Characteristics of the BLM Scaffold

SEM images of the BLM scaffold revealed a dense outer layer of nano-sized PGLA fibers of diameter 857.02 ± 211.81 nm, and a loose microporous alginate hydrogel underneath. The thickness of the PLGA and alginate hydrogel layer were 20 and 100 μm, respectively. The FTIR results of the samples are shown in . The spectrum of raw PLGA data shows absorbance peaks at wavelengths of 753 (CH-bend), 1,082 and 1,170 (CO stretch), 1,388 (CH-bend), 1,750 (CO ester), and 2,847 (CH 2 -bend) cm (), while all spectrum peaks were observed in the PLGA nanofiber layer in BLM. Raw alginate produced a peak at 3,400 cm indicating the presence of hydroxyl groups. Asymmetric and symmetric –COO groups are indicated by the peaks at a wavelength of 1,597 and 1,407 cm, respectively. The CO group is indicated by the peak at a wavelength of 1,027 cm. For alginate hydrogel layer in BLM, peaks were observed at the same sites (). The FTIR results showed that the preparation process does not affect the chemical properties of PLGA and alginate. The moisture retention and vapor transmission rates of the PLGA, alginate hydrogel and BLM scaffold are shown in . While the hydrogel alone showed poor moisture retention and lost most of the water within 24 h, the BLM scaffold retained its moisture for 72 h (). Consistent with this, the PLGA nanofiber and BLM scaffolds had lower vapor transmission rates compared to that of alginate hydrogel (). Thus, the PLGA nanofiber membrane reduces evaporation from the alginate hydrogel. This is critical for wound healing since adequate moisture not only reduces the epithelial cell death but also promotes their migration and proliferation, and helps in tissue regeneration. Furthermore, while only 20% of the PLGA nanofiber degraded within 4 weeks, the degradation rate of alginate was slow in the first 2 weeks, and accelerated to 80% thereafter (), which is consistent with previous reports (Danhier et al., 2012; Lee and Mooney, 2012). The degradation rate of the BLM scaffold was intermediate to that of PLGA and alginate hydrogel. The rapid degradation of the hydrogel layer, which was designed to be in direct contact with the damaged area, is necessary for skin cell growth and tissue regeneration. In contrast, the outer PLGA membrane was designed to cover the wound for an extended period of time in order to prevent wound infection. The mechanical properties of the skin scaffold are critical for wound healing. The tensile stress, elongation at break, and Young's modulus of alginate were 231.51 ± 10.41 KPa, 139.18 ± 12.02% and 24.93 ± 2.45 KPa, respectively, which increased to 2753.58 ± 92.25 KPa, 304.28 ± 15.74% and 531.11 ± 12.64 KPa with the addition of the outer PGLA layer (). Taken together, the PLGA nanofiber layer has high tensile strength and can protect hydrogel while the latter aids in tissue regeneration.

BLM Scaffold Resists Bacterial Invasion and Is Cyto-Compatible

In addition to desirable mechanical properties and the ability to retain the moisture of the hydrogel, PGLA also acted as a barrier to bacterial invasion. As shown in , S. aureus was seeded onto the surface of three scaffolds. After the culture, it was found that the bacteria could infiltrate into the alginate hydrogel, but not into PLGA nanofiber or BLM scaffold. This indicates that the bacteria are able to penetrate into the hydrogel, but not into the PLGA layer, probably due to the high density nanofiber structure. This result provides evidence that the PLGA nanofiber layer can resist bacterial invasion. Furthermore, shows that murine fibroblasts are able to adhere to both the PLGA nanofibers and BLM scaffold regardless of their water content. In contrast, the alginate hydrogel and BLM scaffold had more cells adhered to them, compared with the PLGA nanofibers within the first 12 h. This demonstrates the stronger cell adhesion ability of alginate hydrogel, when it is moist. Therefore, it is essential to prevent water evaporation from the hydrogel to optimize cell adhesion during wound healing. In this regard, the BLM scaffold was more conducive to cell adhesion compared to pure hydrogel due to the protective PLGA layer. The biocompatibility of the different materials was tested by culturing murine L929 cells on the respective surfaces for 1, 3, 5, and 7 days. The cells spread and proliferated rapidly on all materials, indicating their biocompatibility (). However, after 5 and 7 days of incubation, the proliferative rate of the L929 cells growing on the alginate hydrogel and BLM scaffold were significantly higher compared to that cultured on the PLGA nanofibers, likely due to the higher biocompatibility, micro-porosity and water content of the hydrogel.

BLM Scaffold Accelerated in vivo Wound Healing

In vivo wound healing was tested by implanting the respective biomaterials in full thickness wounds made in a rat model. All the animals survived, and showed gradual wound healing without infection, although the BLM scaffold induced the fastest wound closure. As shown in , the wound sizes were similar in the control, PLGA and alginate hydrogel groups after 4 days, whereas the BLM scaffold reduced wound size by 20.8% in the same time period (). On day 8 post wounding, the BLM and alginate hydrogel scaffolds resulted in 47.8 and 72.2% wound closure, respectively, whereas only 11.5 and 15.2% wound closure was observed in the control and PLGA groups (). The wounds treated with BLM scaffolds were completely healed by day 12, while 8.8, 28.5, and 33.7% of the wounds remained unhealed in the alginate hydrogel, PLGA and the control groups, respectively (). Histological examination revealed granulation tissue in the wound bed in all groups on the 4th day after the operation (), which was gradually replaced with new tissue in the BLM and alginate hydrogel-treated groups by day 8 (). On the 12th day, continuous and regular epithelial formation was seen on the alginate and BLM-treated wounds (), with a thicker epidermis thickness in the latter. In contrast, no continuous mature epithelial layer was seen in the control and PLGA groups. Taken together, the soft and porous structure of the alginate hydrogel accelerated wound healing by supporting cell adhesion, proliferation and migration (Lee and Mooney, 2012). In contrast, the hydrophobicity of PGLA prevents cell adhesion and migration (Danhier et al., 2012), which translated to poor wound healing in the absence of a supportive hydrogel layer.

BLM Promotes Inflammation, Vascularization, and Collagen Deposition in the Wound

Wound healing is a complex process which is initiated with the clotting cascade and platelet activation, with the latter releasing pro-inflammatory cytokines like IL-1β and TNF-α. Senapati et al. and Ganeshkumar et al. showed that increased expression of pro-inflammatory cytokines in the early stages promoted wound healing (Senapati et al., 2011; Ganeshkumar et al., 2012) by recruiting leukocytes and activating fibroblasts, thereby promoting neovascularization and collagen deposition (Ganeshkumar et al., 2012). Consistent with this, IL-1β and TNF-α mRNAs were detected at all time points after surgery. The expression of IL-1β and TNF-α were significantly higher in the BLM and alginate hydrogel-treated groups compared to the PLGA and control groups on the 4th day (), and opposite trends were seen on the 8th and 12th day ().

Vascularization is critical for wound healing since that delivers nutrients and oxygen to the newly-formed tissues. In addition, neovascularization accelerates the migration of cellular and humoral factors to the wound (Eming et al., 2014), which in turn promote wound healing via formation of granulation tissue and collagen synthesis (Eming et al., 2014). We observed an increase in CD31-expressing endothelial cells in the BLM and alginate hydrogel-treated wounds on day 4 compared to the untreated control and PLGA-treated groups (). On day 8, the mean number of CD31+ cells in the wounds of the BLM, alginate hydrogel, PLGA and control groups were 9.66, 6.34, 1.36, and 1.5%, respectively (). Furthermore, the blood vessel density was significantly higher in the BLM and hydrogel-treated wounds compared to the untreated and PLGA-treated wounds (). The capacity of BLM and alginate hydrogel to stimulate neovascularization in the early stages of wound healing may be attributed to the 3D porous alginate scaffold. Interestingly, the CD31+ cells and blood vessel density was higher in the BLM-treated compared to the alginate hydrogel group on the 12th day after surgery (), possibly due to the PLGA membrane in the former which maintained the moist microenvironment of the hydrogel and accelerated neovascularization.

Collagen synthesis and deposition play a vital role in wound healing by providing mechanical support to the tissues, and promoting cell adhesion, proliferation and differentiation (Eming et al., 2014). Masson's trichrome staining showed relatively sparse and disordered collagen fibers in the control and PLGA groups, while that in the BLM and alginate hydrogel groups were bundled and neatly arranged on day 4 (). With gradual wound healing, the deposition of collagen fibers increased in all groups. On the 8th and 12th days post wounding, the collagen fibers in the BLM and alginate hydrogel-treated groups were significantly denser compared to that in the control and PLGA groups (). Consistent with this, the relative expression of Col1α1 and Col3α1 were significantly higher in the BLM-treated wounds compared to the untreated and PLGA-treated wounds on days 8 and 12 (). Furthermore, the BLM-treated wounds had higher collagen content compared to the alginate-treated wounds (). The greater capacity of BLM scaffold to promote collagen deposition was likely related to the 3D porous alginate scaffold, as well as the outer protective PLGA membrane. Taken together, the BLM scaffold accelerates wound healing by promoting inflammation in the early stages, neovascularization and collagen synthesis.

Materials

PLGA (Mw = 70–88 kDa, LA: GA = 50:50) was purchased from Jinan Daigang Co. Ltd. (China), and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) from Alfa Aesar Company (Ward Hill, MA, USA). Analytical grade sodium alginate and calcium chloride were obtained from Aladdin Co. Ltd (China).

Fabrication of BLM Scaffold by 3D Printing

To prepare the PLGA nanofiber layer, the copolymer was dissolved in HFIP [w/v(%) = 15%] with constant stirring at room temperature for 4 h. The PLGA solution was placed in a barrel loaded in syringe pump and operated at a speed of 1.5 mL/h via the 3D printer (Tongli micro-nano technology Co., Ltd., Shenzhen, China). A high voltage of 12 kV was generated and connected to the printed needle site. The distance between the needle and the collector was 5–6 cm. The printing needle was set in a circular motion at the speed of 1 mm/s, and the layer height was set to 0.05 μm. PLGA nanofiber membranes of 10 mm diameter and 2 μm thickness were obtained.

The second hydrogel layer was prepared by dissolving sodium alginate in water with constant stirring at 40°C for 4 h. The hydrogel was placed in another barrel which was loaded in air compressor and operated at 0.05 MPa using the same 3D printer. The printing speed was 10 mm/s, and the layer height was set to 100 μm. The hydrogel layer was printed on the surface of the PLGA nanofibers membrane to obtain the BLM scaffold. The latter was then immersed into 5% (w/v) calcium chloride solution for 5 min to crosslink the alginate hydrogel, and then in sterile PBS for hydration. All 3D printing steps were performed under aseptic conditions at 25°C and 40% relative humidity. The equipment was UV-sterilized before 3D printing, and all biomaterials were sterilized through a 0.22 μm filter.

Characterization of BLM Scaffold

The BLM scaffold was freeze-dried for 3 days, and its structure and surface morphology were observed under a scanning electron microscope (SEM, Hitachi TM-100, Japan) fitted with a digital camera (Cannon-50D, Japan). The raw PLGA, raw alginate, PLGA nanofiber layer in BLM scaffold, and alginate hydrogel layer in BLM scaffold were characterized using Fourier transform infrared spectroscopy (FTIR, Avatar 380 FTIR spectrometer). To analyze the moisture retention capacity, alginate hydrogel and BLM scaffold samples were weighed and dried at 37°C for 1, 6, 12, 24, 48, and 72 h in 24-well plates. The dried samples were weighed again, and the residual mass of samples was calculated according to the following formula:

Weight remaining % = 100 % × W m W i (1)

W m is the mass of samples after evaporation (g), W i is the initial mass of samples (g).

To determine the water vapor transmission rate, the pre-weighed samples were sealed and fixed on a utensil. The latter was then placed in a water-filled vessel and incubated at 37°C for 1, 2, 4, 8, and 12 h. The dried samples were weighed and water vapor transmission rate was calculated by the following formula:

Water vapor trasmission rate % = 100 % × W t W 0 (2)

W t is the residual mass after evaporation (g), W i is the initial mass (g).

The degradation of the alginate hydrogel, PLGA nanofiber and BLM scaffold in vitro was also measured by the loss of weight method. The pre-weighed samples were immersed in PBS containing 0.02 % (w/v) sodium azide and incubated at 37°C for 1, 3, 5, 7, 14, 21, and 28 days. The samples were weighed after the incubation period, and the residual mass (weight remaining %) of the samples degraded over time was calculated by the following formula:

Weight remaining % = 100 % × W f W i (3)

W f is the mass of samples after degradation (g), W i is the initial mass of samples (g). The above assays were repeated three times.

The mechanical strength of the samples (n = 5) was measured on a universal materials tester (HY-940FS, China) at room temperature and 60% humidity with stretching speed set at 10 mm per min. The stress-strain curve of each sample was observed, and the corresponding Young's modulus was calculated.

Anti-bacterial Barrier and Mammalian Cell Support Function

BLM samples were fixed on nylon net supports that were then placed into petri dishes. Fifty microliters Staphylococcus aureus suspension containing 1 × 10 colony-forming units (CFU)/ml was dropped on the center of each BLM sample, and the latter were immersed in culture medium. After incubating at 37°C for 24 h, the samples were fixed with 4% paraformaldehyde for 2 h, and dehydrated through an ethanol gradient (50, 70, 90, 95, and 100%). Bacterial invasion was observed by SEM, and the images were pseudo-colored using Photoshop CS 5.0.

To evaluate the surface adhesion of mammalian cells on the different materials, PLGA nanofiber, alginate hydrogel and BLM scaffold were dried for 1, 6, 12, 24, 48, and 72 h, and inoculated with 1 × 10 murine fibroblast L929 cells. After 6 h of incubation, the non-adherent cells were removed with fresh medium, and then the number of adherent cells were counted using the Cell Counting Kit-8 (CCK-8 Beyotime, China). Cell proliferation was also analyzed on days 1, 3, 5 and 7 of culture. Briefly, 10 μL CCK-8 solution was added to each well and incubated for 2 h, and the absorbance was measured at 450 nm using a micro-plate reader. Viability was measured on day 5 using the LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies) according to the manufacturer's instructions.

Establishment of Rat Dorsal Model of Wound Healing

Seventy-two 10-weeks old female Sprague-Dawley (SD) rats weighing 300–350 g were purchased from Xipu'er-bikai Experimental Animal Co. Ltd. (Shanghai, China). The animals were housed in specific pathogen-free conditions and had ad libitum access to food and water. All protocols were approved by the Animal Care and Use Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine approval no. SH9H-2019-A612-1. The rats were randomized into the untreated control, PLGA, alginate hydrogel and BLM scaffold groups (n = 18 each) prior to wound induction. Anesthesia in rats was induced using isoflurane, while the rats were in a chamber (isoflurane/oxygen: 5%).Thereafter the rats were placed in a prone position and allowed to breathe spontaneously through a nasal cone (isoflurane/oxygen: 1.5–2.0%). The backs of the rats were sterilized and shaved prior to surgery. Thereafter, three 10 mm biopsy punch wounds were made on each side of the dorsal midline, and the epidermis, dermis and perichondrium were removed to expose the underlying muscle tissue, as previously described previously (Gilmartin et al., 2016). The respective scaffolds were stitched to the edge of wounds using a 5-0 suture (Polypropylene suture (Ethicon U.S) for PLGA, Green Braided Polyester suture (Tevdek U.S) for alginate hydrogel and Gore-Tex suture (W.L. Gore & Associates U.S) for BLM, and the animals were placed on a 37°C heating plate for recovery.

Wound Healing Evaluation

The wound area was measured on days 0, 4, 8, and 12 after operation (n = 6) using a sterile ruler, and the wound healing rate was calculated as the ratio of the areas of the healed and original wounds. Six animals from each group were sacrificed on days 4, 8, and 12 days after operation, and the wound tissue along with a 5 mm margin of surrounding intact tissue were removed. The tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E), Masson's trichrome and immuno-histochemical reagents (Song et al., 2018).

Quantitative RT-PCR

Total RNA was extracted from the wound tissues, and reverse transcribed with Prime Script™ RT reagent Kit (RR037A, TaKaRa). Quantitative RT-PCR was performed using SYBR Green mixture on a QuantStudio™ 7 Flex Real-time PCR system (Applied Biosystems). The primers are listed in the table below.

Gene Primer sequences IL-1β Sense: 5′-CAGGCTTCGAGATGAACAACAA-3′

Antisense: 5′-ATCACTTGAGAGGTGGTCCCA-3′ TNF-α Sense: 5′-CCCACGTCGTAGCAAACCACCA-3′

Antisense: 5′-CCATTGGCCAGGAGGGCGTTG-3′ Col1α1 Sense: 5′-CTCAAGATGTGCCACTCTGACT-3′

Antisense: 5′-GAGGGAGTTTACAGGAAGCAGAC-3′ Col3α1 Sense: 5′-TGGCGGCTTTTCACCATATT-3′

Antisense: 5′-ACTCTCTATTTGTCCGTTAACAGACTTG-3′ Gapdh Sense: 5′-CCTTCATTGACCTCAACTAC-3′

Antisense: 5′-GGAAGGCCATGCCAGTGAGC-3′

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 5 software for Windows (GraphPad Software Inc., La Jolla, CA, USA), while comparison between multiple groups were made using single factor analysis of variance (ANOVA), and the differences between groups were evaluated using the Bonferroni post-hoc test.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.