Skin injuries, such as burns and trauma, often lead to impaired wound healing, scarring, and significant morbidity and mortality. Current treatments like autologous skin grafts have limitations including donor site morbidity, graft survival issues, and scar formation. Tissue-engineered skin substitutes offer a promising alternative but often suffer from slow vascularization and epithelialization. 3D bioprinting offers a solution by precisely controlling cell distribution and scaffold structure. This study aimed to create and test a 3D-printed skin substitute using hADSCs and a novel biomaterial to accelerate wound healing. The use of hADSCs offers advantages over fibroblasts and keratinocytes due to their easy accessibility from liposuction, high proliferation rate, low immunogenicity, and regenerative potential. The biomaterial is comprised of adipose tissue dECM, which promotes cell growth and tissue repair, GelMA, and HAMA. GelMA provides biocompatibility and cell adhesion, while HAMA enhances mechanical properties and moisture retention. The combination is hypothesized to create a superior scaffold for 3D bioprinting, resulting in faster and higher-quality wound healing.

The literature review highlights the limitations of current skin wound treatment methods, particularly autologous skin grafts and flap transplantation. Existing tissue-engineered skin substitutes, while promising, often necessitate secondary grafting due to slow vascularization and epithelialization. 3D bioprinting is identified as a potential solution, enabling precise control of cell distribution and scaffold architecture. The review discusses the advantages of using ADSCs, including their abundance, proliferative capacity, low immunogenicity, and regenerative potential, compared to traditional skin cells like fibroblasts and keratinocytes. The use of decellularized extracellular matrices (dECMs), specifically adipose tissue dECM, is explored for its biomimetic properties and ability to promote cell growth and tissue repair. Finally, the properties of GelMA and HAMA hydrogels, individually and in combination, are discussed in the context of their suitability for 3D bioprinting of skin substitutes.

The study involved several key steps: 1. **hADSC Culture and Expansion:** hADSCs were cultured and expanded in vitro until passage 4. 2. **Adipose Tissue Decellularization:** Adipose tissue was obtained with patient consent and decellularized using enzymatic digestion and organic solvent extraction (Flynn's method). DNA quantification confirmed successful decellularization. 3. **Preparation of dECM Pre-gel:** Lyophilized dECM was solubilized using hydrochloric acid and pepsin. 4. **Preparation of dECM-GelMA-HAMA Bioink:** dECM pre-gel was mixed with GelMA and HAMA solutions, along with a photoinitiator and hADSCs suspension (1x10^7 cells/mL), to create the bioink. 5. **Rheological Analysis and SEM Imaging:** The rheological properties of the dECM-GelMA-HAMA precursor were analyzed using a rheometer to determine the sol-gel transition temperature. SEM imaging characterized the porosity and pore size of the cross-linked hydrogel. 6. **3D Bioprinting:** The bioink was used to 3D print skin substitutes with a specified nozzle size, pressure, speed, scaffold dimensions, and layer height. UV irradiation photo-crosslinked each printed layer. 7. **In vivo Wound Healing Model:** Full-thickness skin wounds were created in nude mice. Mice were divided into four groups: full-thickness skin graft, 3D-bioprinted skin substitute, microskin graft, and control. Wound healing was assessed by measuring wound area over time using ImageJ software. 8. **Histological Analysis:** HE and Masson staining were used to analyze histological structure, re-epithelialization, and collagen deposition. Immunohistochemistry (CD31) assessed angiogenesis. 9. **Laser Doppler Perfusion Imaging:** Blood perfusion in the wounds was measured using laser Doppler perfusion imaging. 10. **Statistical Analysis:** SPSS 26.0 was used for statistical analysis. Student's t-test and one-way ANOVA were employed to compare groups.

Decellularization successfully removed cellular components from adipose tissue, leaving behind a dECM pre-gel with minimal residual DNA (24.5 ± 7.1 ng/mg). Rheological analysis revealed a gel-sol phase transition at 17.5°C for the dECM-GelMA-HAMA precursor. SEM imaging showed a 3D porous network structure with 65% porosity and an average pore size of 73 ± 18 μm. The 3D-printed skin substitute maintained structural integrity after printing and incubation. In vivo studies showed significantly faster wound healing in the 3D-bioprinted skin substitute group compared to the control group (P < 0.01 on days 7 and 10, P < 0.05 on day 14). Histological analysis revealed complete re-epithelialization and improved collagen deposition and organization in the experimental group. Immunohistochemistry showed significantly increased angiogenesis (CD31 staining) in the experimental group. Laser Doppler perfusion imaging confirmed higher blood perfusion in the experimental group compared to the control group.

The findings demonstrate the efficacy of the 3D-printed dECM-GelMA-HAMA skin substitute loaded with hADSCs in accelerating wound healing. The improved outcomes in re-epithelialization, collagen deposition, angiogenesis, and blood perfusion support the synergistic effect of the novel biomaterial and the hADSCs. The study successfully addressed the limitations of traditional skin substitutes and autologous grafts by providing a readily available, easily fabricated, and effective treatment option. The results have significant implications for the field of tissue engineering and regenerative medicine, particularly in the treatment of large-area skin defects where autologous grafts are limited. The biomaterial's properties, including its thermo-sensitivity, porosity, and biocompatibility, all contributed to the successful integration of the skin substitute and its promotion of tissue regeneration.

This study successfully developed and validated a 3D-printed tissue-engineered skin substitute using a novel dECM-GelMA-HAMA biomaterial loaded with hADSCs. The substitute demonstrated superior wound healing capabilities compared to controls in a nude mouse model. This pre-clinical research lays a strong foundation for future clinical translation of this technology for treating skin defects, particularly in situations with limited donor skin availability. Future research should focus on optimizing biomaterial composition, further investigating the long-term effects of the skin substitute, and exploring the feasibility of incorporating other skin appendages like hair follicles.

The study utilized a nude mouse model, which might not fully reflect the complexity of human wound healing. The sample size was relatively small. Long-term effects of the skin substitute and its potential for complete regeneration of skin appendages were not fully investigated. The study primarily focused on the in vivo efficacy of the skin substitute, and further in vitro studies could provide a deeper understanding of cellular interactions with the biomaterial.