Hypertrophic scar tissue (4 females and 3 males; age range 18–40 years) and wound edge tissue (3 females and 5 males; age range, 18–40 years) samples were obtained from patients who had undergone prior reconstructive burn surgery. Normal skin tissue (4 females and 3 males; age range, 18–40 years) adjacent to the scar excision during surgery was used as a control. Foreskins were also obtained from males undergoing circumcision (10 males; age range, 18–20 years). The present study was approved by the Ethics Committee of the Chinese PLA General Hospital (Beijing, China), and written informed consent was obtained from all individuals prior to obtaining the samples.

Normal skin, wound edge and hypertrophic scar tissue samples were fixed in 10% buffered formalin, dehydrated through ethanol solutions with increased concentration successively, and finally embedded in paraffin, respectively. Paraffin-embedded tissues were cut into 4-μm-thick sections for staining with hematoxylin and eosin (H&E), Masson's trichrome, and methenamine silver (all from Zhongshan Golden Bridge, Beijing, China). Images were captured using an optical microscope (BX53; Olympus, Tokyo, Japan). To perform immunofluorescence staining, the sections were fixed in 4% paraformaldehyde for 30 min, and then permeabilized in 0.2% Triton X-100 (T8787; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 10 min. The sections were incubated with primary anti bodies at 4°C overnight and then with secondary antibodies for 2 h at room temperature. The following primary anti bodies were used: mouse anti-human cytokeratin (CK)10 (1:200, ab111447) and rabbit anti-human CK14 (1:200, ab7800) (both from Abcam, Cambridge, MA, USA), rabbit anti-human CK5 (1:200, ZA-0518; Zhongshan Goldenbridge, Beijing, China), mouse anti-human CK19 (1:200, ab53119; Abcam), rabbit anti-human integrin-β1 (1:200, PB0063; Boster, Wuhan, China); mouse anti-human integrin-β4 (1:200, ab128068; Abcam), mouse anti-human laminin (1:200, ZM-0181; Zhongshan Goldenbridge), rabbit anti-human laminin-5 (1:200, ab14509; Abcam) and mouse anti-human collagen IV (1:200, ZM-0081; Zhongshan Goldenbridge). The following secondary antibodies were used: Alexa-Fluor 488-conjugated anti-mouse (1:200, ab150117) and Alexa-Fluor 594-conjugated anti-rabbit (1:200, ab150080) (both from Abcam). The nuclei were stained with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were captured using a confocal laser scanning microscope (SP8; Leica, Solms, Germany).

Primary human epidermal keratinocytes (HEKs) were isolated from the male foreskins as previously described (13), with minor modifications. The human immortalized keratinocyte (HaCaT) cells were purchased from the China Infrastruture of Cell Line Resources (3111C0001CCC000373; Beijing, China). Both the HEKs and the HaCaT cells were incubated in EpiLife medium (M-EPI-500-CA; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 0.06 mM Ca²⁺, 1% EpiLife defined growth supplement (S-001-5; Invitrogen Life Technologies), and 1% penicillin/streptomycin (P1400; Solarbio, Beijing, China). To investigate the roles of the BM in regulating keratinocyte behavior in vitro, the HEKs and HaCaT cells were plated in 60 mm-dishes coated with MaxGel ECM (E0282) and collagen type IV (C7521) (both from Sigma-Aldrich), respectively. For Ca²⁺ treatment, the cells were incubated in EpiLife medium supplemented with 1.5 mM Ca²⁺ for 0, 12 and 24 h. The MaxGel ECM contains human extracellular matrix (ECM) components including collagens, laminin, fibronectin, tenascin and elastin, as well as a number of proteoglycans and glycosaminoglycans. The cells not treated with Ca²⁺ were used as controls.

Total RNA was isolated from the cells after they were subjected to the various treatments using TRIzol reagent (15596-026; Invitrogen, Carlsbad, CA, USA) and then cDNA was synthesized using GoScript reverse transcriptase (A5001; Promega, Madison, WI, USA) according to the manufacturer's instructions. The primer sequences used for gene amplification are listed in Table I. qPCR reactions were performed with GoTaq qPCR Master Mix (A6001; Promega) using an ABI 7500 Real-Time PCR system and software (Applied Biosystems, Foster City, CA, USA). The reaction protocol consisted of the following cycles: 95°C for 15 sec, 55°C for 30 sec, and 72°C for 30 sec for 40 cycles of PCR amplification.

Total protein was isolated from the cells and 20 μg of protein was dissolved in substrate-soluble buffer. The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (IPFL00010; Millipore, Billerica, MA, USA). After blocking, the blots were probed with the primary antibodies overnight at 4°C. The antibodies used for western blot analysis included CK10 (1:1000, ab111447), CK14 (1:500, ab7800), CK19 (1:500, ab53119), integrin-β4 (1:500, ab128068), and mouse anti-human β-actin antibody (1:5000, ab6276) (all from Abcam). After washing, the membranes were incubated with secondary antibodies, which were goat anti-rabbit and goat anti-mouse IgG conjugated to horseradish peroxidase (sc-2004, 1:1000; sc-2005, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were detected by enhanced chemiluminescence (ECL) kit (PE-0010-100; Solarbio, Beijing, China) and imaged using the ImageQuant LAS 4000 system (GE Healthcare Bio-Sciences, Pittsburgh, USA).

All experiments were repeated at least 3 times, unless otherwise indicated. Data are presented as the means ± standard deviation (SD). Statistical analysis was performed using SPSS 20.0 software. Data comparison was performed using the independent Student's t-test and one-way ANOVA followed by the LSD test. A P-value <0.05 was considered to indicate a statistically significant difference.

Skin wound repair is a precise remodeling process, which is divided classically into four overlapping phases and involves the interaction of many different tissues and cell lineages (5). In chronic wounds, however, the normal healing process is interrupted, which results in a pathological cycle of inflammation and protease release leading to the development of a pathological scar. In this study, to evaluate the morphological changes occurring in the regenerating epidermis during skin wound healing, specimens of normal, wound edge and hypertrophic scar tissues were obtained in order to perform routine H&E and Masson's trichrome staining. In the normal skin samples, the histological structure of the skin was visible, showing layers of the epidermis and dermis. The epidermis contained 4 to 5 layers, in which keratinocytes differentiated and gradually matured into spinous cells, granular cells and cornified cells during their outward passage (Fig. 1A). Moreover, in the underlying dermis, fibroblasts made up loose connective tissue, which was rich in parallel collagenous fibers (Fig. 1D). In the tissues around the wound edge, however, the epidermis proliferated; it was comprised of multiple layers of keratinocytes of distinct morphology and was of visibly increased thickness (Fig. 1B and E). The epidermis and dermis interlocked through finger-like projections (called rete ridges), which increased the surface area of contact between the epidermis and the dermis. In both the normal and wound edge tissues, the basal keratinocytes were arranged as a regimented single layer of cuboidal or low columnar cells, which connected to the spinous cell layers above (Fig. 1A and B). The histological structure of the hypertrophic scar tissue differed from that of normal skin tissue by having a rich blood supply, and a thick epidermal layer (Fig. 1C and F). The dermis was composed largely of dense, disorganized connective tissue that includes tough collagenous fibers of an irregular shape and a larger diameter (Fig. 1F). To further clarify the differences in histological structure among the normal, wound edge and hypertrophic scar tissues, methenamine silver staining was performed to assess the formation of the BM, which indicated that the BM structure was detectable in the sections of the normal and wound edge tissues (Fig. 1G and H). In the scar tissues, however, BM staining was absent (Fig. 1I).

Given the morphological differences which were observed in the normal, wound edge and hypertrophic scar tissues, we examined whether the basal keratinocytes exhibited a different cellular behavior during scar formation. Initially, we performed immunofluorescence staining to detect the expression of CK10, CK14, CK5, CK19 and integrin-β1 in the sections of normal, wound edge and scar tissues. Previous studies have verified that CK10 is a marker of differentiated keratinocytes, that CK14 and CK5 are markers of proliferating basal keratinocytes or TAs, and that CK19 is a putative marker of epidermal progenitor cells or stem cells in the skin (14–17). Integrin-β1 is also thought to be a marker of precursor cells in the skin (18,19). Our results indicated that CK10 was expressed in the outer layer of the epidermis in the normal and wound edge tissues (Fig. 2A and B), and in the suprabasal, terminally differentiating cells in the epidermis of the hypertrophic scar tissue (Fig. 2C). CK14 was expressed in both basal and suprabasal layers in the stratified epidermis of all three tissues (Fig. 2D–F), and there was an extensive distribution of CK14 in the multilayered epidermis of the scar tissue (Fig. 2F). The distribution pattern of CK5 was similar to that of CK14 (Fig. 2G–I), which indicated an increased number of proliferating cells in the hyperproliferative epidermis. Integrin-β1 and CK19 were expressed in the basal layer of the normal epidermis (Fig. 2J and M) and in the basal and supra-basal layers of the wound edge epidermis (Fig. 2K and N), but were not detectable in the epidermis of the hypertrophic scar tissues (Fig. 2L and O). Collectively, these observations indicate that the keratin expression profile of basal keratinocytes differed between the normal, wound edge and hypertrophic scar tissues. Although fewer CK19-expressing cells were detected in the basal and suprabasal layers of the hypertrophic scar tissue (Fig. 2P), a proliferative phenotype was revealed due to the increased expression of CK14 and CK5 in the multilayered epidermis.

As a key component of the stem cell niche, the ECM not only anchors stem cells but also directs their fate (20). As mentioned above, the histological structure of the BM appeared to be absent in the hypertrophic scar tissue samples. Given the differences in the keratin expression profiles in the basal layer of normal, wound edge and hypertrophic scar epidermis, we then speculated that the structural abnormalities of the BM play a role in regulating the cell fate decision of basal keratinocytes during wound healing. To examine this hypothesis, immunofluorescence staining was performed to visualize the components of the BM in the normal, wound edge and hypertrophic scar tissue samples. Our results revealed that collagen IV expression was detected in the BM area in both normal (Fig. 3A) and wound edge (Fig. 3B) tissues. However, the formation of BM-like structures with an absence of collagen IV expression was observed in the epidermis of hypertrophic scar tissues (Fig. 3C). Although the expression of laminin was detected in all three tissue types (Fig. 3D–F), the double-labelling of laminin-5 and its receptor integrin-β4 further verified our observation that the structure of the BM was altered in the epidermis of the hypertrophic scar tissues; negative staining of both laminin-5 and integrin-β4 (Figs. 3I and 4G–I) was observed as compared with that in the normal (Figs. 3G and 4A–C) and wound edge tissues (Figs. 3H and 4D–F). Given these findings, it is likely that the normal BM contributed to the recruitment of integrin-β1- and CK19-expressing cells in the basal layer of the normal and wound edge epidermis. Abnormalities in the BM structure, however, induced the basal keratinocytes to differentiate and adopt a proliferative phenotype during scar pathogenesis.

The mechanical support provided by the BM is determined primarily by its type IV collagen scaffold (21), into which the laminin networks and perlecan oligomers are integrated by the cross-linking nidogens to finally assemble the sheet-like BM complex (22). To further clarify the roles of the BM in the regulation of epidermal keratinocyte behavior, we used the human BM extract-derived ECM to coat the culture dishes in order to control the pattern of cell-matrix adhesion. As shown in Fig. 5, ECM administration resulted in the upregulation of integrin-β4 expression in the HEKs (Fig. 5A) and the HaCaT (Fig. 5B) cells, which was present on the plasma membrane and at the sites of cell-matrix attachment. We also performed western blot analysis to examine the protein levels of integrin-β4 in the epidermal cell lines with or without ECM coating. Our data demonstratd that higher levels of integrin-β4 were detectable in the ECM-treated groups than in the controls, which further confirmed the fluorescent immunostaining findings (Fig. 5C). Moreover, the HEKs and HaCaT cells were switched to medium containing 1.5 mM Ca²⁺ for the indicated periods of time to generate differentiating epidermal cells as previously described (23,24). Following Ca²⁺ treatment, the morphological alterations in both the HEKs (Fig. 6A) and HaCaT cells (Fig. 6B) were observed under a light microscope, which included cell enlargement and flattening. Ca²⁺ induced the expression of CK10 (Figs. 5D, G and J, and 6C and D) and CK14 (Figs. 5E, H and J, and 6C and D) in a time-dependent manner in epidermal cell lines at both the mRNA (Figs. 5D, E, G and H, and 6C and D) and protein (Fig. 5J) levels, with peak levels of CK10 and CK14 expression being observed 24 h after initiating 1.5 mM Ca²⁺ treatment in the HEKs (Figs. 5D, E and J and 6C) and the HaCaT cells (Figs. 5G, H and J and 6D). Following Ca²⁺ treatment, the expression of CK19 was downregulated in the HEKs, reaching its lowest level at 24 h after the start of Ca²⁺ treatment (Figs. 5F and J and 6C). Similar results were observed in the HaCaT cells (Figs. 5I and J and 6D). ECM treatment, however, reduced the differentiating responses of keratinocytes associated with Ca²⁺ administration in the HEKs and HaCaT cells, and enhanced the expression of CK19 at 12 h and 24 h after Ca²⁺ treatment (Fig. 5F, I and J). To further determine the potential role of the BM in the regulation of epidermal keratinocyte behavior, type IV collagen was used to form a BM-like structure in vitro. Collagen IV treatment contributed to CK19 expression and reduced the expression of CK10 and CK14 at the mRNA and protein levels in both the HEKs (Fig. 7A–C and G) and the HaCaT cells (Fig. 7D–F and G). These results further validated our in vivo findings that the BM appears to be a positive regulator of the recruitment of precursor-like cells in the basal layer of the epidermis. Alterations in the BM structure promoted the development of a proliferative phenotype in basal keratinocytes as indicated by the enhanced expression of the markers associated with cell proliferation, e.g., CK14 and CK5.