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Background In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion. Methods Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses. Results Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group. Conclusions The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.

BackgroundAging skin is characterized by a disturbed structure and lack of blood supply, which makes it difficult to heal once injured. ADSCs secrete large amounts of cytokines, which promote wound healing and vascular regeneration through paracrine secretion, and the number of cytokines can be elevated by hypoxic pretreating. However, the components of ADSCs are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel synthesized from gelatin and has recently emerged as a potentially attractive material for tissue engineering applications. GelMA loaded with concentrated hypoxic pretreated ADSCs conditioned medium could provide a new method of treating wounds in aged skin.MethodsPrimary ADSCs were isolated from human adipose tissue and characterized by flow cytometry and differentiation test. ADSCs in passages 4-6 were pretreated in the hypoxic and normoxic environments to collect conditioned medium, the conditioned medium was then concentrated to prepare concentrated ADSCs conditioned medium(cADSC-CM)(the one collected from ADSCs under hypoxia was called hypo-CM ,and the one from normoxia was called nor-CM). The concentration of cytokines was detected. After treated with cADSC-CM, the abilities of proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs) were assayed, and Akt/mTOR and MAPK signal pathway was detected using western blotting. GelMA+hypo-CM hydrogel was prepared, and a comprehensive evaluation of morphology, protein release efficiency, degradation rate, mechanical properties, and rheology properties were performed. Full-thickness skin wounds were created on the backs of 20-month-old mice. After surgery, GelMA, GelMA+F12, GelMA+hypo-CM, and GelMA+nor-CM were applied to the wound surface respectively. H&E, Masson, and immunohistochemistry staining were performed, and a laser Doppler perfusion imager was used to evaluate the blood perfusion. The student’s t-test was used for analysis between two groups and a one-way analysis of variance (ANOVA) was used for analysis among multi groups.ResultsOur results revealed that 1) wounds in aged skin healed more slowly than that in young skin and exhibited poorer perfusion; 2) hypoxic pretreated ADSCs secreted more cytokines including VEGF by activating HIF1α; 3) hypo-CM promoted proliferation and migration of HUVECs through VEGF/Akt/mTOR and MAPK signal pathway; 4) GelMA-hypoCM accelerated wound healing and angiogenesis in aged skin in vivo.ConclusionGelMA loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells conditioned medium could accelerate wound healing in aged skin by promoting angiogenesis.

Adipose-derived stem cells (ADSCs) have raised big interest in therapeutic applications in regenerative medicine and appear to fulfill the criteria for a successful cell therapy. Their low immunogenicity and their ability to self-renew, to differentiate into different tissue-specific progenitors, to migrate into damaged sites, and to act through autocrine and paracrine pathways have been altogether testified as the main mechanisms whereby cell repair and regeneration occur. The absence of standardization protocols in cell management within laboratories or facilities added to the new technologies improved at patient’s bedside and the discrepancies in cell outcomes and engraftment increase the limitations on their widespread use by balancing their real benefit versus the patient safety and security. Also, comparisons across pooled patients are particularly difficult in the fact that multiple medical devices are used and there is absence of harmonized assessment assays despite meeting regulations agencies and efficient GMP protocols. Moreover, the emergence of the COVID-19 breakdown added to the complexity of implementing standardization. Cell- and tissue-based therapies are completely dependent on the biological manifestations and parameters associated to and induced by this virus where the scope is still unknown. The initial flow chart identified for stem cell therapies should be reformulated and updated to overcome patient infection and avoid significant variability, thus enabling more patient safety and therapeutic efficiency. The aim of this work is to highlight the major guidelines and differences in ADSC processing meeting the current good manufacturing practices (cGMP) and the cellular therapy-related policies. Specific insights on standardization of ADSCs proceeding at different check points are also presented as a setup for the cord blood and bone marrow.

Repair of large soft tissue defects by tissue expansion often faces difficulties in skin expansion. Particularly in those who have lost a significant amount of skin and subcutaneous tissue due to total mastectomy, tissue expansion may result in skin breakdown and exposure of the expander. Subcutaneous fat construction by autologous fat grafting before expansion seems to assist skin expansion. We hypothesize that it may be related to the adipose‐derived stem cells (ADSCs) in subcutaneous fat. In this study, we confirmed this phenomenon through animal experiments and provided a preliminary investigation of the possible mechanisms involved. Four groups were designed for the experiment, experimental group (EG) for autologous fat flap transfer and tissue expansion. Fat grafting control group (FGCG), where only autologous fat flap transfer was performed without tissue expansion. Tissue expansion control group (TECG) did not perform autologous fat flap transfer but only skin tissue expansion on the back. Blank control group (BCG) has not received any surgery. In each group of 20 rats, skin, and fat flaps of the dilated area were sampled at four time points (7, 14, 21, and 28 days, n = 5), and the immediate skin retraction rate of the expanded area in the EG and TECG groups was measured to evaluate the expansion effect. GFP + ADSCs were observed to demonstrate whether they were facilitated by tissue expansion and migrated from autologous fat flaps to skin. Our study supported that fat layer construction prior to skin expansion helps to promote skin growth. The promotion may be related to the migration of ADSCs from adipose to dermis by compression, and ADSCs migrating to dermis further promote skin stretching through direct differentiation or paracrine cytokines to promote cell proliferation and collagen synthesis.

Background Exosomes derived from adipose-derived stem cells (ADSC-Exos) have emerged as promising therapeutic agents for promoting wound healing through paracrine mechanisms. While adult ADSC-Exos have been extensively studied, those derived from infant ADSCs have received limited attention, despite their potentially superior regenerative capacity. In addition, hypoxia preconditioning has been shown to enhance the therapeutic efficacy of stem cells and their secretome; however, its influence on exosome function across different donor ages remains unclear. This study aimed to evaluate and compare the wound healing effects of ADSC-Exos from adult and infant sources expanded under hypoxic and normoxic conditions in a diabetic mouse model. Methods ADSC-Exos were isolated from adult and infant donors cultured under normoxic and hypoxic conditions. Exosomes were characterized by morphology, transmission electron microscopy (TEM) imaging, nanoparticle tracking analysis (NTA), and Western blotting. RNA sequencing was performed to profile miRNAs encapsulated in the exosomes. In vitro assays assessed the effects of exosomes on proliferation, migration, and wound healing in human dermal fibroblasts (HDFs) under high-glucose conditions. In vivo efficacy was evaluated using full-thickness wounds in db/db diabetic mice to assess the therapeutic effects of the exosomes. Results Normoxic infant ADSC-Exos exhibited a broader size distribution and a larger mean particle size. Several wound-healing-related miRNAs were upregulated in hypoxic ADSC-Exos from both adult and infant sources. Normoxic infant ADSC-Exos significantly enhanced HDF proliferation and migration, whereas hypoxic adult ADSC-Exos exhibited superior early wound closure. In vivo, normoxic adult ADSC-Exos achieved the fastest wound closure at day 7, and normoxic infant ADSC-Exos showed significantly greater wound closure by day 10. All ADSC-Exos promoted dermal regeneration, with hypoxic adult ADSC-Exos showing the most pronounced effects. Conclusion Infant ADSC-Exos demonstrated inherent advantages under normoxic conditions, including enhanced effects on cell proliferation, migration and wound regeneration. While hypoxic preconditioning enhanced the efficacy of adult ADSC-Exos, it provided limited additional benefit to infant-derived exosomes, highlighting their intrinsic suitability for regenerative therapies.

Background The therapeutic effects of adipose-derived mesenchymal stromal cells (ADSCs) may be mainly mediated by their paracrine effects. The ADSC-secretome can ameliorate hepatic ischemia–reperfusion injury (IRI). We explored the therapeutic effect of the ADSC-secretome from the perspective of excessive hepatocyte autophagy induced by hepatic IRI. Methods We established a miniature pig model of hepatic ischemia–reperfusion (I/R) and hepatectomy using a laparoscopic technique and transplanted ADSCs and the ADSC-secretome into the liver parenchyma immediately after surgery. Liver injury and hepatocyte autophagy were evaluated by histopathological examination and assessment of relevant cytokines and other factors. Results The results showed that the ADSC-secretome alleviated the pathological changes of liver tissue and the microstructural damage of hepatocytes after IRI. Moreover, the expression levels of autophagy-related markers including Beclin-1, ATG5, ATG12, and LC3II/LC3I decreased, whereas those of p62 increased during phagophore expansion. Furthermore, the expression levels of markers related to the autophagy inhibition pathway phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR), including PI3K, Akt, and mTOR, increased. Conclusion The ADSC-secretome attenuates hepatic I/R and hepatectomy-induced liver damage by inhibiting autophagy, which is possibly mediated by activation of the PI3K/Akt/mTOR signaling pathway. In addition, there was no significant difference between ADSCs and the ADSC-secretome in the regulation of hepatocyte autophagy. Therefore, ADSCs may improve the excessive autophagy-induced injury of hepatocytes in hepatic I/R and hepatectomy through paracrine effect. Our findings provide new insight into the therapeutic potential of cell-free products, which could replace cell therapy in liver diseases.

Rotator cuff tears often require surgical reconstruction; however, outcomes remain suboptimal, primarily because of the challenges in fully restoring the tendon–bone interface (TBI). The normal fibrocartilaginous transition zone is often replaced by fibrous scar tissue, thereby increasing the risk of retears. A porous hyaluronic acid methacrylate hydrogel is developed to encapsulate adipose‐derived stem cells (ADSCs) and bone morphogenetic protein‐2 (BMP‐2). Gelatin microspheres served as porogens to create micropores within the hyaluronic acid methacrylate hydrogel (HMs), and hollow gelatin methacryloyl hydrogel microspheres are used to encapsulate BMP‐2 (GMBs). In vitro experiments confirm substantial proliferation and homing of ADSCs within the porous hydrogel system. When the ADSCs‐loaded composite hydrogel (HMs/MBs/ADSCs) is applied to a rat rotator cuff injury model, it effectively reconstructs the fibrocartilage transition zone and promotes TBI healing. Transcriptome sequencing is performed to elucidate the mechanisms underlying BMP‐2‐induced fibrocartilage reconstruction. Furthermore, integrated in vitro sequencing reveals that HMs/MBs/ADSCs modulated the tissue microenvironment to enhance tissue regeneration, primarily through BMP‐2′s regulation of the paracrine effects of ADSCs. This study explores the comprehensive pathway through which HMs/MBs/ADSCs enhance tissue regeneration, emphasizing the synergistic effects of BMP‐2 and ADSCs, and offers novel insights into the mechanisms underlying TBI healing. This study designs an injectable porous hydrogel (HMs/MBs/ADSCs) integrating hollow microspheres for sustained BMP‐2 release to enhance adipose‐derived stem cells' paracrine effect. It accelerates tendon‐bone healing in rat rotator cuff injuries via spatiotemporal regulation: early AMPK‐mediated mitochondrial protection and late TGF‐β/Smad2/3‐driven fibrocartilage regeneration. By integrating material design and mechanistic exploration, this study enhances the application potential of tissue‐engineered hydrogels.

Background Air pollution and obesity are crucial risk factors for cardiovascular disease (CVD), with epidemiological evidence indicating that air pollution exacerbates obesity-induced cardiac damage. Treatment with adipose-derived stem cells (ADSCs) attenuates cardiac damage by releasing paracrine factors. However, the effects of ADSCs on air pollution- and obesity-induced cardiomyocyte apoptosis and the related mechanisms are still unclear. Methods Palmitic acid (PA) and a high-fat diet (HFD) were used to cause obesity, and particulate matter (PM) was used to simulate air pollution in the study. We studied the impact of conditioned medium from adipose-derived stem cells (ADSC-CM) on the apoptosis of PA + PM-treated H9c2 cells and HFD + PM-treated mouse cardiomyocytes and the underlying mechanisms involved. Results The levels of apoptosis-related proteins (PUMA and cleaved caspase-3) were significantly increased in PA + PM-treated H9c2 cells and HFD + PM-treated mouse cardiomyocytes, whereas the antiapoptotic protein Bcl-2 expression was reduced. However, ADSC-CM treatment effectively reduced the PUMA and cleaved caspase-3 expression but increased the Bcl-2 expression. ADSC-CM significantly reduced PA + PM- and HFD + PM-induced cardiomyocyte apoptosis, as detected by the TUNEL assay. RT-qPCR revealed that PA + PM and HFD + PM significantly reduced miR221/222 levels, whereas ADSC-CM treatment increased miR221/222 levels. Furthermore, knockout (KO) and transgenic (TG) mice were used to demonstrate that miR221/222 in ADSC-CM ameliorated cardiac apoptosis that was induced by HFD + PM treatment. Furthermore, PA + PM treatment increased the reactive oxygen species (ROS) production, which triggered mitochondrial fission and contributed to apoptosis. However, ADSC-CM effectively reduced ROS levels and regulated mitochondrial fission, alleviating cellular apoptosis. Conclusions Our findings demonstrated that ADSC-CM attenuated PA + PM-induced cardiomyocyte apoptosis by modulating miR221/222 levels and suppressing ROS production.

The functional recovery of peripheral nerve injury (PNI) is unsatisfactory, whereas diabetes mellitus (DM) and its related complications further attenuate the restoration of diabetic PNI (DPNI). Adipose-derived stem cells (ADSCs) are promising candidates for treatment of DPNI due to their abundant source, excellent differentiation and paracrine ability. Our results showed that ADSCs remarkably enhanced the proliferation and migration of Schwann cells and endothelial cells, and tube formation. Mechanistically, ADSCs could regulate Nrf2/HO-1, NF- κ B and PI3K/AKT/mTOR signaling pathways, showing multiple functions in reducing oxidative stress and inflammation, and regulating cell metabolism, growth, survival, proliferation, angiogenesis, differentiation of Schwann cell and myelin formation. In current study, novel graphene foam (GF)/hydrogel-based scaffold was developed to deliver ADSCs for treatment of DPNI. GF/hydrogel scaffold exhibited excellent mechanical strength, suitable porous network, superior electrical conductivity, and good biocompatibility. In vitro results revealed that GF/hydrogel scaffold could obviously accelerate proliferation of Schwann cells. Moreover, in vivo experiments demonstrated that ADSCs-loaded GF/hydrogel scaffold significantly promoted the recovery of DPNI and inhibited the atrophy of targeted muscles, thus providing a novel and attractive therapeutic approach for DPNI patients.

The influence and underlying mechanisms of human adipose-derived stem cells (Hu-ADSCs) on breast cancer cells in the tumor microenvironment remain unclear. Understanding the association between Hu-ADSCs and cancer cells may provide targets for breast cancer treatment and reference for the clinical application of stem cells. Therefore, a Hu-ADSC and breast cancer MCF7 cell coculture system was established to investigate the paracrine effects of Hu-ADSCs on MCF7 cell migration and invasion, in addition to the potential mechanism of action by reverse transcription-quantitative polymerase chain reaction and western blotting. Hu-ADSCs enhanced MCF7 cell migration and invasion by decreasing the expression of epithelial marker E-cadherin, and increasing the expression of interstitial marker N-cadherin and epithelial-mesenchymal transition (EMT) transcription factors in vitro. The EMT effect of cocultured MCF7 cells was inhibited with the addition of anti-transforming growth factor (TGF)-β1 or phosphoinositide 3-kinase (PI3K) inhibitor LY294002, accompanied by a significant decrease in phosphorylated (p)-mothers against decapentaplegic homolog (Smad) and p-protein kinase B (AKT) expression. The data suggested that the paracrine effect of Hu-ADSCs in the tumor microenvironment promoted the EMT of MCF7 cells by cross interacting with the TGF-β/Smad and PI3K/AKT pathways.