1. Introduction
Restoring the structural integrity and functionality of the skin after various injuries, such as burns, chronic wounds, and surgical incisions, is a major challenge [
1]. Standard skin repair methods employ autologous skin grafting, which is a surgical procedure that utilizes the patient’s own skin to repair damaged or lost skin tissue [
2]. However, this approach has inherent limitations, including limited availability of healthy donor sites, donor site morbidity, and the risks of graft failure and complications [
3], [
4]. Therefore, development of new strategies for tissue engineering of skin (STE) to efficiently repair damaged skin tissue and restore barrier function is urgently needed. Recently, efforts have been made to address these limitations through advancements in STE, including the development of artificial skin substitutes and regenerative therapies [
5], [
6], [
7]. Various materials, such as hydrogels, collagen, biodegradable polymers, natural cellulose, and decellularized dermal matrix, have been employed as artificial skin substitutes to mimic the structure and function of native skin tissue [
8], [
9], [
10]. Among these materials, hydrogels with three-dimensional (3D) networks of hydrophilic polymers are notable due to their high water-absorbing capacity, exceptional biocompatibility, and biodegradability. These traits allow hydrogels to closely mimic the extracellular matrix (ECM) of native soft tissues [
11]. Hydrogels can also effectively create a moist environment, provide a protective barrier, facilitate cell migration and proliferation, and deliver therapeutics, making them valuable assets in the field of skin wound healing [
12]. However, a notable discrepancy in mechanical properties persists between hydrogel scaffolds and natural skin. Overcoming this issue and enhancing the mechanical properties of hydrogel-based scaffolds for STE remain major challenges.
The mechanical properties of hydrogel-based scaffolds are crucial for determining their functionality and compatibility with native skin tissue [
13]. A mechanical mismatch between the hydrogel scaffold and the adjacent skin can obstruct proper integration and functional recovery, limiting the natural movement and deformation of the repaired skin, and adversely affecting the overall functionality and aesthetic appearance of the regenerated tissue. Furthermore, the mechanical properties of hydrogels may affect cellular behaviors and responses. Biological cells in native skin are accustomed to a specific mechanical environment (e.g., stiffness, elasticity, and stress distribution), and the mismatched mechanical properties of hydrogels may affect cell adhesion, migration, proliferation, and differentiation as well as the integration of hydrogel-based scaffolds with the surrounding tissue, ultimately impacting the quality and functionality of the regenerated skin tissue [
14]. To address these challenges, researchers have extensively focused on developing hydrogels with improved mechanical properties that closely mimic those of natural skin via the use of engineered hydrogel formulations with appropriate stiffness, elasticity, and viscoelastic behavior to match the dynamic mechanical characteristics of native skin [
15]. Various approaches, such as incorporating reinforcing materials, adjusting the crosslinking density, or designing composite structures, have been widely explored to enhance the mechanical performance of hydrogel-based skin scaffolds [
16]. The mechanical properties of hydrogels can be optimized by constructing interpenetrating network (IPN) hydrogels, which possess a combination of mechanical properties derived from multiple polymer networks. The mechanical properties of these materials can be tailored by adjusting the ratio and molecular weight of the polymers, the crosslinking density, and the processing conditions [
17]. Through meticulous design of the composition and structure of the IPNs, specific mechanical properties can be customized to meet the demands of various applications. On the basis of our previous study, we demonstrated that a supramolecular-polymer IPN hydrogel consisting of self-assembled peptide supramolecular networks and covalent polymer networks exhibited excellent mechanical properties and was used to estimate cartilage regeneration [
18].
In addition to mechanical properties, several other biological properties (e.g., anti-inflammatory and proangiogenic properties) are essential for increasing integration, functionality, and long-term success in skin wound healing. Various efforts, including ① incorporating anti-inflammatory drugs or growth factors and cytokines, ② performing surface functionalization of specific functional groups or bioactive substances (e.g., peptides or polysaccharides), ③ preparing bioactive agents as nanoparticles and suspending them within hydrogels using nanotechnology, increasing surface area and bioactivity, and ④ integrating biological cells with hydrogel-based scaffolds using tissue engineering principles to create bioactive skin repair scaffolds, have been made to achieve better anti-inflammatory and angiogenic effects [
19]. These methods can endow hydrogel-based skin repair scaffolds with improved anti-inflammatory and angiogenic effects but have drawbacks, such as exorbitant price and relatively complex preprocessing methods. A recent report showed that surface modification with polydopamine (PDA) can easily and quickly endow scaffolds with an excellent biocompatible adhesive ability [
20]. In addition, PDA has been used as a surface modification agent for skin repair scaffolds to enhance their biological functions [
21]. For example, when mesenchymal stem cells (MSCs) are seeded onto PDA-coated biomaterial surfaces, several effects on their paracrine activities, such as increased secretion of trophic factors, immunomodulatory effects, and cell survival and proliferation, are observed [
22]. Li et al. [
23] reported that the secretome of adipose-derived MSCs (AD-MSCs) cultured on mussel-inspired nanostructures exhibited increased expression of immunomodulatory and proangiogenic factors, demonstrating functional effects
in vitro and promising efficacy in treating a diabetic model of skin wound healing.
In particular, researchers have been able to precisely control the architecture and composition of hydrogel-based skin repair scaffolds by integrating 3D bioprinting techniques, allowing the fabrication of scaffolds with tunable mechanical properties, such as elasticity, stiffness, and porosity, that closely match those of native skin tissue [
24]. Additionally, 3D-printed scaffolds have shown major benefits in enhancing biological functions during the process of skin regeneration. These porous scaffolds create a 3D microenvironment that promotes cellular migration and ingrowth. A pore size ranging from 20 to 500 μm has been found to be suitable for STE [
25]. The structural features of the scaffolds can also influence angiogenesis. Through incorporation of platelet-rich plasma [
26], AD-MSCs [
27], and multicomponent bioinks [
28], the rate of epithelization can be significantly increased. Moreover, the predesigned vessel-like channels within the scaffolds facilitate the vascularization process, while the precise microstructure achieved through 3D printing closely mimics that of native human skin [
29]. The addition of normal human epidermal keratinocytes/human dermal microvascular endothelial cells [
30] or endothelial progenitor cells/AD-MSCs [
31] to bioink further promoted vascularization. Notably, the 3D printing technique offers a major advantage in fabricating hair follicle-like structures, which are integral parts of the dermis and subcutaneous tissue. By alternately printing multiple cell types in different layers, this technique can replicate the microstructure of the dermal/epidermal structure in the scaffold [
32]. Therefore, 3D-printed scaffolds show good potential for accelerating wound healing and controlling cellular functions by modifying both the structure and bioink components. Hence, PDA surface modification may endow 3D bioprinting hydrogel-based skin repair scaffolds with enhanced immune modulatory and angiogenic properties through increased paracrine secretion of MSCs.
In this study, PDA-modified polyvinyl alcohol (PVA)-based IPN hydrogels (Dopa-gel) were prepared and used as bioinks for 3D bioprinting to construct skin repair scaffolds. The morphology and mechanical properties of the Dopa-gel were systematically characterized. Subsequently, the
in vitro biocompatibility and angiogenic properties of the Dopa-gel were assessed by analyzing cell viability, the cytoskeleton and angiogenesis-related gene expression. The skin repair performance of the Dopa-gel was evaluated using a rat model of skin wound healing
in vivo through analyses of wound closure, vascularization, and macrophage polarization
. Furthermore, the modulatory effects of PDA modification of the Dopa-gel on paracrine behaviors, such as the production of proangiogenic and anti-inflammatory factors, were evaluated. The signaling pathways involved in Dopa-gel-mediated paracrine signaling were further investigated. We believe that the Dopa-gel, which has mechanical properties similar to those of native skin tissue and enhances the immunomodulatory/angiogenic properties induced by paracrine signaling, could be a potential repair scaffold for skin tissue regeneration (
Fig. 1).
2. Materials and methods
2.1. Materials
Bisacrylamide, acrylamide, alginate, ammonium persulphate, PVA, dopamine, CaCl2, and NaOH were purchased from Aladdin Reagent (Shanghai) Co., Ltd. (China). The rat tendon fibroblast line CP-R237 was purchased from Procell Life Science & Technology Co., Ltd. (China). The murine macrophage lines RAW264.7 and AD-MSCs were obtained from the Shanghai Institute for Biological Science, Chinese Academy of Sciences (China). The L929 cell line was kindly provided by Professors Hongli Mao and Zhongwei Gu from Nanjing Tech University. Deionized water was obtained using a water purification system (UNIQUE-R10; RSJ Water Purification Technology, Co., Ltd., China).
2.2. Fabrication of the Dopa-gel
Bisacrylamide (50 mg∙mL-1), acrylamide (225 mg∙mL-1), and ammonium persulphate (6 mg∙mL-1) were solubilized in 5% w/v PVA solution, followed by the addition of alginate (6% w/v) to prepare the 3D bioprinting ink. 3D bioprinted scaffolds were constructed using a Bio-Architect® WS 3D printer (Regenovo Biotechnology Co., Ltd., China). During the printing process, photopolymerization of the bioprinting scaffolds was performed under ultraviolet (UV) light (365 nm). The as-prepared scaffolds were subsequently immersed in CaCl2 solution (80 mmol∙L-1) for 10 min. After three washes with phosphate-buffered saline (PBS), the 3D bioprinted scaffolds were treated with NaOH solution (6 mol∙L-1) for 20 min and subsequently washed with PBS for three times. Afterward, the scaffolds were frozen (storage at -80 °C for 1 h) and thawed (storage at room temperature for 6 h) for ten cycles. For dopamine treatment, the as-prepared scaffolds were immersed in 2-morpholinoethanesulphonic acid (MES) buffer solution containing dopamine (2 mg∙mL-1) for 40 min, washed with PBS for three times and subsequently stored at 4 °C until further use. For comparison, a gel was prepared according to the above protocol, except for the absence of PVA hybridization and the freeze-thaw process.
2.3. Characterization of the Dopa-gel
The morphologies of the 3D bioprinted scaffolds were observed using a field-emission scanning electron microscope (FESEM; Zeiss Supra 40 Gemini, Germany). The printing resolution was calculated based on the images observed under an optical microscope (IMT-2; Olympus, Japan). The tensile mechanical properties of the gel and Dopa-gel (length: 20 mm, width: 8 mm, thickness: 2 mm) were assessed using an Instron-5944 universal instrument equipped with a 2 kN sensor in air at room temperature. The tensile rate was set to 2 mm∙min-1 in the tensile crack and tensile relaxation cycle tests. In the stress-relaxation test, the strain for each tensile process was set to 10%, and the test was repeated for five times. Rat skin of the same size was used as a control for analysis of tensile and mechanical properties. The rheological properties of the gel and Dopa-gel (diameter: 8 mm, thickness: 2 mm) were evaluated using an HAAKE MARS Modular Advanced Rheometer System (Thermo Fisher Scientific, USA). For the frequency sweep experiment, the frequency was set from 10 to 50 rad∙s-1 at 1% strain. For the strain sweep experiment, the strain was set from 5% to 30% strain at 6.28 rad∙s-1. For the recovery experiments, we measured the storage modulus (G′) and the loss modulus (G′′) with a 0.1% strain and a 6.28 rad∙s-1 frequency for 300 s. Afterward, the gels were destroyed with a 300% strain and a 100 Hz frequency for 60 s and immediately switched back to a 0.1% strain and 6.28 rad∙s-1 to measure the recovery of the mechanical properties for 300 s. The temperature was set to 20 °C for all the rheological measurements.
2.4. Cell experiments
Cell culture. The CP-R237 cell line derived from rat tendons was cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
Cytotoxicity assay. The gel and Dopa-gel were tailored into cylinders (diameter: 8 mm, thickness: 2 mm), washed with PBS for three times and sterilized using UV light for at least 24 h. Afterward, the samples were transferred to 24-well plates, and a suspension of CP-R237 cells was added to the surface of the gels and cultured for 1, 3 or 7 d. A cell viability assay was performed by transferring 100 μL of culture medium into 96-well plates, after which 10 μL of cell counting kit-8 (CCK-8) reagent was added for 1 h of reaction. Cell viability was calculated based on the optical density at 450 nm (OD450) on a microplate reader. For live/dead staining, CP-R237 cells were washed with PBS for three times and stained with calcein-AM/propidium iodide (PI) for 45 min. The dyed cells were observed using a laser scanning confocal microscope (141 FV3000; Olympus).
Cell adhesion. After the samples were tailored and sterilized as described above, the suspensions of CP-R237 cells were added to the surfaces of the plates. After 7 d of culture, the cells were fixed with paraformaldehyde for 10 min, treated with 0.1% Triton X-100, washed with PBS for three times and stained with phalloidin and 4′,6-diamidino-2-phenylindole (DAPI; ab104139; Abcam, USA). The dyed cells were observed using a laser scanning confocal microscope.
Quantitative real-time polymerase chain reaction (qPCR). CP-R237 cells were cultured on the surface of the samples for 7 d. Total RNA was then isolated using an RNA-quick purification kit, followed by reverse transcription into complementary DNA (cDNA) using HiScriptIIQ RT SuperMix and qPCR using a 7300 real-time PCR system (Applied Biosystems, USA). The fibrogenesis-related primers (fibronectin (FN), decorin (DCN), type III collagen (COL3), and type I collagen (COL1)), angiogenesis-related primers (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (BFGF), hepatocyte growth factor (HGF), and angiopoietin-1 (ANG-1)), and inflammation-related primers (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1ra), interleukin-10 (IL-10), and arginase-1 (ARG-1)) are listed in Tables S1-S3 in Appendix A. All the data were normalized to that of the internal reference (2-ΔΔCT method).
Western blot analysis
. CP-R237 cells were cultured on the surface of the samples for 7 d, after which the proteins were collected and extracted for Western blotting as previously described [
18]. The blots were processed with primary antibodies and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The proteins were detected using enhanced chemiluminescence (ECL) plus a Western blotting detection system.
Assessment of paracrine behaviors. AD-MSCs were used as a model for the evaluation of paracrine behaviors. Briefly, AD-MSCs were seeded on the surface of tailored samples and cultured for 24 h. Afterward, the cells were washed with PBS for three times and cultured in serum-free medium for 24 h. Angiogenic-related gene expression, including that of VEGF, BFGF, HGF, and ANG-1, in AD-MSCs was assessed using qPCR. The supernatants were collected to assess immunomodulatory factors, including IL-1ra, transforming growth factor-β (TGF-β), and prostaglandin E2 (PGE-2), using enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s instructions. RAW264.7 cells were cultured with the above supernatant, after which total RNA was extracted. The messenger RNA (mRNA) expression of PGE-2, monocyte chemoattractant protein-1 (MCP-1), IL-1ra, and TGF-β was determined via qPCR. For further analysis of the effects of paracrine behaviors of AD-MSCs induced by Dopa-gel treatment of macrophages, RAW264.7 cells were treated with lipopolysaccharide (LPS, 500 μg∙mL-1) for 1 h, and the cells were cultured under five conditions for 8 h: the blank group (pure culture medium), NS group (culture medium + normal saline), non-gel group (culture medium + supernatants of AD-MSCs cultured on culture dish), gel group (culture medium + supernatants of AD-MSCs cultured on the surface of gel), and Dopa-gel group (culture medium + supernatants of AD-MSCs cultured on the surface of Dopa-gel). The expression of proinflammatory genes (TNF-α, IL-1β, and IL-6) and anti-inflammatory genes (IL-1ra, IL-10, and ARG-1) was determined via qPCR. Afterward, the cells were stained with inducible nitric oxide synthase (iNOS, an M1 macrophage marker) and cluster of differentiation 206 (CD206, an M2 macrophage marker). The intensities of CD206+ cells and iNOS+ cells were quantified using semiquantitative analysis. For α-smooth muscle actin (α-SMA) immunofluorescence staining, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 30 min. After two additional washes with PBS, the cells were subjected to α-SMA immunofluorescence staining according to the manufacturer’s instructions. For visualization of the nuclei, mounting medium containing DAPI was used, and the stained cells were observed under a fluorescence microscope.
To further investigate the correlation between tensile stress and paracrine activity, we conducted a cyclic tensile strain test using the L929 cell line, which is a fibroblast line derived from the subcutaneous tissue of mice. Initially, the L929 cells were seeded onto the surface of the gel/Dopa-gel and allowed to culture for 24 h. Subsequently, the cells were washed three times with PBS and divided into two groups. The first group, referred to as the static group (named as blank, gel, and Dopa-gel), was cultured in serum-free medium for an additional 24 h in an incubator. The second group, known as the cyclic tensile strain group (named as gel-tension and Dopa-gel-tension), was cultured in a Flexcell-5000 system (Flexcell, USA) using serum-free medium. This group was subjected to a cyclic tensile strain of 7% at a frequency of 1 Hz for 24 h [
33]. To assess the effects of tensile stress on paracrine function, we collected the supernatants from both groups and performed ELISAs following the manufacturer's instructions to measure the levels of VEGF and IL-10. Additionally, we used qPCR to evaluate the expression levels of Rho-associated kinase-1 (
ROCK-1),
ROCK-2, and hypoxia-inducible factor-1α (
Hif-1α) (primers are presented in Table S4 in Appendix A). To further validate our findings, we conducted Western blot analysis to assess the expression levels of ROCK-1, ROCK-2, VEGF, and Hif-1α.
2.5. In vivo study
Eighteen male Sprague-Dawley rats (weighing ∼250 g) were obtained from the Laboratory Animal Center of Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University (China). All the experimental protocols in this study were approved by the Committee of Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University. A full-thickness skin defect (10 mm × 10 mm) was made on the back of the animals for skin regeneration assessment. Eighteen rats were divided into three groups (n = 6 per group): the blank group (full-thickness defect without scaffold implantation), gel group (full-thickness defect implanted with a gel scaffold), and Dopa-gel group (full-thickness defect implanted with the Dopa-gel scaffold). Images of the wounds were captured at days 0, 7 and 14, and subsequently, the wound areas were analyzed with ImageJ software. After 14 d of treatment, the rats were sacrificed, and the tissues surrounding the wounds were harvested for in vivo skin regeneration assessment.
Histological analysis and immunofluorescence staining
. The harvested tissues were fixed using paraformaldehyde for 1 d and subsequently embedded in polymethylmethacrylate (PMMA). The embedded tissues were subsequently cut into 30-μm-thick slices for hematoxylin and eosin (H&E) staining, Masson staining, and α-SMA immunohistochemical staining. The modified Vancouver scar scale and wound healing score were used to evaluate
in vivo scar formation (Table S5 in Appendix A) [
34], [
35]. CD31 and α-SMA immunofluorescence staining was performed to assess angiogenesis. CD68 and CD206 immunofluorescence staining was performed to assess macrophage polarization. The stained sections were observed using a microscope equipped with a CCD camera (Olympus).
2.6. Statistical analysis
The differences between groups were determined using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc analysis. p < 0.05 indicated statistical significance. All the statistical analyses were performed using SPSS 19 software (IBM, USA).
3. Results and discussion
3.1. Design, preparation, and characterization of the Dopa-gel
IPN hydrogels, which have double-network (DN) or triple-network (TN) structures, have been extensively studied in tissue regeneration due to their ability to balance stiffness and toughness. In these IPN hydrogels, one brittle and rigid network dissipates energy through easy rupture, while another softer, ductile network enhances stretchability [
36]. To fabricate superior hydrogels with mechanical properties similar to those of skin tissue, we fabricated superior TN hydrogels consisting of three networks (
Fig. 2(a)), namely, ① a crosslinking polymer network constructed from acrylamide monomers and bis-acrylamide as a covalent crosslinker through free radical polymerization in the presence of ammonium persulfate (APS) as an initiator to serve as model hydrogels [
37]; ② an ionic crosslinking network using CaCl
2 to create ionic bonds between alginate polymers, forming a Ca
2+-alginate ionic network to further increase the mechanical performance of hydrogels [
38]; and ③ a crosslinking polymer network constructed from frozen-thawed PVA by forming ice crystals at -80 °C and crystallizing PVA chains at 25 °C to further enhance the elastic properties and excellent tensile strength [
39]. Previous reports have shown that surface modification of PDA can endow biomaterials with enhanced roughness and hydrophilicity and facilitate several specific biological activities, such as cell proliferation, differentiation, and paracrine functions of MSCs [
40], [
41]. Hence, the as-prepared TN hydrogels were surface modified with PDA, and their microstructures were observed via scanning electron microscope (SEM). As shown in
Fig. 2(b), a uniform nanolayer could be observed on the surface of the Dopa-gel, whereas the gel demonstrated a smooth and cracked nanostructure (Fig. S1 in Appendix A). The elemental mapping images (Fig. S2(a) in Appendix A) and semiquantitative determination of the corresponding elemental composition (Table S6 in Appendix A) revealed that C, O, N, Na, and S were uniformly distributed in both the gel and Dopa-gel. The S content in the Dopa-gel was significantly greater than that in the gel, which could be attributed to the surface modification of PDA (Fig. S2(b) in Appendix A). Surprisingly, the Dopa-gel could support relatively large weights (75 times its own weight) for at least 10 s and subsequently return to the original state, exhibiting an excellent capacity for deformation and antitensile properties (
Fig. 2(c)). In contrast, the gel fractured upon being stretched to 175%, indicating its relatively poor tensile resistance (Fig. S3(a) in Appendix A). Accumulating evidence has shown that scaffolds fabricated by 3D bioprinting technology may have applications in tissue regeneration due to their designed 3D structure. The as-prepared gel could be further used as a bioink for 3D bioprinting with the desired structure. The digital model and 3D bioprinting images of the Dopa-gel scaffold observed by optical microscopy are shown in Fig. S3(b) in Appendix A. The precision of the 3D-printed lattice structure was notable, indicating high accuracy in its intricate details. A quantitative comparison between the digital model and the 3D bioprinting scaffold was conducted, as shown in Fig. S3(c) in Appendix A. No notable discrepancies were observed in the dimensions of the contour, large pores, or small pores, demonstrating the high printing accuracy. Furthermore, we prepared 3D bioprinting scaffolds with more complex structures (Fig. S3(d) in Appendix A).
3.2. Mechanical behaviors of the Dopa-gel
For promotion of skin regeneration, the scaffold should have a mechanical stability comparable to that of native skin tissue. The stretch properties of biomaterials were reported to play a pivotal role in modulating the fibrotic response of biological cells [
42]. Mechanical stretching serves as a dynamic stimulant, affecting cellular behaviors such as cell morphology and alignment, which are involved in cellular fibrosis [
43], [
44]. Cells embedded within biomaterials that exhibit favorable stretch properties may experience increased proliferation, migration, and ECM remodeling, contributing to a profibrotic environment. Hence, we initially evaluated the dynamic mechanical properties of the hydrogel samples using a rheometer. Frequency-sweep tests (Fig. S4 (a) in Appendix A) and strain-sweep tests (Fig. S4(b) in Appendix A) were performed. We found that the storage modulus (G′) of the as-prepared hydrogels exceeded their loss modulus (G′′), indicating solid-like behavior. In the recovery test (Fig. S4(c) in Appendix A), both the gel and the Dopa-gel exhibited complete and rapid recovery during five cycles of breaking and reforming, transitioning from a high-magnitude strain (300%) to a low strain (0.1%). In addition, to further assess the mechanical behaviors of the rat skin, gel, and Dopa-gel, we performed tensile stress-strain tests and tensile-relaxation tests. Unlike most fast-gelation bioinks, which tend to be rigid and fragile, our bioink exhibited good mechanical stability. The stress-strain curve is shown in
Fig. 3(a). The rat skin tissues exhibited the greatest breaking strength and elongation at break, as indicated by the tensile modulus ((11.65 ± 1.61) kPa) and toughness ((376.05 ± 29.58) kJ∙m
-3). However, the Dopa-gel displayed a greater tensile modulus ((2.19 ± 0.23) kPa) and toughness ((13.01 ± 1.52) kJ∙m
-3) than did the gel (
Figs. 3(b) and
(c),
Table 1), indicating the potential of the Dopa-gel as a skin regenerative scaffold. Uninterrupted tensile-relaxation cycles were conducted on the rat skin, gel, and Dopa-gel (20 cycles with no time elapsed between cycles).
Figs. 3(d) and
(e) reveal that the rat skin possessed the highest tensile limit and energy dissipation, while the Dopa-gel exhibited a superior tensile limit and energy dissipation compared to those of the gel. The original curves of the tensile-relaxation cycles are shown in Fig. S4(d) in Appendix A. In addition, the amount of energy dissipated and the recovery rate of the samples are shown in
Fig. 3(f). The Dopa-gel exhibited the highest recovery ability, with the amount of energy dissipated decreasing to 39.24% and the recovery rate remaining above 98.50% after 20 cycles; these values are greater than those of the rat skin and the gel. In addition, the stress-relaxation curve, as shown in
Fig. 3(g), demonstrated the stress-relaxation behavior observed in the gel, Dopa-gel, and rat skin (
Fig. 3(g), inset). Local magnification of the first fourth stress-relaxation curve is shown in Fig. S4(e) in Appendix A. The characteristic relaxation time scales at different strains of the above three samples are presented in
Fig. 3(h), which shows that the relaxation time decreased in the Dopa-gel compared with that in the gel. The toughness and recovery capacity of the Dopa-gel under tension strongly resemble those of rat skin, which may be attributed to the robust covalent and ionic crosslinking within the interpenetrating polymer networks.
3.3. In vitro biocompatibility and biological functions
To assess the potential biotoxicity of the gel and Dopa-gel
in vitro, we used rat tendon fibroblast CP-R237 cells as a model. As shown in Fig. S5(a) in Appendix A, no obvious changes in cell morphology were observed after the cells were seeded on the gel or the Dopa-gel. In addition, we conducted live/dead staining and CCK-8 assays on CP-R237 cells treated with the gel or Dopa-gel. Cell proliferation (
Fig. 4(a)) and cell viability (
Fig. 4(b)) were evaluated after 7 d of culture. The results showed that the proliferation and viability of the CP-R237 cells treated with the gel or Dopa-gel were not affected, and no significant differences were observed between the groups. A similar trend was found for the cells treated with the gel or Dopa-gel for 1 or 3 d (Figs. S5(b) and S5(c) in Appendix A). Furthermore, no obvious damage to the cytoskeleton of CP-R237 cells was observed in the Dopa-gel group compared to the other two groups according to the phalloidin/DAPI staining (
Fig. 4(c)). Additionally, we conducted a live/dead assay and phalloidin/DAPI staining on days 1 and 3 using the L929 cell line derived from the subcutaneous tissue of mice. The results (Fig. S6 in Appendix A) demonstrated the satisfactory biocompatibility of the Dopa-gel. In addition to biocompatibility, biodegradation is an important property of tissue repair scaffolds, which may provide enough time for the biological cells in the scaffold to generate new tissue [
45]. The degradation process over 7 d (Fig. S7 in Appendix A) was examined, and the results showed that the degradation rate of the gel and Dopa-gel was maintained at 20% within 7 d, which may provide a proper environment for cell growth. Accumulating evidence has shown that angiogenesis plays a critical role in promoting skin repair by facilitating the formation of new blood vessels, supplying oxygen and nutrients to damaged tissue, and promoting the recruitment of essential cells involved in the healing process [
46]. Hence, we further examined the fibrogenic effects of the Dopa-gel on CP-R237 cells. α-SMA expression in skin defects was reported to indicate the activation and presence of myofibroblasts, which play a crucial role in the contraction and remodeling of the ECM during wound healing [
47]. Immunofluorescence staining was performed to assess the expression of α-SMA in CP-R237 cells (
Fig. 4(d)). Quantitative analysis demonstrated that, compared with those in the blank and gel groups, the CP-R237 cells in the Dopa-gel group exhibited significantly greater α-SMA expression (
Fig. 4(e)). Moreover, the results of immunohistochemical staining indicated that keratin K10 (K10) expression was significantly upregulated in the Dopa-gel group (
Fig. 4(f)), and the statistical analysis revealed a substantial increase in K10 expression compared to that in the blank and gel groups (
Fig. 4(g)). Furthermore, we investigated the expression of fibroblast-related indicators, including
FN,
DCN,
COL3, and
COL1. Compared to those in the other two groups, the expression levels of these factors were greater in the Dopa-gel group (
Fig. 4(h)). These findings revealed that the Dopa-gel not only exhibited strong biocompatibility but also effectively upregulated fibroblast-related genes in CP-R237 cells.
3.4. In vivo skin wound healing assessment
To evaluate the healing effect of Dopa-gel scaffolds, we created a full-thickness wound defect on the dorsal region of mice. Representative photographs showing the progressive stages of wound healing in each group were taken at three specific time intervals: 0 (immediately after the operation), 7, and 14 d post-operation (
Fig. 5(a)). These images provided visual insight into the healing process and allowed comparative analysis across the various groups. Wound size was decreased in all three groups, particularly in the Dopa-gel group, in which more pronounced promotion of healing was observed on day 7. Moreover, notably, the blank and gel groups still exhibited a relatively greater residual wound area than the Dopa-gel groups on day 14 (Fig. S8(a) in Appendix A). The remaining wound area in the Dopa-gel group were (31.3 ± 12.4) and (16.4 ± 1.9) mm
2 at days 7 and 14, respectively; these values were significantly lower than those observed in the gel group (
Fig. 5(b)). Furthermore, H&E, Masson, and Sirius Red staining were used for histomorphological examination of wound regeneration. H&E staining (Fig. S8(b) in Appendix A) revealed that complete epithelialization could be observed in the wounded area of the gel and Dopa-gel groups by day 14, whereas partial wound defects persisted in the blank group. Local magnification of the α-SMA immunohistochemical staining images is shown in Fig. S8(c) in Appendix A. Compared to the blank and gel groups, the Dopa-gel group exhibited reduced scar formation at the wound site (Fig. S9 in Appendix A). The color of the wound site in the Dopa-gel group was lighter and more similar to that of normal skin. Additionally, the Dopa-gel group exhibited higher-quality granulation tissue, more extensive deposition of ECM, and better reconstruction of dermal architecture than the other groups. As illustrated in
Fig. 5(c), Masson staining revealed that the dermis had more collagen deposited in a fine mesh structure in the Dopa-gel group, which was remarkably similar to that in healthy dermal tissue. In addition, many appendage-like structures were clearly visible in the Dopa-gel group, while the gel group exhibited a lower abundance of these structures. Conversely, the blank group showed distinct reductions in both the length and thickness of the newly formed epithelium after 14 d, accompanied by reduced collagen deposition and a lack of appendage-like structures. Similar results were observed with Sirius Red staining. Skin sections were further subjected to immunohistochemistry staining to assess the expression of α-SMA, which can be used to visualize the distribution of smooth muscle cells within blood vessel walls [
48]. Compared to those in the blank and gel groups, the α-SMA level in the Dopa-gel group was significantly increased (white array). The upregulation of α-SMA suggested that the Dopa-gel can promote angiogenesis, and the presence of α-SMA-positive smooth muscle cells was crucial for effective wound healing, as these cells contributed to tissue remodeling, deposition of ECM components (e.g., collagen), and revascularization of the wound bed [
49]. As shown in
Fig. 5(d), the semiquantitative analysis determined the percentage of collagen deposition as follows: blank group ((39.6 ± 7.0)%), gel group ((71.5 ± 8.4)%), and Dopa-gel group ((86.9 ± 10.3)%). As expected, both Masson staining and Sirius Red staining revealed a greater presence of vascular-like structures in the Dopa-gel group than in the gel group, which exhibited a significantly greater number of vessels than that in the blank group. Quantitatively, the Dopa-gel group exhibited (55 ± 7) vessels, which was significantly greater than the (27 ± 4) vessels observed in the gel group and the (11 ± 3) vessels observed in the blank group (
Fig. 5(e)). Angiogenesis was clearly shown through immunofluorescence staining for CD31 and α-SMA (
Fig. 5(f)). Compared to those in the gel and blank groups, the Dope-gel group exhibited a greater number of vascular-like structures that clearly contained vascular smooth muscle and endothelial cells. These results indicated that Dopa-gel exhibited more pronounced effects on facilitating angiogenesis, which not only provided oxygen and nutrients but also promoted cell migration and tissue reconstruction for skin regeneration.
Furthermore, macrophages are known to play a pivotal role in intercellular communication, acting as primary mediators in the regulation of both inflammatory and regenerative processes [
50]. To further validate the polarization of macrophages in the skin defect area following treatment with the gel or Dopa-gel, we used a double-labeling immunofluorescence staining approach involving the simultaneous staining of two specific markers: CD68, a pan-macrophage marker, and CD206, an M2 marker. As displayed in
Fig. 6(a), all three groups exhibited different levels of positive staining for both M1 macrophages (red fluorescence) and M2 macrophages (green fluorescence). Notably, the blank group exhibited significantly greater expression of CD68 than the other two groups, suggesting greater infiltration of macrophages into the wound defect. Furthermore, both the gel and Dopa-gel groups demonstrated notably greater levels of CD206 than the blank groups. In particular, the Dopa-gel group exhibited the highest level of CD206 expression among the three groups, indicating the presence of M2 macrophages. Quantitatively, there were (962.3 ± 105.8) total macrophages (CD68
+) in the blank group, while further increases in the total number of macrophages were observed in the Dopa-gel and gel groups ((499.7 ± 87.7) and (645.3 ± 109.2), respectively) (
Fig. 6(b)). In contrast, the total number of macrophages (CD206
+) exhibited the opposite trend in all three groups (
Fig. 6(c)). Moreover, the M1 phenotype/total macrophage ratio in the Dopa-gel group was significantly lower than that in the blank and gel groups (
Fig. 6(d)). In contrast, the M2 phenotype/total macrophage ratio in the Dopa-gel group was (33.1 ± 5.2)%, which was significantly greater than that in the blank ((15.3 ± 2.9)%) and gel ((24.6 ± 4.3)%) groups (
Fig. 6(e)). These results indicated that Dopa-gel exhibited enhanced efficacy in modulating macrophage phenotypes, promoting an anti-inflammatory environment, and facilitating tissue repair and regeneration.
3.5. Mechanisms of Dopa-gel-induced skin defect repair
When the skin is damaged, neutrophils are among the first immune cells recruited to the wound, followed by proinflammatory M1-like macrophages. During the healing process, M1-like macrophages are gradually transformed into M2-like anti-inflammatory macrophages to regulate tissue regeneration [
51], [
52]. In addition, neutrophils are first polarized into the proinflammatory N1 phenotype and secrete cytokines. Activated platelets express CD40 ligands, which interact with receptors on the surface of macrophages and stimulate macrophages to secrete vascular endothelial growth factor, thereby promoting angiogenesis [
53], [
54]. To investigate the biological function of Dopa-gel in skin regeneration, we examined the expression of paracrine factors related to inflammation, including IL-1ra, TGF-β, and PGE-2, in AD-MSCs cultured with Dopa-gel using ELISAs. The ELISA results for the conditioned medium from AD-MSCs demonstrated significant upregulation of the mRNA expression of IL-1ra, TGF-β, and PGE-2 in the Dopa-gel group compared to those in the gel group and blank group (
Fig. 7(a)). Furthermore, we examined the expression of angiogenic factors, such as
VEGF,
BFGF,
HGF, and
ANG-1, and found greater expression in the Dopa-gel group than in the gel group and blank group (
Fig. 7(b)). These findings suggested that AD-MSCs cultured on Dopa-gel can modulate the release of inflammation-related cytokines and angiogenic factors, which are crucial for skin regeneration. To further investigate the anti-inflammatory and proangiogenic functions of the factors secreted from AD-MSCs, we added conditioned medium derived from AD-MSCs to both the gel and the Dopa-gel and incubated the samples for a period of 14 d. Afterward, the harvested conditioned medium was utilized for culture of RAW264.7 macrophages. Immunofluorescence staining was carried out, and the results are shown in Fig. S10(a) in Appendix A. The secreted factors present in the Dopa-gel group strongly promoted M2 macrophage polarization, as evidenced by the increased expression of CD206. The gel also affected CD206 expression, albeit to a lesser extent. The lowest expression level of CD206 was found in the blank group, indicating little M2 macrophage activation in this group. Conversely, the expression of iNOS, an M1 macrophage marker, exhibited the opposite changes across all three groups, whereas the Dopa-gel group exhibited the lowest levels and the gel group showed intermediate levels of iNOS expression. Analysis of the immunofluorescence intensity validated the aforementioned findings, supporting the observed trends in CD206 and iNOS expression. In the Dopa-gel group, a notable decrease in the level of iNOS, which was approximately 0.69-fold lower than that in the blank group, was found (Fig. S10(b) in Appendix A). In contrast, we found a significant increase in CD206 expression—approximately 1.75-fold greater than that in the blank group (Fig. S10(c) in Appendix A). Moreover, the M1/M2 phenotype ratio in the Dopa-gel group was (20.8 ± 3.2)%, which was significantly lower than that in the gel ((43.3 ± 6.6)%) and blank ((53.7 ± 7.8)%) groups (Fig. S10(d) in Appendix A). Moreover, we further investigated whether the active factors of AD-MSCs have anti-inflammatory functions. The results showed that the mRNA levels of proinflammatory factors (
TNF-α,
IL-1β, and
IL-6) were dramatically decreased, while the mRNA expression of the anti-inflammatory factors (
IL-1ra,
IL-10, and
ARG-1) demonstrated the opposite trend in the Dopa-gel group compared with the other groups (
Fig. 7(c)). These results demonstrated that the active factors secreted by AD-MSCs cultured on Dopa-gel could change the morphology of M1 macrophages to M2 macrophages and decrease the level of inflammation.
Various signaling pathways are involved in the regulation of angiogenesis. For example, angiopoietin specifically binds to receptor tyrosine kinases (TIE2/Tek-RTK) [
55], [
56]. Cellular activity is mediated primarily by activation of the phosphatidylinositol-3 kinase (PI3K) pathway or the Ras pathway. Among these cytokines, angiotensin 1 (Ang1) can promote the survival of endothelial cells by activating the PI3K pathway, and angiotensin 2 (Ang2) acts as an agonist of TIE2 receptors in the absence of Ang1 and activates the PI3K-AKT pathway. Ang1 stimulates the phosphorylation of endothelial cell TIE2, AKT and endothelial nitric oxide synthase (eNOS) in a PI3K-dependent manner [
57], [
58]. Therefore, the focal adhesion kinase (FAK) signaling pathway was investigated, as previous studies showed that activation of this pathway promotes myosin light chain (MLC) phosphorylation and regulates integrin expression, facilitating integrin-induced aggregation. [
59]. FAK signaling is involved in integrin-mediated downstream signaling, resulting in changes in vascular morphology, cell germination, cell migration, and vascular bed formation. In addition, activation of the extracellular signal-related kinase1/2 (ERK1/2) pathway plays a crucial role in promoting angiogenesis by regulating the production of proangiogenic factors, facilitating endothelial cell migration, and modulating the remodeling of the ECM [
60]. Our results revealed that the Dopa-gel induced the phosphorylation of ERK1/2, which are downstream molecules, through cascade-mediated activation of FAK signaling pathways, thereby promoting angiogenesis and regulating immune responses. The signaling molecule FAK, which is associated with mechanical force transduction, has been reported to undergo integrin-mediated phosphorylation at Tyr397, leading to its interaction with the Src family. Src binds to FAK, resulting in phosphorylation of Tyr925 on FAK. The FAK/Src complex can activate multiple signaling pathways, one of which is the Ras-dependent mitogen-activated protein kinase (MAPK) pathway. When activated, MAPK phosphorylates and activates transcription factors. To further investigate the underlying mechanism by which the Dopa-gel promotes the release of inflammatory factors from AD-MSCs, we assessed the protein expression levels of FAK and its autophosphorylation site Tyr397. These results demonstrated the significant upregulation of both proteins in the Dopa-gel group compared to the other groups (
Fig. 7(d)). This upregulation led to the formation of the FAK/Src complex, resulting in increased phosphorylation of the downstream protein Tyr925, consistent with the protein analysis findings. Phosphorylated Tyr925 can bind to the linker protein growth receptor-bound protein 2 (Grb2), where the peptide motif YYNN, containing Tyr925, provides a binding site for another linker protein (Grb2). The SH3 domain of Grb2 can bind to Son of Sevenless (SOS), which is a guanine nucleotide exchange factor for Ras. Through Grb2, FAK can activate the Ras pathway and MAPK. ERK1/2, a member of the MAPK family, is associated with signaling networks involved in regulating cell growth, development, and division. Therefore, the protein levels of ERK1/2, along with the level of ERK2 phosphorylated at the Thr202/Tyr204 phosphorylation sites, were examined in the Dopa-gel group (
Fig. 7(d)). The semiquantitative results demonstrated a significant increase in the protein expression and phosphorylation of ERK1/2 (
Fig. 7(e)). These findings indicated that the FAK-ERK signaling pathway may be involved in the mechanism of PDA modification, thereby enhancing the immunomodulatory function of AD-MSCs.
3.6. Mechanisms of tensile stress in regulating paracrine function
Due to the considerable deformation observed in the skin on the backs of rats in various postures, the scaffold must possess excellent mechanical properties and generate strain that aligns with the surrounding skin to ensure effective wound regeneration (Fig. S11 in Appendix A). Therefore, we further explored the relationship between tensile stress and paracrine function. Previous studies have reported that tensile strain can mediate the signal transduction of endothelial cells by increased the secretion of Ang2 and platelet-derived growth factor (PDGF) [
33], [
61]. Additionally, tensile stress can trigger the activation of PI3K, p21, and
Ras homolog gene family member A (RhoA) signaling during vascular remodeling [
62], [
63], [
64]. To investigate whether tensile stress can affect inflammatory reactions, we assessed the paracrine effects of IL-10 (a representative anti-inflammatory factor) and VEGF (a representative angiogenesis-related factor). As shown in
Fig. 8(a), both the static group and the cyclic tensile strain group exhibited increased secretion of IL-10. No significant difference was found between the two groups. However, the cyclic tensile strain group presented a greater level of VEGF than the static group and blank group. Within the cyclic tensile strain group, a significant difference also existed between the gel and Dopa-gel groups, indicating that better mechanical properties result in a greater secretion level of VEGF. Therefore, we believe that tensile stress mainly regulates paracrine effects rather than anti-inflammatory effects by affecting the process of vascularization. ROCK signaling has been shown to play a pivotal role in mechanical stress-mediated vascularization [
64]. To confirm this inference, we assessed the expression of angiogenesis-related genes (
Fig. 8(b)). Similar to the results for VEGF secretion, the Dopa-gel presented a greater expression level in the cyclic tensile strain group. The cyclic tensile strain group exhibited significantly greater
ROCK-2 expression, but the expression of
ROCK-1 did not significantly differ among these groups. These results were similar to those of previous studies in which tensile force induced the expression of
ROCK-2 but not
ROCK-1, and the ROCK inhibitor prevented tensile force-induced VEGF expression in a dose-dependent manner [
65]. Hif-1α, another classic factor that has been proven to regulate VEGF, was also assessed. The expression trend was the same as that of
ROCK-1, indicating that mechanical stress and surface modification with dopamine cannot activate this signaling pathway. The results of the Western blot and related semiquantitative analyses provided a clearer demonstration of this phenomenon (
Figs. 8(c) and
(d)). Specifically, the group subjected to cyclic tensile strain exhibited increased expression levels of ROCK-2 and VEGF. Interestingly, the Dopa-gel group displayed the highest expression of both proteins. However, no significant difference in the expression of ROCK-1 or Hif-1α was observed among the groups. Therefore, tensile stress can regulate paracrine function through ROCK-2-induced VEGF-mediated angiogenesis during the wound regeneration process.
4. Conclusions
In summary, a PDA-modified 3D bioprinted scaffold exhibited mechanical properties that closely matched those of native skin tissue. Moreover, the Dopa-gel demonstrated a superior ability to modulate paracrine function due to tensile stress through surface modification with PDA by promoting the secretion of immunomodulatory and proangiogenic factors. In vivo, the Dopa-gel accelerated wound closure, enhanced vascularization, and prompted a phenotypic shift in macrophages from the M1 state to the M2 state within the wound area. Mechanistic investigations suggested that the FAK signaling pathway might play a role in the Dopa-gel-mediated promotion of skin defect healing by increasing the paracrine effects of immunomodulatory and proangiogenic factors. The mechanical properties can exert synergistic effects on regulating paracrine behaviors through ROCK-2/VEGF-mediated angiogenesis during tensile stress. Overall, the multifunctional Dopa-gel, which has appropriate mechanical properties resembling native skin tissue and enhanced immunomodulatory and angiogenic characteristics, has potential as a scaffold for skin tissue regeneration.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (32271413 and 32271408), the National Basic Research Program of China (2021YFA1201404), the Natural Science Foundation of Jiangsu Province (BK20232023), the Science Program of Jiangsu Province Administration for Market Regulation (KJ2024010), the Jiangsu Provincial Key Medical Center Foundation, and the Jiangsu Provincial Medical Outstanding Talent Foundation.
Compliance with ethics guidelines
Peng Wang, Liping Qian, Huixin Liang, Jianhao Huang, Jing Jin, Chunmei Xie, Bin Xue, Jiancheng Lai, Yibo Zhang, Lifeng Jiang, Lan Li, and Qing Jiang declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2024.02.005.
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