3D-printing flexible PLGA scaffold modified with bioorthogonal IGF-1 for skin regeneration

Abstract

Introduction

3D-printing implants have attracted increasing attention for enhancing the regeneration of various wound types. The rigidity of traditional 3D printing materials limits their applicability in skin wound repair. In addition, improving the bioactivity of polymeric materials remains an urgent challenge to be addressed. Insulin-like growth factor-1 (IGF-1) is widely recognized for its efficacy in skin wound repair. However, the potency of IGF-1 in wound regeneration is limited by its unstable binding to the implants.

Methods

In this study, A phase inversion-based deposition modeling 3D printing technique was employed to construct flexible poly(lactic-co-glycolic acid) (PLGA) scaffold. Additionally, a novel 3D-printing flexible skin implantation functionalized with bioorthogonal IGF-1 (DA-IGF-1) was fabricated. The adhesion, proliferation and relative mRNA expression of cells on PLGA scaffold were assessed. Meanwhile, a full-thickness skin defect regeneration model was established on the rat back to evaluate the wound repair efficacy of the implanted scaffolds.

Results

The DA-IGF-1 exhibited outstanding binding ability on the scaffold. The resulting PLGA flexible scaffold modified with DA-IGF-1 showed significantly enhanced hydrophilicity and biocompatibility compared with the control group materials (p < 0.05). The expression of wound regeneration related genes was also upregulated significantly in the DA-IGF-1 group (p < 0.05). In vivo study indicated that bioactive implants effectively promoted healing and neovascularization of full-thickness skin defects in rats.

Conclusion

These results demonstrated that the 3D-printing flexible PLGA scaffold modified with bioorthogonal IGF-1 holds great potential for application as a novel therapeutic strategy for wound regeneration.

1. Introduction

Full-thickness skin injury resulting from burns and chronic wounds represents considerable challenges for wound healing. In such cases, acute damage disrupts the endogenous skin regeneration system, impairing spontaneous repair. Autologous skin grafting, the most commonly used clinical treatment, may induce secondary damage and scarring, and therefore not suitable for extensive wounds involving more than 2% of the total body surface area [1]. The application of allogeneic and xenogeneic grafts is expected to overcome these disadvantages. However, the side effects arising from immune rejection cannot be neglected [[2], [3], [4], [5]]. Thanks to advances in tissue engineering, skin substitutes have offered novel treatment avenues for full-thickness skin injury, particularly those with extensive wounds. However, few ideal skin substitutes have been developed yet due to the complexity of the wound healing process, which involves neovascularization, granulation tissue formation, and tissue remodeling (see Scheme 1).

Scheme 1.

Schematic diagram of the 3D-printing flexible PLGA scaffold modified with bioorthogonal IGF-1 for skin regeneration.

Due to the diversity of wound morphology, customized skin substitutes are necessary for clinical application. 3D printing technology is widely applied in the customization of bioengineering scaffolds, which provide sufficient space to support the growth of cells and tissues. Hydrogels, as inks for direct-write 3D printing, can be fabricated into flexible extracellular matrix (ECM) -mimicking materials, yet they typically exhibit insufficient mechanical strength. Nevertheless, hydrogels used as printing inks must possess temperature sensitivity, cross-linkability, or thixotropic behavior to retain their shape and achieve in-situ solidification under specific printing conditions. Cells-laden hydrogels can also be printed using direct-write 3D printing technology. Nevertheless, cells encapsulated within hydrogels face challenges in secreting sufficient ECM and efficiently migrating to form cell-cell junctions. Synthetic polymer materials represented by polyesters such as poly-lactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) are considered to be excellent raw materials for 3D printing. Their favorable processability facilitates the fabrication of skin defect repair scaffolds with ideal structure using polyester materials. However, rigid scaffolds 3D-printing via traditional fused deposition modeling (FDM) using polyesters are unsuitable to mimic the pliability of native skin, making them unsuitable for skin repair applications. Additionally, the high temperatures and viscosities involved in the FDM process may lead to thermal degradation of polymers. Consequently, there is an urgent need to develop a simplified 3D printing process capable of fabricating flexible, biodegradable polyesters with appropriate strength and topology to support cell and tissue growth.

The biological activity of the implants is another problem need to be addressed. Many growth factors were administered to improve the therapeutic effects of the implants because of their positive effect on epidermal and dermal cells. Wound regeneration involves multiple growth factors, such as basic fibroblast growth factor (bFGF) [6], epidermal growth factor (EGF) [7], and insulin-like growth factor 1 (IGF-1) [8]. And the IGF-1 is identified as a critical factor in skin regeneration. It enhances the adhesion between keratinocytes by promoting the proliferation, migration, and differentiation of epidermal cells via PI3K/AKT/mTOR and ERK pathway [9], further improves the formation of the stratum corneum. Additionally, it stimulates cells to synthesize extracellular matrix [10], which is essential for skin reconstruction. Furthermore, IGF-1 is important for promoting angiogenesis. It was reported that the IGF-1R antagonist suppressed neovascularization in vivo by inhibiting vascular endothelial growth factor signaling [11]. However, the potency of IGF-1 is limited when applied due to its shot half-life and rapid release from the wound site. For this reason, growing interest has arisen in IGF-1 maintaining on biomaterials. At present, the strategies for immobilizing IGF-1 on tissue grafts include physical adsorption, chemical conjugation, and specific adhesion. Physical adsorption stands out for its simply and extensive application. Quint [12] present a functionalized gelatin methacrylate (GelMA) hydrogel which was capable of controlling the release of IGF-1 by utilizing the electrostatic interactions with LAPONITE(R) nanoclays (NCs). Physiologically relevant levels of IGF-1 were maintained during a controlled release over two weeks. The NC was able to retain 50% of the released IGF-1 within the hydrogel niche, significantly improving cellular proliferation and differentiation compared to control hydrogels. However, this approach suffers from low stability of IGF-1 and a high risk of burst release. By comparison, chemical conjugation ensures stable binding of IGF-1. Wei et al. [13] developed a novel supramolecular self-assembling nanofibers, Nap-FFG-IGF1, engineered from IGF-1. This biomaterial accelerated diabetic wound healing by regulating neutrophil extracellular traps formation. Yet chemical conjugation suffers from a complicated process and poor usability. Specific adhesion features strong binding and convenient application. In Zhang's report [14], a collagen-coated PLGA conduits loaded with CBD-IGF-1 were designed. The results showed that the CBD-IGF-1 could be released slowly from the PLGA/col/CBD-IGF-1 conduit to play a long-term role in promoting peripheral nerve injury repair. However, this approach is only applicable to specific materials and lacks general applicability.

Recently, the adhesion of active molecules inspired by dopamine and dihydroxyphenylalanine (l-DOPA) was proposed. l-DOPA (a precursor of dopamine), in the league with lysine-enriched proteins nearby, is the major source of extraordinary attachment to solid surfaces of mussels. In vivo, tyrosine is turned from tyrosine by two different processes, as shown in Fig. 1(A). To prevent the further oxidation of l-DOPA to dopaquinone by tyrosinase, a hydrogen donor such as ascorbic acid, NADH or NADPH is always needed [15,16]. Messersmith's group has made an outstanding contribution to the molecular mechanics of mussel adhesion. They originally designed an anchoring 5-mer peptide (DOPA-Lys-DOPA-Lys-DOPA) mimicking M. edulis foot protein (Mefp)-5 [17,18]. In Zhang's report [19], A pentapeptides composed of Tyr-lys-Tyr-lys-Tyr (YKYKY) were combined with IGF-1. This tag was then converted into DOPA-Lys-DOPA-Lys-DOPA by tyrosinase. The bioorthogonal IGF-1 (DA-IGF-1) exhibited high affinity for titanium substrates and significantly promoted the growth of NIH3T3 cells. In our previous work, surface modification of PLGA films with DA-IGF-1 was performed to promote the paracrine activity of human umbilical cord mesenchymal stem cells (hUCMSCs) by secreting neurotrophic factors. Subsequently, the acquired neurotrophic factors secreted by the hUCMSCs cultured on the DA-IGF-1@PLGA films obviously enhanced neurite outgrowth of PC12 cells [20]. In another research from our team, hUCMSCs and DA-IGF-1 were simultaneously applied to enhance the bioactivity of PLGA scaffolds as nerve implants. The potential of hUCMSC-seeded, DA-IGF-1-modified PLGA implants as promising candidates for promoting axonal regeneration and motor functional recovery in spinal cord injury treatment were demonstrated [21]. Notably, DA-IGF-1 offers straightforward operation, robust binding, and exhibits universal adhesive properties across most substrate materials. While DA-IGF-1 has been demonstrated to exert a therapeutic effect, its application in skin repair remains unexplored. Moreover, dopamine has been reported to exhibit anti-angiogenic activity [22]. Whether DOPA, an analog of dopamine, impairs angiogenesis in wound remains to be elucidated, making the investigation of DA-IGF-1's efficacy in skin repair highly significant for broadening its biomedical utility.

Fig. 1.

(A) The schematic showing the metabolic pathway of tyrosine hydroxylation. The part in the block was the tyrosine hydroxylation governed in this study. (B) The diagram of YK-IGF-1 encoding frame. The pentapeptide YKYKY was the precursor of adhesion domain. (C) SDS-PAGE analysis of recombinant IGF-1. Lane M: marker; lane 1: whole bacteria before induction; lane 1: whole bacteria after induction; lane 3: solubilized recombinant protein in dissolution buffer; lane 4: flow of purification; lane 5: elution of purification; lane 6: the renatured protein; lane 7-9: target protein purified by molecular sieve. (D) Cricular Dichroism (CD) spectrum of IGF-1, YK-IGF-1 and DA-IGF-1. (E) The CCK-8 test showed the bioactivity of IGF-1, YK-IGF-1 and DA-IGF-1 with gradient concentrations on the proliferation of NIH-3T3 cells. n = 3.

In the present study, as a continuation of our previous work, the recombinant DA-IGF-1 was prepared to fabricate a novel bioactive scaffold for skin regeneration. A deposition modeling 3D printing technology based on the phase inversion method was used to construct flexible PLGA scaffold with regular porous structure, which is beneficial for growth of cell. The adhesion, proliferation and relative mRNA expression of cell on PLGA scaffold was evaluated. Meanwhile, the regeneration of full skin injury in the rat's back was carried out to evaluate the ability of the implantation for wound repair. The purpose of this study was to establish a novel skin implantation to promote wound regeneration.

2. Methods

2.1. Preparation of DA-IGF-1

The gene sequence encoding YK-IGF-1 was inserted into the pET15b plasmid (Novagen) through restriction enzyme cloning to generate a recombinant expression vector. This plasmid construct was subsequently introduced into chemically competent Escherichia coli BL21(DE3) cells via thermal shock transformation to establish a prokaryotic expression platform. A single transformant colony was selected and pre-cultured in 10 mL of LB medium supplemented with 100 μg/mL ampicillin at 37 °C with orbital shaking (150 rpm) for 12 h to achieve logarithmic growth. The primary culture was then inoculated at 1% (v/v) ratio into 1 L of YTA medium (containing 1% tryptone, 0.5% yeast extract, and 0.5% NaCl) under identical incubation conditions until the optical density at 600 nm (OD₆₀₀) reached 1.2, indicating mid-log phase bacterial growth. Protein expression was induced by supplementing the culture with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by further incubation at 28 °C with continuous agitation (150 rpm) for 12 h.

The bacterial cells were harvested via centrifugation at 4000×g for 15 min at 4 °C and subsequently resuspended in lysis buffer (50 mM PBS, 8 mM urea, pH 8.0). Cellular lysis was performed using an ultrasonic disruptor operating at 200 W output power with a duty cycle of 10 s-on/10 s-off for a total duration of 5 min under ice-cooled conditions. Following centrifugation at 12,000×g for 20 min at 4 °C, inclusion bodies were selectively isolated from the pellet fraction. Prior to solubilization in 30 mL of dissolution buffer [50 mM PBS, 8 M urea, 5 mM dithiothreitol (DTT), pH 8.0], the inclusion body fraction underwent two sequential wash cycles with wash buffer (50 mM PBS, 2 M urea, pH 8.0) to remove residual contaminants. The clarified supernatant containing the solubilized recombinant protein was subjected to immobilized metal affinity chromatography (IMAC) purification using Ni-NTA resin (QIAGEN). Target protein YK-IGF-1 was specifically eluted with elution buffer (50 mM PBS, 0.5 M NaCl, 8 M urea, 5 mM DTT, 200 mM imidazole, pH 8.0). A two-stage refolding strategy was implemented: Primary refolding was achieved through controlled gradient dilution (100 μL/min infusion rate) of the eluate with 10 vol of renaturation solution [50 mM Tris, 400 mM l-arginine, 2 mM EDTA, 2 M urea, 5 mM reduced glutathione (GSH), 0.5 mM oxidized glutathione (GSSG), 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0]. Subsequently, the refolded protein solution was dialyzed against deionized water (4 °C, 12 h with three buffer exchanges) to eliminate residual urea, imidazole, and DTT. Protein identity and purity were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.

Tyrosine hydroxylation was performed using tyrosinase (50 U μL⁻¹, 1 μL) (Sigma Aldrich, USA), ascorbic acid (5 mg mL⁻¹, 500 μL, pH 7.2), YK-IGF-1 (100 μg mL⁻¹, 500 μL, pH 7.2), and PBS (1 mL, pH 7.2) at 4 °C for 4 h. The circular dichroism (CD) spectrum and bioactivity assay of IGF-1, YK-IGF-1 and DA-IGF-1 were performed to explore whether the structure and bioactivity of IGF-1 was influenced through preparation and hydroxylation.

2.2. Fabrication of 3D-printing PLGA scaffold

The flexible PLGA (lactide/glycolide ratio = 75:25, MW = 80 kDa) scaffolds were fabricated via phase inversion deposition modeling (PIDM)-based 3D printing technology. PLGA obtained from Changchun Institute of Applied Chemistry was dissolved in N-methyl-2-pyrrolidone (NMP, Aladdin Biochemical Technology Co., Ltd.) to prepare a 40% (w/v) polymer solution as printing ink. Scaffold fabrication was performed using a custom-built 3D bioprinter (Bio-Fabrication Plus, Ubbiotech) controlled by SolidWorks® CAD software (Dassault Systèmes), with the following processing parameters: an extrusion nozzle diameter of 200 μm, an ambient temperature of 25 °C, and a layer thickness of 100 μm. The ink was pneumatically extruded at 0.2 MPa onto 75% (v/v) ethanol-moistened filter paper, where immediate phase inversion occurred through solvent-nonsolvent exchange. After printing, the scaffolds were subjected to sequential solvent removal by immersion in 50% ethanol (4 °C, 48 h) followed by vacuum freeze-drying (−80 °C, 0.05 mbar, 48 h) to eliminate residual NMP.

For DA-IGF-1 functionalization, scaffolds were incubated in protein solution (pH 8.5) for 12 h at 37 °C under gentle agitation (50 rpm), followed by extensive washing with PBS (pH 7.4) to remove unbound proteins. For morphological characterization, the scaffolds were sputter-coated with 15 nm of gold prior to observation via field-emission scanning electron microscopy (FE-SEM, SU8010, Hitachi) at an accelerating voltage of 5 kV. Surface chemical composition analysis was performed using X-ray photoelectron spectroscopy (XPS, Axis Supra, Kratos Analytical) with monochromatic Al Kα radiation (1486.6 eV). Hydrophilicity assessment was conducted via sessile drop method using a contact angle goniometer (DSA100, Krüss GmbH) with 3 μL droplets of deionized water under ambient conditions (25 °C, 60% RH). The tensile strength of hydrogels was test by a universal testing machine (Instron 5982, INSTRON, US).

2.3. Adhesion and stability of DA-IGF-1

The protein conjugation efficiency was evaluated using an optimized ligand immobilization protocol. Scaffolds were fixed in 24-well culture plates and incubated with 1 mL DA-IGF-1 solution (100 ng/mL in PBS, pH 7.4) at 4 °C for 12 h to allow sufficient immobilization. The initial supernatant was collected after centrifugation (400×g, 5 min) and designated as S1. Subsequently, three consecutive washing cycles were performed using 1 mL of PBS-T buffer (0.1% Tween 20, pH 7.2) under orbital shaking (150 rpm, 5 min/cycle) to remove non-specifically adsorbed proteins. The wash supernatants from the second to fourth cycles (S2–S4) sequentially after each washing step. The IGF-1 content in all supernatants was quantitatively analyzed using a commercially available ELISA kit (COIBO BIO, Shanghai) according to the manufacturer's standardized protocol. The absorbance was measured at 450 nm using a microplate reader. The standard calibration curve of the IGF-1 ELISA kit was shown in Fig. S2. The adhesion amount of fusion protein was calculated using the following formula:

$$\text{Adhesion}\text{Amount}\left(\text{AA}\right)=\text{Initial}\text{Amount}\left(\text{IA}\right)-\text{Sample}\text{Amount}\left(\text{SA}\right)$$

IA was the total protein amount in solution. SA was the total amount of unconjugated protein on the microspheres (SA =S1+S2+S3+S4).

The dynamic release kinetics of IGF-1 were quantitatively assessed using a modified sample-and-replace protocol under physiological simulation conditions (37 °C, 100 rpm). Scaffolds (100 mg) were loaded into 24-well plates containing 1 mL PBS (pH 7.2) maintained at 37 °C using a thermostatic shaker (TS-100C, Biosan). At predetermined intervals (10 min −72 h), 200 μL of supernatant was collected for analysis and replaced with fresh PBS (pH 7.2). The stability of IGF-1 is determined by the percentage of the protein retained on the scaffold to the total adhesion protein. Commercial IGF-1 was used as a control.

2.4. Bioactivity test in vitro

The NIH/3T3 cells (China Typical Model Cultivation Center, CTMCC) were expanded in high-glucose Dulbecco's Modified Eagle Medium (HG-DMEM; Servicebio) supplemented with 10% fetal bovine serum (FBS; BI) under standard culture conditions (37 °C, 5% CO₂). Second-passage (P2) cells were enzymatically dissociated using 0.25% trypsin-EDTA solution and subsequently seeded onto different scaffolds at a density of 1.5 × 10⁴ cells/well. Scaffold modification involved 12 h co-incubation with either 200 ng/mL IGF-1, DA-IGF-1, or PBS control (pH 7.2) under static conditions. The culture medium was replenished every 48 h with pre-warmed (37 °C) complete medium.

On day 4 and 7, in order to evaluate cell adhesion, Calcein AM (Beyotime) was used to stain the cells. After the cells were washed with PBS, the morphology of the cells was observed by a fluorescence microscope. At the same time, CCK-8 assay was used to determine cell proliferation. On day 7, total RNA was extracted using TRIzol Reagent (Invitrogen). And the reverse transcription was governed using PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time, TaKaRa). The expressions of genes related to skin regeneration were detected via RT-PCR. All the primers using in this study were listed in Table S1. Specificity of listed oligonucleotides were checked by Basic Local Alignment Search Tool (BLASTN) against the homo RefSeq RNA database at NCBI.

2.5. In vivo wound healing assessment

Ten female Wistar rats (8 weeks, 200-240 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rats were acclimatized under specific pathogen-free (SPF) conditions for 7 days prior to the experiment. All procedures were performed in strict compliance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Jilin University. A full-thickness skin defect model was established to evaluate the ability of the scaffolds to accelerate wound regeneration. Briefly, all animals were anaesthetized via intraperitoneal injection of pentobarbital sodium (50 mg kg⁻¹). The back hair was removed with a razor and hair removal cream. Then, a full-thickness skin defect with a diameter of 15 mm was created on the back of the rat. Different scaffolds (PLGA, IGF-1@PLGA, and DA-IGF-1@PLGA) were then implanted into the defects and fixed with gauze. Wound healing was photographed and observed using a digital camera (EOS R5, Canon) at 0-, 3-, 7-, and 10-days post-implantation. The healing rate of the wound was calculated according to the following formula:

$$\text{Wound}\text{closure}\text{rate}(\%)=\left(A_0-A_t\right)/A_0\times 100\%$$

A₀ represents the initial area of the wound, and A_t represents the area of the wound on stated time points.

At the final time point, the rats were sacrificed via overdose anesthesia. The samples were cut and fixed with 4% paraformaldehyde. After dehydrated with ethanol, the samples were embedded in paraffin blocks and sliced. The tissue sections were stained with H&E and Masson trichrome. Meanwhile, the immunohistochemical staining of CD31 and alpha smooth muscle actin (α-SMA) was performed on wound sections to evaluate angiogenesis. Finally, the staining sections was observed under a microscope (Axio Observer Apotome 3, ZEISS).

2.6. Statistical analysis

The statistical analysis was performed using ANOVA, followed by Tukey's honestly significant difference (HSD) for multiple comparisons. The differences were considered statistically significant at p < 0.05. Three or four independent replicates of each experiment were conducted.

3. Results

3.1. Preparation and characterization of fused protein

After induction with 1 mM IPTG at 25 °C for 16 h, the fused protein was produced by E. coli containing pET15b-YK-IGF-1. As shown in Fig. 1(B), the recombinant protein was designed to comprise natural IGF-1, YKYKY, and His-tag (for affinity purification). The SDS–PAGE photos in Fig. 1(C) depict the expression and purification process of YK-IGF-1. At the desired molecular weight position near 8.5 kD, a distinct band was observed in lane 9, which corresponds to the final product of YK-IGF-1. Few visible contaminating proteins was observed in this lane.

After hydroxylation by tyrosinase, YK-IGF-1 was converted to DA-IGF-1. To explore whether the structure and bioactivity of IGF-1 were affected by the preparation and hydroxylation processes, CD spectrum and bioactivity assay of IGF-1, YK-IGF-1 and DA-IGF-1 were performed. The CD spectral profiles were smoothed 10 times and shown in Fig. 1(D). Positive peaks near the wavelength of 192 nm occurred in all the fused proteins to represent α-helices. However, the peak height and half-peak width were slightly different. A weak positive peak corresponding to β-sheets was detected at 200 nm for IGF-1, YK-IGF-1, and DA-IGF-1. No obvious negative peek was observed at 198 nm. This result indicated that there were few random coils in the structures of fused proteins. In the wavelength ranged from 208 nm to 250 nm, no significant peak was detected at the characteristic wavelength. Positive peaks representing aromatic amino acid residues were observed in all the fused proteins. Overall, there was no significant difference among the CD spectrum atlases of fused IGF-1s.

Growth factors are critical in skin regeneration. Many reports have confirmed that IGF-1 is an effective growth factor to promote skin regeneration [[23], [24], [25]]. However, it cannot be neglected that growth factor has a short half-life and easily diffuses with body fluids [26]. In this work, a novel adhesive IGF-1 with a bionic nonspecific adhesion domain, which was first mentioned by P. B. Messersmith's group to mimic Mefp-5 [17,18], was designed and prepared. DOPA was regarded as the key agent in Mefp-5 for adhesion. In Chen's report [19], a mussel-inspired biorthogonal approach was used to design a DOPA-containing recombinant insulin-like growth-factor-1 (DA-IGF-1) using a combination of gene recombinant technology and tyrosinase treatment for the surface modification of titanium. DA-IGF-1 exhibited stronger adhesion to titanium and significantly accelerated the growth of NIH-3T3 cells on the surface of titanium. Based on this method, in our previous work, surface modification of PLGA films with DA-IGF-1 was performed to promote neurite outgrowth of PC12 cells [20], further promote axonal regeneration and motor functional recovery in spinal cord injury [21]. However, the effect of DA-IGF-1 in skin tissue engineering has not been investigated yet. In the present study, YK-IGF-1 was achieved and further transformed into DA-IGF-1. Proliferation of NIH-3T3 in the presence of soluble IGF-1s was carried out to evaluate the bioactivity. The results in Fig. 1(E) proved that the fused IGF-1s (YK-IGF-1 and DA-IGF-1) were as bioactive as natural IGF-1. The CD spectrum atlases of the fused IGF-1s, which were used to reveal the secondary structures, were barely different. The CD spectrum atlas results indicated that the addition of the adhesion domain seldom altered the secondary structure of IGF-1. The spatial structure of IGF-1 was determined to govern the conjugation between IGF-1 and the insulin-like growth factor-1 receptor (IGF-1R). By binding to IGF-1R, IGF-1 phosphorylates and activates downstream signaling pathways such as PI3K-Akt [27], Tau [28], and ERK pathway [29], subsequently affecting the angiogenesis, development, and cell growth and apoptosis [30]. Consequently, the conserved spatial structure contributed to the good bioactivity of DA-IGF-1.

3.2. Characterization of modified scaffolds

The PLGA scaffold prepared by 3D printing exhibited the expected flexible properties (Fig. S1). The SEM photos of scaffolds are performed in Fig. 2(A), mainly showing the surface morphology of the composite scaffolds. As can be seen, the filaments and pores of the scaffold were uniform and neat, with a pore size of approximately 400 μm. The structures provide a channel for exchange of nutrient and body fluid, mimicking the structure of bone trabeculae, and also serve as a framework for tissue growth. Unlike the FDM 3D-printing scaffold reported before [31], The surface of the PIDM 3D-printing scaffold is composed of many micro and nano pores and gullies, which render the scaffold a rougher surface and a larger specific surface area. No significant differences in surface morphology were observed between the PLGA and IGF-1@PLGA scaffolds. Unlike the other two groups, certain areas on the surface of the DA-IGF-1@PLGA scaffold were covered with proteins.

Fig. 2.

(A) The SEM photos of flexible PLGA scaffolds in different groups. (B) X-ray photoelectron spectroscopy (XPS, N1s) of the 3D-printing flexible PLGA scaffold modified with PBS, IGF-1 and DA-IGF-1. (C) The hydrophilia performance of the 3D-printing flexible PLGA scaffold modified with PBS, IGF-1 and DA-IGF-1. (D) The tensile strength of the 3D-printing flexible PLGA scaffold modified with PBS, IGF-1 and DA-IGF-1. (E) Bar = 200 μm, n = 3, ∗p < 0.05.

The XPS results are shown in Fig. 2(B). According to the high-resolution spectrogram of N1s, superposed peaks consisting of the characteristic peaks were found at 398.3 eV, 399.8 eV and 402.1 eV, which indicated amine (–NH–), imine (=N-), and (N⁺) in the spectrograms of IGF-1@PLGA and DA-IGF-1@PLGA groups, respectively. It was found that the characteristic peaks on DA-IGF-1@PLGA scaffold were slightly higher than that on IGF-1@PLGA. The full width at half maximum (FWHM) of the characteristic peak on DA-IGF-1@PLGA scaffold was slightly wider than that of IGF-1@PLGA. This result indicated that the immobilization of IGF-1 was successfully achieved via the DOPA-containing binding domain.

Fig. 2(C) shows the hydrophilia performance of the modified scaffolds. As expected, the hydrophilicity of the scaffolds improved with the adhesion of different proteins. However, the IGF-1@PLGA scaffold was still hydrophobic, while the scaffold modified by DA-IGF-1 was hydrophilic. The uniaxial static tensile testing was performed using a universal testing machine to evaluate the tensile properties of the scaffolds. Samples were stretched longitudinally at a constant displacement rate of 1 mm/min until fracture. The maximum tensile strength under strain was recorded throughout the process. The tensile strengths of each group of scaffolds were basically the similar, ranging around 2.1-2.2 MPa. No statistically significant difference was observed across groups (p > 0.05; Fig. 2(D)).

The adhesion and release of DA-IGF-1 were quantitatively detected via the ELISA method. According to the result in Table S2, the adhesion rate of DA-IGF-1 on scaffold was 79.12 ± 2.63 %, while that of IGF-1 was 42.35 ± 5.28 %. The adhesion capacity of DA-IGF-1 to the scaffold was significantly higher than that of IGF-1 (p < 0.05). As shown in Fig. 2(E), DA-IGF-1 was released from the scaffold at an extremely slow rate over time. After 72 h, 79.98 ± 1.33% of DA-IGF-1 remained on the scaffold. In contrast, IGF-1 exhibited a rapid release profile from the scaffold. After 72 h, only 21.96 ± 2.56% of IGF-1 remained.

An appropriate material is very important for skin tissue engineering. Many kinds of natural materials, such as collagen [32]and chitosan [33], as well as synthetic materials, including polylactic acid (PLA) [34] and poly(gamma-glutamic acid) (PGA) [35], have been demonstrated to promote skin regeneration. PLGA is one of the most extensively studied biodegradable materials. Due to its good biocompatibility, low immunogenicity and low toxicity, it has been widely applied in tissue engineering. However, its hydrophobicity and lack of bioactivity seriously limit its biological applications. In this work, PLGA was selected as the basic scaffold material. SEM images clearly demonstrated that the scaffold possessed a rough topography and porous structure, which are believed to facilitate cell adhesion, growth, and migration. Benefitting from DA-IGF-1 modification, the hydrophobic PLGA scaffold was absolutely improved to become hydrophilic. Enhanced hydrophilicity is generally recognized as an advantage for cellular interaction with materials [[36], [37], [38]], which is very important in skin regeneration.

3.3. Cell morphology and proliferation on scaffold

Enhancement of cell adhesion and proliferation is critical for evaluating the biocompatibility of biomaterials and facilitates skin regeneration [39]. Therefore, the cell morphology and proliferation on all groups of scaffold materials were investigated to determine whether the biocompatibility of the biomaterial was improved (Fig. 3A-B). In this study, the scaffold material modified with DA-IGF-1 exhibited superior cytocompatibility, as the number of cells growing on it was significantly higher than that in the other groups. Compared with the PLGA group, the cell proliferation in each IGF-1-containing group was significantly accelerated at 3 and 7 days (p < 0.05). In addition, the DA-IGF-1-modified scaffold exerted a significantly positive effect on the proliferation of NIH-3T3 cells compared with IGF-1 group at 3 and 7 days, with a statistically significant difference (p < 0.05). Since the number of cells growing on the bottom of the well (rather than on the scaffold) was essentially the same across all groups (Fig. S3), the observed intergroup differences were solely attributed to the different growth states of cells on the scaffolds. This result indicated that not only the adhesion but also the proliferation of NIH-3T3 cells was enhanced on the scaffold modified by DA-IGF-1. The biocompatibility of the PLGA scaffold was adequately improved by DA-IGF-1.

Fig. 3.

Cytocompatibility and bioactivity of different scaffolds. (A) Calcein-AM staining photos and (B) proliferation of NIH-3T3 cells on scaffold in each group. (C) The relative mRNA expression of bFGF, Col1, integrin β, and VEGF in NIH-3T3 cells on different scaffolds at 7th day. Bar = 200 μm, n = 3, ∗p < 0.05.

In this study, a viable skin implant based on a bioactive PLGA 3D-printing scaffold modified by DA-IGF-1 was developed. It is well known that skin wound healing is a dynamic and interactive process, in which cell proliferation plays a crucial role in the re-epithelialization of the wound area [[40], [41], [42]]. It has been widely recommended that IGF-1 treatment can increase cell proliferation and collagen production while reducing collagenase activity [43]. However, the effect of IGF-1 cannot be sustained unless the its rapid loss is controlled in vivo. Benefiting from DOPA-mediated adhesion, DA-IGF-1 bound stably to the material, enabling the sustained stimulatory effect of IGF-1 on fibroblast proliferation. On the other hand, the scaffold was proven to become hydrophilic when modified by DA-IGF-1, which was considered to be conducive to cell adhesion and proliferation [20,21]. The promoted cell adhesion and proliferation ability will assist the scaffold material to promote the repair of skin damage.

3.4. Related gene expression by qRT–PCR tests

In the comparative study evaluating the effects of different materials on gene expression in NIH-3T3 cells, the expression levels of four key genes (bFGF, Col1, integrin β, and VEGF) were quantified by qRT–PCR (Fig. 3(C)). The expression intensity of each gene was normalized to the expression level of GAPDH.

Basic fibroblast growth factor (bFGF), a potent mitogen, accelerates skin repair by stimulating fibroblast proliferation, keratinocyte migration, and extracellular matrix (ECM) synthesis. Upon binding to FGFR1, it activates MAPK and PI3K/Akt pathways, thereby enhancing collagen production. Additionally, bFGF promotes angiogenesis through endothelial cell activation, upregulates MMPs for ECM remodeling, and enhance re-epithelialization and granulation tissue formation during wound healing [44]. According to the results shown in Fig. 3(C), the bFGF gene expression in NIH-3T3 cells cultured on the DA-IGF-1@PLGA scaffold was significantly upregulated compared with that in cells cultured on PLGA and IGF-1@PLGA groups (p < 0.05). Furthermore, the bFGF expression level in the IGF-1@PLGA group was significantly higher than that in the PLGA group (p < 0.05).

Collagen type I (Col1), the predominant structural protein in the dermal ECM, is essential for restoring the mechanical integrity during skin repair. Col1 provides tensile strength to regenerated tissue by forming cross-linked fibrils that resist deformation. It facilitates fibroblast migration, proliferation, and differentiation via integrin-mediated signaling, while synergizing with growth factors to promote ECM deposition [45]. During the tissue remodeling, Col1 gradually replaces Col3 to drive the maturation of scar tissue [46]. At 7 days, compared with the other two groups, the expression of Col1 in DA-IGF-1@PLGA group was remarkably increased (p < 0.05). Cells on DA-IGF-1@PLGA scaffold exhibited superior collagen synthesis.

Integrin β is a key factor to mediates ECM adhesion. It facilitates fibroblast migration, thereby further regulating wound contraction and ECM remodeling [47]. At 7 days, the expression of integrin β in the IGF-1@PLGA group was significantly higher than that in the PLGA group (p < 0.05). Notably, the expression level of the integrin β gene in the DA-IGF-1@PLGA group was highest among all groups and significantly upregulated (p < 0.05).

Vascular endothelial growth factor (VEGF) is a pivotal mediator of angiogenesis during skin repair, driving the formation of new blood vessels to restore oxygen and nutrient supply to hypoxic wound beds [48,49]. VEGF also synergizes with bFGF to stabilize nascent vessels via pericyte recruitment [50]. In the present study, VEGF gene expression was markedly upregulated in the DA-IGF-1 group, and was absolutely higher than that of the PLGA group (p < 0.05). These results fully indicate that the DA-IGF-1-modified graft is capable of promoting angiogenesis.

Skin regeneration requires the coordinated activation of cellular proliferation, ECM synthesis, and angiogenesis. Tissue-engineered scaffolds incorporating growth factors such as IGF-1 have demonstrated great potential. However, challenges including burst release and low bioavailability remain unresolved. Dopamine (DA) modification, known for enhancing interfacial adhesion and enabling controlled release, was hypothesized to optimize IGF-1 delivery and fibroblast activation. The DA-IGF-1@PLGA scaffold outperformed the other groups by integrating material functionalization and biological signaling.

3.5. Wound healing effect of skin implantation in SD rats

The healing effect of skin implantation was verified using a dorsal skin wound model in SD rats. Fig. 4(A) shows the wound appearance in each group at different time points. As time progressed, the wound area of each group gradually decreased. Although the scaffold acted as a factor hindering skin contraction and thus impeded wound closure, the DA-IGF-1@PLGA group still exhibited a superior ability to promote wound closure compared with the control group at different stages of wound repair (p < 0.05). On day 10, the skin wound in the DA-IGF-1@PLGA group was the smallest among all groups. The wound closure rate of each group was subsequently measured. The results are shown in Fig. 4(B). At all time points, the wound closure rate of DA-IGF-1@PLGA group was significantly higher than that of the other groups (p < 0.05). On day 10, the relative reduction in wound size of the DA-IGF-1@PLGA group nearly reached 100%, whereas that of the other groups were less than 90%.

Fig. 4.

(A) The appearance of the reduction in wound size in different groups of rats treated with various scaffolds for 10 days. (B) The wound size reduction (%) with time. Bar = 5 mm, n = 3, ∗p < 0.05.

Traditional synthetic polymer-based wound repair materials are primarily fabricated via electrospinning, solution casting, and thermal stretching et al. Such materials are either non-porous or possess merely dense micro/nano-scale pore structures [51]. The 3D-printing scaffold is capable of providing a spacious microenvironment to support cellular proliferation and granulation tissue neoformation [52,53]. Thus, developing a 3D printing method to prepare flexible biodegradable polymer scaffold for cell and tissue growth is essential. In this study, a DA-IGF-1@PLGA membrane was fabricated via the combined use of solution casting and the PIDM method as a control for in vivo investigations. The appearances of the reduction in wound size treated with DA-IGF-1@PLGA membrane for 10 days was shown in Fig. S4. The results demonstrated that the wound closure rate of the DA-IGF-1@PLGA membrane group reached 67.6 ± 12.5% at day 10, which was significantly lower than that of the DA-IGF-1@PLGA (3D-printing) group (p < 0.05). Moreover, the DA-IGF-1@PLGA (3D-printing) group exhibited a significantly superior wound repair efficacy over the DA-IGF-1@PLGA membrane group at all preceding time points (p < 0.05). The DA-IGF-1@PLGA membrane occupied the wound bed and thereby impeded wound re-epithelialization and closure.

The degradation rate of the material depends not only on the molecular weight and feed ratio of the raw materials, but also on the processing technology, material topography and microenvironment [54,55]. The molecular weight of PLGA raw materials decrease during the dissolution and 3D printing processes [56]. In addition, the 3D-printing scaffolds fabricated via the method established in this study possessed hierarchical microstructures, including micron-scale pores, micro/nano-scale pores and surface grooves. Meanwhile, the incorporation of DA-IGF-1 significantly enhanced the hydrophilicity of the scaffolds. These synergistic factors facilitated the infiltration of bodily fluids into the scaffolds following in vivo implantation. And the scaffolds further acted in concert with various enzymes in the bodily fluids to accelerate the degradation rate [55]. Although complete degradation was not achieved, the overall structural integrity of the scaffolds was compromised, thus precluding any impediment to wound closure and repair. The residual material fragments were partially eliminated and exfoliated from the wound site. A similar phenomenon of structural incompleteness was clearly observed in the DA-IGF-1@PLGA membrane group at day 10 (Fig. S4). In contrast, the PLGA scaffolds remained structurally intact at day 10. Furthermore, empty cavities left by the space-occupying scaffolds were clearly visible in the stained tissue sections. In Chen's report [57], a 3D fibrous scaffolds of poly(lactic acid-co-glycolic acid) (PLGA) were successfully fabricated by liquid-collecting electrospinning. Surface modification with the native ECM component aims at providing biological recognition for cell growth. In vivo research showed that the scaffold degraded and contracted progressively during wound healing, and sloughed off by day 10. Zhang et al. [58] engineered a PLGA electrospun nanofiber membrane loaded with citric acid (CA) and iron (Fe) nanoparticles (Fe@PLGA + CA). The Fe@PLGA + CA membrane significantly promotes vessel formation, wound-healing and accelerates skin regeneration. Consistent with our findings, the material gradually degraded, shrank and was eliminated from the wound before the experimental endpoint. The results of present study underscore the advantage of DA-IGF-1 modification in regulating the in vivo degradation of the scaffolds, which enables the degradation process to be more well-matched to the natural course of cutaneous wound repair.

The histological characteristics of each group were evaluated by H&E and Masson trichromatic staining. According to the H&E photos (Fig. 5), the histological morphology of the epidermis and dermis was varied in different groups. In PLGA group, the structure of newly formed dermal tissue was loose. The boundary between epidermis and dermis was not clear. A large number of inflammatory cells infiltrated the interstitium of the regenerate tissue. In contrast to the PLGA group, the control and IGF-1@PLGA groups exhibited a clear boundary between the epidermis and dermis. Although the new dermis structure was still somewhat loose, it was better than that of PLGA group. Additionally, fewer inflammatory cells were observed in the interstitium. Compared with the other groups, the dermal tissue in the DA-IGF-1@PLGA group was the densest. In addition, the regenerated epidermal layer in the DA-IGF-1@PLGA group was the thickest and densest among all groups.

Fig. 5.

The photos of H&E staining of specimens at 10 days postoperatively. Bar = 100 μm.

The result of Masson trichromatic staining (Fig. 6) was strong evidence of H&E result. The blue-dyed collagen fibers in PLGA group were loosely arranged. Compared with other groups, there were more blue-dyed collagen fibers in DA-IGF-1@PLGA group and they were arranged closely. This indicates that the new dermal tissue in DA-IGF-1@PLGA group was more mature. Briefly, compared with the other groups, skin regeneration was more obvious after the transplantation of DA-IGF-1@PLGA scaffold.

Fig. 6.

The photos of Masson trichromatic staining of specimens at 10 days postoperatively. Bar = 100 μm.

The immunohistochemical staining of CD31 was shown in Fig. 7. There were only some sporadic positive cells in PLGA groups. In contrast, it was revealed that numerous CD31-positive cells were present in the control, IGF-1@PLGA, and DA-IGF-1@PLGA groups, most of which formed tubes (neovasculature), at 10 days after transplantation. However, compared with the control group and the IGF-1@PLGA group, the number of CD31-positive cells in the DA-IGF-1@PLGA group was the highest.

Fig. 7.

The immunohistochemical staining of CD31 of specimens at 10 days postoperatively. Positively stained neovascularization is pointed by black arrows. Bar = 100 μm.

It was reported that the IGF-1 could induce maturation of vascular network with pericytes [59,60]. Fig. 8 presented the immunohistochemical staining results for α-SMA. The control and PLGA groups showed only a small number of scattered α-SMA-positive cells. In contrast, abundant α-SMA-positive cells were found in the IGF-1@PLGA and DA-IGF-1@PLGA groups at 10 days post-transplantation. A significantly higher density of α-SMA-positive cells was detected in the DA-IGF-1@PLGA group than in both the control and IGF-1@PLGA groups. This indicated a higher vascular maturation at the wound sites in the DA-IGF-1@PLGA group.

Fig. 8.

The immunohistochemical staining of α-SMA of specimens at 10 days postoperatively. Positively staining is pointed by black arrows. Bar = 100 μm.

In the present study, it was found that wound healing was obviously promoted in the DA-IGF-1@PLGA group, while H&E and Masson trichromatic staining showed that the dermis was dense and the regenerated epidermal layer was the thick in the DA-IGF-1@PLGA group. The research also found that CD31 and α-SMA were significantly increased in wound tissues with DA-IGF-1-modified scaffold transplantation. Consistently, a previous study [61] indicated that IGF-1 could promote the healing of diabetic ulcers by neovascularization. Abundant extracellular matrix (ECM) could play an extremely positive role in skin regeneration. As the major component of the skin ECM, Col1 can facilitate cell proliferation, adhesion and migration. In the previous study in this report, the expression of Col1 in seed cells has been evaluated. The results indicated that the DA-IGF-1-modified scaffold excellently benefited the deposition of Col1. This would benefit the interconnection among multiple cells settled on the implantation. Angiogenesis nourished the cambium and removed wastes from the damaged skin. Therefore, the neovasculature is very important for wound regeneration. While DA-IGF-1 has been demonstrated to exert a therapeutic effect on neural repair in our prior research [21], its application in skin repair remains unexplored. Moreover, dopamine has been reported to exhibit anti-angiogenic activity [22]. It remains unclear whether DOPA, a dopamine analog, has a detrimental impact on cutaneous wound angiogenesis. The results of this study fully demonstrate that DA-IGF-1 can effectively promote wound angiogenesis. This also confirms, for the first time, that DOPA does not impair the biological activity of IGF-1. The bFGF and VEGF were considered the keys to vascularization in skin regeneration [62]. According to our study, the DA-IGF-1-modified scaffold could significantly induce bFGF and VEGF expression in the cells. The up-regulation of the expression of important genes and proteins directly affected the wound repair effect of stents in vivo.

4. Conclusion

In the present study, in order to obtain a novel skin repair material, a bioorthogonal IGF-1 (DA-IGF-1) with an adhesive domain derived from the mussel adhesive protein containing l-DOPA was achieved to modify the 3D-printing flexible PLGA scaffold. The DA-IGF-1 exhibited outstanding binding ability to the scaffold. Moreover, DA-IGF-1 also enhance the hydrophilicity, biocompatibility, and bioactivity of the scaffold. In vivo study indicated that the bioactive implants promoted healing and neovascularization of full-thickness skin defects in rats. These results demonstrated that the 3D-printing flexible PLGA scaffold modified with bioorthogonal IGF-1 has a potential application as a novel strategy for wound regeneration.

Author contributions

YZ performed the experiments and wrote the manuscript. QF performed the experiments. XY contributed to the study conception and design. All authors contributed to the article and approved the submitted version.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This research was supported by the Jilin Province Science and Technology Development Plan Item (No. 212558JC010489607).

Declaration of competing interest

The authors declare that they have no conflict of interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article: