cells of EGF in the dermal tissue per group. (Each bar represents the mean ± SD. \* *p* < 0.05: significantly different from the control group.).

**Figure 4.** Immunohistochemical analysis of fibroblast growth factor (FGF) expression in wounded tissues (Magnification, ×100); the histogram shows the total of positively stained cells of FGF in the dermal tissue per group. (Each bar represents the mean ± SD \* *p* < 0.05: significantly different from the control group.

**Figure 5.** Immunohistochemical analysis of CD31 expression in wounded tissues (Magnification, ×200). The histogram summarizes the microvessel density (MVD), which was determined by immunohistochemical staining for CD31. (Each bar represents the mean ± SD. \* *p* < 0.05: significantly different from the control group.).

#### **3. Discussion**

As the structural and functional component of dermal extracellular matrix, collagen plays a vital role in wound healing process [18,30–32]. In the present study, PCS, ACS, and electrospun bovine skin collagen nanofibers were successfully fabricated. Due to its unique perforated structure, controllable fiber diameter (50 nm–5 μm), large surface area, high porosity and unique biological property [33], PCS, ACS and BCE I, BCE II were examined by using the full-thickness wound model in SD rats and the total protein, hydroxyproline content, H&E, and immunohistochemical examinations were assessed with control group. In full-thickness wounds in SD rats, PCS, ACS, BCE I and BCE II treated groups revealed significantly higher wound healing ability, total protein and hydroxyproline content, fibroblasts proliferation and collagen synthesis when compared to control, BCS and woundplast groups.

Based on the earlier findings, the possible reasons for accelerating wound healing of PCS, ACS, BCE I, and BCE II were due to its unique structures and the high porosity of the matrix, which could induce fibroblasts proliferation and collagen synthesis, and play crucial roles in re-epithelialization and vascularization in the wound healing process [34,35]. In addition, PCS, ACS, BCE I, and BCE II exhibited good biocompatibility to support the adhesion and proliferation of fibroblasts that lead the deposition and maturation of collagen [9]. Collagen could be completely degraded and absorbed by the wound that helps to formation of fibroblasts and collagen fibers over time in epidermal tissue and therefore, the wounds were replaced with regenerated dermis.

Histopathological and immunohistochemical examinations indicate that PCS, ACS, BCE I, BCE II had a positive effect on neovascularization, inducing fibroblasts proliferation, collagen synthesis, re-epithelialization and regeneration of skin appendages, and consequently led to an increased wound healing ability compared with control, BCS and woundplast groups. Besides, PCS, ACS, BCE I and BCE II were similar to normal skin because of smooth surface along with loose collagen fiber. PCS, ACS, BCE I, and BCE II could provide a proper microenvironment for fibroblasts attachment in skin due to its three-dimensional features and high porosity.

Vascular endothelial marker CD31, growth factors EGF and FGF are involved in wound healing process [36]. It has been confirmed that PCS, ACS, BCE I, and BCE II significantly induce EGF and FGF expression, which can promote proliferation and differentiation of fibroblasts and keratinocytes. EGF can induce keratinocytes proliferation to impel re-epithelialization of the wound. As chemotactic agent and mitogenic agent of fibroblasts, EGF can promote the proliferation and differentiation of fibroblasts to synthesize collagen. The increased expression of CD31 and FGF revealed the vascularization and wound healing properties of collagens.

In this work, PCS and ACS groups revealed significantly better wound healing ability than woundplast and control groups, slightly higher than BCS group, which might be due to the three-dimensional features and high porosity of PCS and ACS than BCS. However, ACS had a more positive effect than PCS, which was similar to previous report [22]. There was no much difference between BCE I and BCE II treated groups. However, there was less inflammatory cells in BCE II treated group than BCE I, which may be related to the bactericidal effect of chitosan. These effects were probably owed to the biomimetic structure, unique biological property and high porosity of the collagen nanofibers.
