Adipose‐Derived Mesenchymal Stem Cell‐Derived Exosomes Biopotentiated Extracellular Matrix Hydrogels Accelerate Diabetic Wound Healing and Skin Regeneration

Abstract Wound healing is an urgent clinical challenge, particularly in the case of chronic wounds. Traditional approaches to wound healing have limited therapeutic efficacy due to lengthy healing times, risk of immune rejection, and susceptibility to infection. Recently, adipose‐derived mesenchymal stem cell‐derived exosomes (ADSC‐exos) have emerged as a promising modality for tissue regeneration and wound repair. In this study, the development of a novel extracellular matrix hydrogel@exosomes (ECM@exo) is reported, which entails incorporation of ADSC‐exos into an extracellular matrix hydrogel (ECM hydrogel). This solution forms a hydrogel at physiological temperature (≈37 °C) upon local injection into the wound site. ECM@exo enables sustained release of ADSC‐exos from the ECM hydrogel, which maintains high local concentrations at the wound site. The ECM hydrogel displays good biocompatibility and biodegradability. The in vivo and in vitro results demonstrate that ECM@exo treatment effectively reduces inflammation and promotes angiogenesis, collagen deposition, cell proliferation, and migration, thereby accelerating the wound healing process. Overall, this innovative therapeutic approach offers a new avenue for wound healing via a biological hydrogel with controlled exosome release.

Swelling ratio (%) =(W0-Wt)/Wt × 100 Erosion test.The erosion property of ECM hydrogels was determined by adding PBS (pH=7.4 and 5.5).The fresh hydrogel samples were weighed and described as W0.Then, the hydrogels were immersed in 1 mL PBS and placed in a shaker at 37°C with a speed of 60 rpm.At each measurement time point, PBS was removed, and the weight was recorded as Wt until the hydrogel dissolved completely.The erosion ratio was calculated according to the following formula.
Erosion ratio (%) =(W0-Wt)/Wt × 100 In vitro degradation study.To measure the enzymatic degradation property of the ECM hydrogel, the hydrogel samples were placed in 5 U/mL and 10 U/mL collagenase type I solution (Worthington, USA) and incubated at 37℃ with a speed of 60 rpm.At the desired timepoint, the hydrogel samples were removed from the degradation buffer, blotted dry and weighed as Wt.W0 is the initial weight.The degradation percent of the ECM hydrogel was computed by using the formula below.
HaCaT cells at a density of 50000 cells/mL were cultured in 96-well plates and incubated overnight at 37°C with 5% CO2.Then, the cell culture medium was removed and replaced with 200 μL fresh medium containing various quantities of ECM hydrogel (0, 1, 5, 10, 25, 50, 100 mg/mL).After incubation for 24, 48, 72 h, 20 μL of 5 mg/mL MTT solution was added to each well for 4 h at 37°C.Then, the culture medium was removed, and 150 μL dimethyl sulfoxide (DMSO) was used to dissolve the purple formazan crystals for 15 min with constant shaking.The result was measured at a wavelength of 562 nm.The calculation of cell viability was performed with the following formula.
Cell viability (%) =(At-Ab)/(Ac-Ab) ×100 At and Ac are the OD values for cells cultured in hydrogel medium and normal medium, respectively.Ab is the OD value for the blank group.From left to right, the concentrations were 5 mg/mL, 8 mg/mL, 10 mg/mL, 12 mg/mL, 15 mg/mL, and 20 mg/mL.These results indicated that the 5 mg/mL ECM solution could not form a hydrogel, and the 20 mg/mL ECM solution was too sticky to flow freely.8 mg/mL ECM solution could form a gel in 3 min at 37°C with poor mechanical strength.ECM solutions (10, 12, and 15 mg/mL) could form hydrogels in 2 min at 37°C with good mechanical strength.Eventually, we chose 10 mg/mL ECM solution for the following experiments to save materials.

Figure S2
The influence of pH and ionic strength on ECM hydrogel formation.From left to right, group 1): digestion solution; group 2): digestion solution with physiological salt concentration (1X PBS); group 3): digestion solution with pH=7.4; group 4): digestion solution with pH=7.4 and physiological salt concentration (1X PBS).Groups 3 and 4 formed hydrogels, while groups 1 and 2 remained liquid.These results showed that pH had a greater effect on ECM hydrogel formation than ionic strength.

Figure S1
Figure S1 The influence of different concentrations on ECM hydrogel formation.A) ECM solution with different concentrations.B) ECM hydrogel formation with different concentrations.

Figure S3
Figure S3The influence of temperature on ECM hydrogel formation.A) ECM solution at 25℃. group B) ECM solution at 37℃.C) ECM solution at 45℃.The ECM solutions at 25℃ and 45℃ were liquid, while the ECM solution at 37℃ formed a hydrogel.These results confirmed the temperature sensitivity of the ECM hydrogel.It could not form a hydrogel at 45°C, which may be caused by protein denaturation at higher temperatures.

Figure S5 BFigure S6
Figure S5Weight changes in BALB/c mice with different treatments for 14 days.

Figure S7
Figure S7Weight changes of diabetic ICR mice with different treatments for 14 days.