Sodium alginate/gelatin hydrogels loaded with adipose‐derived mesenchymal stem cells promote wound healing in diabetic rats

Chronic refractory wounds are a common complication in diabetic patients. Adipose‐derived mesenchymal stem cells (ASCs) have been shown to play an essential role in diabetic wound repair.

A range of studies have validated the role of stem cells during wound repair. 5 Adipose-derived mesenchymal stem cells (ASCs) are pivotal candidates for the treatment of chronic wounds. During the healing process, ASCs repair damaged cells through the proliferation and differentiation into skin cells, and activate cell regeneration through autocrine or paracrine pathways. 6 ASCs enhance diabetic wound healing through the release of angiogenic cytokines and increased granulation tissue formation and epithelialization. 7 In diabetic rats, the transplantation of ASCs accelerates wound healing and angiogenesis. 8 Delivery of ASCs leads to increased levels of vascular endothelial growth factor (VEGF)-A, endothelial cell density and macrophages, and accelerates wound healing in diabetic mice. 9 Hydrogels are favorable materials for tissue engineering due to their high capability for cell loading, excellent biocompatibility, and high water content. 10 Cell adhesion, proliferation, and differentiation are also influenced by the hydrophilic properties of hydrogels. 11 Hydrogels crosslinked by gelatin show outstanding biocompatibility and are extensively employed as scaffolds for cell adhesion and growth factor transport. 12 Sodium alginate has been widely applied in bioprinting and its gelation can be induced by multivalent cationic transfer. 13 Sodium alginate and gelatin type A coacervate have been proposed as an innovative delivery system for epidermal growth factor (EGF), with improved efficiency for chronic diabetic wounds. 14 The feasibility of delivering mesenchymal stem cells from gelatin-alginate hydrogels to the stomach wall has been demonstrated in stomach explant mouse models. 15 In this study, we report the development of sodium alginate/ gelatin (Gel-Al) hydrogels with good biocompatibility. We document the beneficial effects of the Gel-Al loaded-ASCs on wound healing, epidermal regeneration, collagen recovery, angiogenesis, antiinflammation, and M2 macrophage polarization in diabetic rats. We therefore reveal the therapeutic potential of Gel-Al loaded-ASCs in diabetic wounds.

| Preparation of sodium alginate/gelatin (Gel-Al) hydrogels
Following disinfection by ultraviolet, 3% (w/v) sodium alginate (Al, Aladdin Chemical, Shanghai, China), and 5% (w/v) gelatin (Gel, Aladdin Chemical) were dissolved in sterilized phosphate buffer solution (PBS). Sodium alginate and gelatin solutions were then mixed at a volume-volume ratio of 40:60, and prepared into a hydrogel mixture through stirring at 600 rpm at 50°C for 2 h. The hydrogel mixture was then added to a 6-well plate (1 ml per well) before cooling on ice. CaCl 2 was mixed in deionized water to a final concentration of 100 mM, followed by high-pressure steam sterilization and storage at 4°C. CaCl 2 solution (3 ml) was added dropwise into each well, followed by soaking at room temperature for 10 min. After washing in PBS (x2), Gel-Al hydrogels were obtained.

| Isolation of ASCs
The skin of Sprague-Dawley (SD) rats aged 4 days was disinfected in 75% alcohol and an incision was performed along the abdominal line. Subcutaneous groin fat was removed and adipose tissue was diced and washed in PBS. Samples were digested in 0.05% trypsin (Gibco) and 0.1% type I collagenase (Gibco), and centrifuged for 10 min at 1500 rpm. Pellets were resuspended in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco), and cultured in 5% CO 2 at 37°C for 48 h and media was replaced following the removal of non-adherent cells. ASCs at passage three were selected for subsequent experiments. transforming growth factor beta1 (TGFβ1), interleukin-10 (IL-10), interleukin-4 (IL-4) and interleukin-13 (IL-13) expression, and increased M2 macrophage polarization.
Conclusions: Gel-Al hydrogels loaded with ASCs accelerate diabetic wound healing.
The Gel-Al hydrogel-based ASC system therefore represents an innovative therapeutic strategy for diabetic wound repair.

K E Y W O R D S
adipose-derived mesenchymal stem cells, diabetes, sodium alginate/gelatin hydrogel, wound healing

| Identification of ASCs by flow cytometry
ASCs at passage three were grown to 80%-90% confluence, passed through a 100 μm filter, and resuspended in PBS at a density of

| Diabetic models and wound creation
Streptozotocin (100 mg/kg, Sigma) was dissolved in citrate buffer (0.01 M, pH 4.5). During the fasting stage, rats were intraperitoneally injected with streptozotocin solution twice weekly to induce diabetes.
Blood glucose were measured following streptozotocin injection using an Accu-Check Active glucometer (Roche, Lyon, France) every 2 days.
When blood glucose reached ≥250 mg/dL, animals were considered diabetic. Following diabetes induction, rats were anesthetized through the inhalation of isoflurane (2%). Full thickness excisional wounds on the dorsal skin of rats were created using a 10 mm biopsy punch.

| Histological staining
Full-thickness wound margins were fixed in 10% formaldehyde, paraffin embedded, and sectioned (4 μm-thick) perpendicular to the epidermal surface. Sections were deparaffinized in xylene and stained in hematoxylin and eosin and Masson solution. Epidermal reconstruction and collagen recovery were then examined.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was obtained following Trizol extraction (Takara, Tokyo, Japan) and cDNA synthesis was performed using commercial reverse transcription kits (Takara). RT-PCRs were performed using

| Statistical analysis
Data were analyzed using GraphPad Prism software and are shown as the mean ± SD. A one-way ANOVA followed by Tukey's post hoc test was used for multiple group comparisons. p < 0.05 was considered statistically significant.

| Effects of the Gel-Al hydrogels on the viability of ASCs
Alginate hydrogels were obtained following the addition of CaCl 2 solution (100 mM) through Ca 2+ -crosslinking at room temperature for 10 min to obtain a white translucent Gel-Al hydrogel ( Figure 1A).
Primary ASCs were isolated and labeled with CD90, CD73, CD105, CD45, and CD34 antibodies to detect their immune phenotype.
CD90, CD73, and CD105 were positively expressed in cells, while CD45 and CD34 were negatively expressed ( Figure 1B). The effects of Gel-Al on ASC viability and cytotoxicity were assessed through Calcein-AM/PI staining following 3 days of incubation. ASCs were seeded on Gel-Al wells and allowed to proliferate on the surface of the hydrogel. The viability of ASCs implanted on the hydrogel were unaltered compared to ASCs grown in dishes ( Figure 1C). These data suggest that the constructed Gel-Al had good biocompatibility.

| Gel-Al loaded-ASCs promote wound repair in diabetic rats
We next explored the efficacy of Gel-Al loaded-ASCs on cutaneous wound healing. Full-thickness diabetic wounds were treated with Gel-Al alone or Gel-Al loaded-ASCs. After 11, 14, and 18 days, the wound sizes in all treated groups significantly decreased. The control group displayed the slowest decrease in wound size, while Gel-Al loaded-ASCs showed the most effective wound healing ( Figure 2A). Compared to pure Gel-Al, wound healing improved following treatment with Gel-Al loaded-ASCs, suggesting that the loading of the ASCs enhanced the wound healing process. Wounds treated with Gel-Al loaded-ASCs healed more rapidly at day 14, with skin appendages observed through hematoxylin and eosin staining ( Figure 2B). Masson staining showed that Gel-Al loaded-ASCs could improve collagen deposition onto the wound site ( Figure 2C).
Collectively, these data indicate that Gel-Al loaded-ASCs promote favorable healing outcomes.

| Gel-Al loaded-ASCs enhance angiogenesis in diabetic wounds
Compared to control wounds, wounds treated with Gel-Al alone or Gel-Al loaded-ASCs exhibited increased EGF, PDGF, and VEGF levels. In addition, EGF, PDGF, and VEGF levels were higher in the Gel-Al loaded-ASC group compared to the Gel-Al alone group ( Figure 3A-C). Immunohistochemical staining of CD31, a biomarker of endothelial cells, was performed to assess angiogenic capacity during wound healing. Compared to the control group, wounds in the Gel-Al alone group or Gel-Al loaded-ASCs group exhibited elevated CD31 staining at day 14. Importantly, Gel-Al loaded-ASCs exhibited the strongest CD31 staining in comparison to other groups ( Figure 3D). These data suggest that Gel-Al loaded-ASCs exert a synergic effect with the hydrogel, leading to faster angiogenesis and wound healing.

| DISCUSS ION
It is now well accepted that ASCs can accelerate the wound healing process during diabetes. 9,16 In this study, a composite of Gel-Al hydrogel and ASCs were constructed to verify its effects on wound healing in diabetic rat cutaneous wounds in vivo. Gel-Al hydrogels showed good biocompatibility and maintained the viability of ASCs.
Importantly, the Gel-Al hydrogel/ASCs composite promoted the healing of diabetic wounds.
A number of hydrogels have been shown to facilitate chronic wound healing. Examples include bioactive antibacterial silica-based nanocomposite hydrogels that significantly accelerate skin tissue regeneration and wound healing. 17 Drug-loaded reactive oxygen species (ROS)-scavenging hydrogels can also be used to efficiently treat difficult-to-heal diabetic wounds. 18 Various natural polymers, including gelatin and alginate, have been used to treat diabetic foot ulcers and have been utilized as hydrogels. 19 PolyP-containing alginate/oxidized-alginate-gelatin hydrogels provide an appropriate regenerative active matrix for the healing of chronic wounds. 20 Diabetic wound healing has been shown to be accelerated by ASCs planted on Pluronic F-127 hydrogels. 21 Our results showed that the constructed Gel-Al hydrogels possessed a moderate gel time.
Moreover, the Gel-Al hydrogels could encapsulate ASCs and maintain their survival. We therefore propose that the hydrogel provides the correct microenvironment for the survival of ASCs.
ASCs show promise for regenerative therapy for chronic diabetic wounds. 22,23 Diabetic wound healing can be promoted by ASCs seeded in Pluronic F-127 hydrogels. 21 ASCs promote granulation tissue formation and facilitate diabetic wound healing. 24 Furthermore, ASCs have been shown to improve wound healing through promoting collagen synthesis. 25,26 To our knowledge, the effects of Gel-Al hydrogels and ASC complexes in diabetic wound healing have not been elucidated. In this study, we selected ASCs in combination with Gel-Al hydrogel for the treatment of chronic diabetic wound. Both Gel-Al hydrogels alone and the Gel-Al hydrogel/ASC combination reduced the wound area in vivo. Furthermore, the Gel-Al hydrogel/ ASC combination induced more rapid wound healing compared to the pure hydrogel or control treatment. Together, the complex of the Gel-Al hydrogel and ASCs on the wound surface could accelerate healing. Of note, skin appendages could be observed and collagen deposition was improved in wounds treated with Gel-Al hydrogel loaded-ASCs. We hypothesize that in the Gel-Al hydrogel/ASC combination group, the viability of ASCs was maintained by the Gel-Al hydrogels, promoting rapid wound healing.
Insufficient angiogenic capacity plays an important role in impaired wound healing in diabetes. 1 ASCs can increase neovascularization to accelerate diabetic wound healing. 24 ASCs planted in Pluronic F-127 hydrogels in rats could enhance angiogenesis (CD31 expression) in diabetic wounds. 21 In this study, CD31 expression was detected at the site of injury, suggesting that angiogenesis could be promoted in both Gel-Al alone and Gel-Al/ASCs combination groups. Furthermore, compared to the Gel-Al alone group, the Gel-Al/ASC combination improved angiogenesis, indicating that the ASCs played a crucial role during hydrogel-based transport to the wounds. This reveals the ability of the Gel-Al hydrogel and ASC combination to evoke angiogenic responses, therefore accelerating diabetic wound healing. Co-cultures of ASCs and DAT increased the secretion of local growth factors, including platelet-derived growth factor (PDGF), VEGF, and EGF, 27 all of which are involved in angiogenesis. 28 ASC-conditioned medium could promote the secretion of VEGF to facilitate wound healing in diabetic rats. 29 Moreover, key angiogenesis growth factors, including VEGF were upregulated in wounds treated with ASCs loaded in Pluronic F-127 hydrogels. 21 Here, VEGF, PDGF and EGF levels increased in both the Gel-Al/ASCs combination and Gel-Al alone groups, most notably in the Gel-Al/ASCs combination group. These results were consistent with the density of the blood vessels detected through CD31 staining.
ASCs regulate the release of inflammatory factors, such as IL-10, IL-4, and IL-13, which possess anti-inflammatory effects. 30 ASCs decrease the expression of inflammatory factors such as TNFα, IL-10, and IL-6, and accelerate diabetic wound closure. 31 The combination of acupuncture and stem cell-seeded cryogel/ hydrogel biomaterials on wounds produces immunomodulatory effects, partially mediated by the reduced expression of proinflammatory cytokines (IL-1β and TNFα). 32 ASC-conditioned medium can promote diabetic wound healing through increasing TGF-β1 secretion. 29 Consistently, TGF-β1, a key wound healing growth factor, is upregulated in wounds treated with ASCs loaded in Pluronic F-127 hydrogels. 21 We found that IL-6 levels decreased, but IL-10 and TGFβ1 levels increased in the Gel-Al alone group compared to the control group. In the ASCs/Gel-Al combination group, IL-1β levels decreased, while IL-13, IL-4, IL-10, and TGFβ1 levels increased, compared to those in the control group or Gel-Al alone group. IL-6 levels decreased in the ASCs/Gel-Al combination group, in comparison to the control group. This further demonstrates how the combination of Gel-Al hydrogels and ASCs can regulate the release of inflammatory cytokines, more efficiently F I G U R E 3 Angiogenesis in wounds treated with Gel-Al loaded-ASCs. (A-C) EGF, PDGF, and VEGF levels detected by qRT-PCR in wounds at postoperative day 7. Data are the mean ± SD. **p < 0.01, ***p < 0.001. (D) Blood vessels stained with DAPI (blue) and CD31 (green) in wounds at postoperative day 7 than pure Gel-Al hydrogels. In diabetic rats, conditioned medium obtained from ASCs seeded onto polydopamine-modified bioceramic scaffolds could promote macrophage switching from the M1 to M2 phenotype, enhancing vascularization and the rate of wound closure. 33 In situ-formed adhesive hyaluronic acid hydrogels encapsulating amnion-derived conditioned medium could regulate F I G U R E 4 Inflammatory cytokines and M2 macrophage polarization in wounds treated with Gel-Al loaded-ASCs. (A) Inflammatory cytokines including IL-13, IL-10, IL-6, IL-4, IL-1β, and TGFβ1 in wounds at postoperative day 3. Data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Wounds stained with the M2 macrophage marker CD163 (red), macrophage marker F4/80 (green), and DAPI (blue) at postoperative day 3 macrophage polarization and promote angiogenesis to accelerate diabetic wound healing. 34 We found that M2 macrophages in the wounds increased in the ASCs/Gel-Al combination group, in comparison to control group or Gel-Al alone group. These outcomes were consistent with wound healing performance, histological data, angiogenesis and inflammatory cytokine expression.
In summary, we have successfully constructed Gel-Al hydrogels and ASC composites for the treatment of chronic diabetic wounds.
Gel-Al hydrogels were appropriate for the delivery of ASC onto the wound surface to promote angiogenesis and M2 macrophage polarization. These hydrogels enabled the growth of ASCs could promote diabetic wound healing.

ACK N OWLED G M ENTS
None.

FU N D I N G I N FO R M ATI O N
This work was supported by National Natural Science Foundation of China Youth Fund (81801927).

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
All experimental procedures were approved by the Animal Ethics Committee in the Fifth Medical Center, Chinese PLA General Hospital (S2018-110-01).