Ultrasound-Driven Healing: Unleashing the Potential of Chondrocyte-Derived Extracellular Vesicles for Chondrogenesis in Adipose-Derived Stem Cells.

Repairing cartilage defects represents a significant clinical challenge. While adipose-derived stem cell (ADSC)-based strategies hold promise for cartilage regeneration, their inherent chondrogenic potential is limited. Extracellular vesicles (EVs) derived from chondrocytes (CC-EVs) have shown potential in enhancing chondrogenesis, but their role in promoting chondrogenic differentiation of ADSCs remains poorly understood. Moreover, the clinical application of EVs faces limitations due to insufficient quantities for in vivo use, necessitating the development of effective methods for extracting significant amounts of CC-EVs. Our previous study demonstrated that low-intensity ultrasound (LIUS) stimulation enhances EV secretion from mesenchymal stem cells. Here, we identified a specific LIUS parameter for chondrocytes that increased EV secretion by 16-fold. CC-EVs were found to enhance cell activity, proliferation, migration, and 21-day chondrogenic differentiation of ADSCs in vitro, while EVs secreted by chondrocytes following LIUS stimulation (US-CC-EVs) exhibited superior efficacy. miRNA-seq revealed that US-CC-EVs were enriched in cartilage-regeneration-related miRNAs, contributing to chondrogenesis in various biological processes. In conclusion, we found that CC-EVs can enhance the chondrogenesis of ADSCs in vitro. In addition, our study introduces ultrasound-driven healing as an innovative method to enhance the quantity and quality of CC-EVs, meeting clinical demand and addressing the limited chondrogenic potential of ADSCs. The ultrasound-driven healing unleashes the potential of CC-EVs for chondrogenesis possibly through the enrichment of cartilage-regeneration-associated miRNAs in EVs, suggesting their potential role in cartilage reconstruction. These findings hold promise for advancing cartilage regeneration strategies and may pave the way for novel therapeutic interventions in regenerative medicine.

Preparation of therapy-grade extracellular vesicles from adipose tissue to promote diabetic wound healing.

Background: Treatment of diabetic wounds is a major challenge in clinical practice. Extracellular vesicles (EVs) from adipose-derived stem cells have shown effectiveness in diabetic wound models. However, obtaining ADSC-EVs requires culturing vast numbers of cells, which is hampered by the need for expensive equipment and reagents, extended time cost, and complicated procedures before commercialization. Therefore, methods to extract EVs from discarded tissue need to be developed, for immediate application during surgery. For this reason, mechanical, collagenase-digestive, and constant in-vitro-collective methods were designed and compared for preparing therapy-grade EVs directly from adipose tissue. Methods: Characteristics and quantities of EVs were detected by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting firstly. To investigate the biological effects of EVs on diabetic wound healing, angiogenesis, proliferation, migration, and inflammation-regulation assays were then evaluated in vitro, along with a diabetic wound healing mouse model in vivo. To further explore the potential therapeutic mechanism of EVs, miRNA expression profile of EVs were also identified and analyzed. Results: The adipose tissue derived EVs (AT-EVs) were showed to qualify ISEV identification by nanoparticle tracking analysis and Western blotting and the AT-EVs yield from three methods was equal. EVs also showed promoting effects on biological processes related to diabetic wound healing, which depend on fibroblasts, keratinocytes, endothelial cells, and macrophages both in vitro and in vivo. We also observed enrichment of overlapping or unique miRNAs originate from different types of AT-EVs associated with diabetic wound healing for further investigation. Conclusion: After comparative analyses, a mechanical method was proposed for preparing immediate clinical applicable EVs from adipose tissue that would result in reduced preparation time and lower cost, which could have promising application potential in treating diabetic wounds.

Production and Biological Effects of Extracellular Vesicles from Adipose-Derived Stem Cells Were Markedly Increased by Low-Intensity Ultrasound Stimulation for Promoting Diabetic Wound Healing.

Diabetic wound treatment has posed a significant challenge in clinical practice. As a kind of cell-derived nanoparticles, extracellular vesicles produced by adipose-derived stem cells (ADSC-EVs) have been reported to be potential agents for diabetic wound treatment. However, ADSC-EV yield is insufficient to meet the demands of clinical therapy. In this study, a novel method involving the use of low-intensity ultrasound stimulation on ADSCs is developed to promote EV secretion for clinical use. A proper low-intensity ultrasound stimulation parameter which significantly increases ADSC-EV quantity has been found. In addition, EVs secreted by ADSCs following low-intensity ultrasound stimulation (US-EVs) are enriched in wound healing-related miRNAs. Moreover, US-EVs promote the biological functions of fibroblasts, keratinocytes, and endothelial cells in vitro, and promote diabetic wound healing in db/db mice in vivo through re-epithelialization, collagen production, cell proliferation, keratinocyte differentiation and migration, and angiogenesis. This study proposes low-intensity ultrasound stimulation as a new method for promoting significant EV secretion by ADSCs and for improving the diabetic wound-healing potential of EVs, which will meet the clinical needs for these nanoparticles. The production of extracellular vesicles of adipose-derived stem cells is obviously promoted by a low-intensity ultrasound stimulation method, and the biological effects of promoting diabetic wound healing were markedly increased in vitro and in vivo.

Regional-Controlled Tissue Expanders Increase Skin Expansion and Thickness Compared to Standard Tissue Expanders in a Rat Model.

BACKGROUND: Skin and soft-tissue expansion are widely used. However, the existing methods cannot expand targeted areas on the top flap. Thus, the authors developed a new expander with a partially thickened top. The authors hypothesized that pressure differences would lead to higher growth near nonthickened regions and lower growth near thickened regions, allowing targeted expansion. METHODS: Eighteen male Sprague Dawley rats (8 weeks old) were used; 20-ml rectangular regional-controlled expanders ( n = 12) and ordinary expanders ( n = 6) were implanted. Flaps on regional-controlled expanders were divided into nonthickened and thickened regions and tattooed. Discontinuous inflation began 14 days postoperatively, 3 ml every 3 days, until the volume reached 50 ml. Tattooed skin area and thickness were measured. Immunofluorescence staining detected cell proliferation (proliferating cell nuclear antigen-positive) and vascular density (CD31 + ). Growth factors (transforming growth factor-ß, epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) were assessed by enzyme-linked immunosorbent assay. RESULTS: The expanded skin area of regional-controlled expansion nonthickened regions (396.2 ± 41.4 mm 2 ) was 33.8 ± 10.0 percent larger than that of thickened regions (297.8 ± 38.9 mm 2 ). Thickened regions had a 28.9 ± 14.6 percent thicker dermal layer (942.4 ± 55.5 µm) than nonthickened regions (737.1 ± 64.5 µm). Nonthickened regions had 295.0 ± 145.0 percent more proliferating cell nuclear antigen-positive cells (92.4 ± 16.2/mm 2 ) than thickened regions (25.6 ± 7.4/mm 2 ). The vascular density was 133.0 ± 61.7 percent higher in thickened regions (24.8 ± 4.7/mm 2 ) than in nonthickened regions (11.1 ± 2.7/mm 2 ) (all above, p < 0.05). CONCLUSIONS: Regional-controlled expansion specifically expands only the targeted area, causing thicker skin flaps with abundant vessels for defect repair. Although this technique has great clinical potential, it should be further validated with large animals and humans. ( Plast. Reconstr. Surg. 150: 1273, 2022.). CLINICAL RELEVANCE STATEMENT: This study presents the newly developed regional-controlled expansion technique that realized the targeted expansion. It is suitable for repairing defects and would contribute to shortening the expansion process and reducing complication rates.

Long-Term Tri-Modal In Vivo Tracking of Engrafted Cartilage-Derived Stem/Progenitor Cells Based on Upconversion Nanoparticles.

Cartilage-derived stem/progenitor cells (CSPCs) are a potential choice for seed cells in osteal and chondral regeneration, and the outcomes of their survival and position distribution in vivo form the basis for the investigation of their mechanism. However, the current use of in vivo stem cell tracing techniques in laboratories is relatively limited, owing to their high operating costs and cytotoxicity. Herein, we performed tri-modal in vivo imaging of CSPCs during subcutaneous chondrogenesis using upconversion nanoparticles (UCNPs) for 28 days. Distinctive signals at accurate positions were acquired without signal noise from X-ray computed tomography, magnetic resonance imaging, and upconversion luminescence. The measured intensities were all significantly proportional to the cell numbers, thereby enabling real-time in vivo quantification of the implanted cells. However, limitations of the detectable range of cell numbers were also observed, owing to the imaging shortcomings of UCNPs, which requires further improvement of the nanoparticles. Our study explores the application value of upconversion nanomaterials in the tri-modal monitoring of implanted stem cells and provides new perspectives for future clinical translation.

A randomized, controlled clinical trial of autologous stromal vascular fraction cells transplantation to promote mechanical stretch-induced skin regeneration.

BACKGROUND: The regeneration response of the skin to mechanical stretching in vivo has been explored in reconstructive surgery to repair large-scale deformities. The ability of the skin to regenerate limits the reconstructive outcome. Here, we propose an approach in which autologous stromal vascular fraction (SVF) cells and mechanical stretching are combined to overcome this limitation and promote skin regeneration. METHODS: This randomized, blinded, placebo-controlled clinical trial screened 22 participants undergoing tissue expansion with exhausted regeneration. Twenty eligible participants received intradermal injections of the SVF or placebo treatments. Follow-ups were conducted at 4, 8, and 12 weeks to assess efficacy and at 2 years to assess safety. The primary endpoint was the expanded skin thickness at 12 weeks. The secondary endpoints included skin thickness at 4 and 8 weeks, the expansion index (EI), and the skin texture score at 12 weeks. RESULTS: The skin thickness of the SVF group was significantly higher than that of the control group at both 8 weeks (mean difference 0.78 [95% CI - 1.43 to - 0.11]; p = 0.018) and 12 weeks (0.65 [95% CI - 1.30 to - 0.01]; p = 0.046). In the SVF group, the increase in skin thickness was significant at 4 weeks (0.49 [95% CI - 0.80 to - 0.06]; p = 0.010) to 8 weeks (0.45 [95% CI - 0.92 to 0.02]; p = 0.026) and maintained after 12 weeks, whereas that in the control group was reduced after 8 weeks (0.42 [95% CI - 0.07 to 0.91]; p = 0.037). The SVF group showed greater EI increases than the control group (0.50 [95% CI - 0.00 to 0.99]; p = 0.047). The skin texture scores in the SVF group were greater than those in the control group at 12 weeks. Histologically, SVF-treated expanded skin showed more proliferating cells and blood vessels, and the extracellular matrix volume increased. No severe adverse events occurred. CONCLUSIONS: Transplantation of SVF cells can expedite the potency of mechanical stretch-induced skin regeneration and provide clinical reconstruction with plentiful tissue. TRIAL REGISTRATION: This trial was registered with the Chinese Clinical Trial Registry, ChiCTR2000039317 (registered 23 October 2020-retrospectively registered).

A Comparative Study of Three-Dimensional Simulation in Nonsurgical Rhinoplasty With Hyaluronic Acid Fillers.

BACKGROUND: The use of 3-dimensional computer imaging has grown steadily over the past decade, especially with cosmetic facial surgery. The technological advance has influenced how we counsel patients, perform procedures, and assess outcomes. The purpose of this study was to analyze the feasibility of quantifying simulated versus actual outcomes for nonsurgical rhinoplasty with hyaluronic acid. METHODS: A retrospective review of 3-dimensional images (LifeViz Inc, France) for rhinoplasty patients was performed. Randomized preoperative, simulated, and actual images were rated by a blinded panel of physicians (1 = poor, 5 = excellent). In addition, a quantitative assessment of nasofrontal angle and nasolabial angle was conducted where paired and 2-sample t tests were performed (P < 0.05 as significant). RESULTS: Twenty-five patients were included in this comparison study. Fifty-six percent of preoperative images were rated as poor (mean, 1.7). The simulation received a mean score of 3.4 (good in 60% of cases), and 80% of actual cases were rated good to excellent (mean, 3.7). Mean nasofrontal angle decreased from 147.1 ± 1.2° preinjection to 143.3 ± 1.6° posttreatment, a mean change of 3.8 ± 2.0°. The mean nasolabial angle decreased from 125.5 ± 1.6° pretreatment to 117.5 ± 1.5° posttreatment. Average volume of actual dosage was 1.74 ± 0.18 mL. CONCLUSION: Three-dimensional simulation for patients undergoing nonsurgical rhinoplasty is helpful for surgical planning and patient communications. It provides a mechanism for critical self-evaluation and helps set patients with realistic expectations about rhinoplasty.

Effect of tissue expansion on chondrocyte sheets in cartilage composite reconstruction.

Flap prelamination has been successfully established in tissue engineering; however, cartilage generation through combination of an expanded flap and chondrocyte sheets has not been reported. Herein, we investigate the effect of tissue expansion on chondrocyte sheets in prelaminating an expanded chondrocutaneous flap. Chondrocyte sheets were implanted into a tissue expander capsule following which capsule inflation was performed weekly. At 4 and 12 weeks post implantation, the specimens were examined with histology and immunohistochemistry analyses. Extracellular matrix (ECM) formation and type II collagen deposition in the regenerated cartilage tissue in vivo were also examined. After 4 weeks of implantation, the generated cartilage was phenotypically stable with minimal hypertrophy, while that formed after the 12-week expansion showed visible hypertrophic differentiation. To evaluate the effect of static pressure and/or hypoxic conditions generated by the expanding tissue, static pressure and/or hypoxic conditions were reproduced in vitro. The chondrocyte sheets stimulated by mechanical static pressure and hypoxia maintained their chondrogenic phenotype. The expression of aggrecan, collagen II, Sox-9, and HIF-1α was increased in chondrocyte sheets cultured in 2% oxygen (hypoxia); however, aggrecan, collagen II, and Sox-9 were downregulated in the static pressure/normoxia group. These results suggest that the expanded environment promoted cartilage formation by the chondrocyte cell sheets, while mechanical forces and hypoxic conditions in vitro allowed chondrocyte cell sheets to retain their chondrogenic phenotype.

Developing a Strontium-Releasing Graphene Oxide-/Collagen-Based Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway.

Significant efforts have been dedicated to fabricating favorable biomaterial-based bone substitutes for the repair of large bone defects. However, the development of bone biomaterials with suitable physiochemical and osteoinductive properties remains a challenge. Here, novel strontium-graphene oxide (Sr-GO) nanocomposites that allow long-term release of Sr ions are fabricated, which are used to reinforce collagen (Col) scaffolds through covalent cross-linking. The prepared Sr-GO-Col scaffold demonstrates significantly high water retention rates and excellent mechanical properties compared with unmodified Col scaffolds. The Sr-GO-modified Col scaffolds display a strong effect on adipose-derived stem cells by facilitating cell adhesion and osteogenic differentiation and by promoting the secretion of angiogenic factors to stimulate the in vitro tube formation of endothelial cells. Additionally, the secretion of angiogenic VEGF and osteogenic BMP-2 proteins is increased, which may be attributed to the synergistic effects of GO and Sr on the activation of the MAPK signaling pathway. The Sr-GO-Col constructs were then transplanted into rat critical-size calvarial bone defects, which showed the best bone regeneration and angiogenesis outcome at 12 weeks. Moreover, histological staining results show that the Sr-GO-Col group achieved complete defect bridging with the newly formed bone tissue and the residual Sr-GO nanoparticles are phagocytosed and degraded by multinucleated giant cells. These findings reveal that the incorporation of inorganic Sr-GO nanocomposites into biocompatible Col scaffolds is a viable strategy for fabricating favorable substitutes that enhance the regeneration of large bone defects.

Intravenous Hyaluronidase with Urokinase as Treatment for Rabbit Retinal Artery Hyaluronic Acid Embolism.

BACKGROUND: Although various salvage methods have been proposed to treat intraretinal artery hyaluronic acid embolism, their applications are still limited by various factors. The authors investigated the effectiveness of intravenous hyaluronidase with urokinase for resolving retinal artery hyaluronic acid embolism. METHODS: The anatomy of rabbit ophthalmic and fundus arteries (retinal and choroid artery) was studied. Approximately 0.35 ml of hyaluronic acid was injected into the ophthalmic artery to create a retinal artery embolism model. The rabbits were grouped randomly (groups A, B, C, D, E, and F) and given hyaluronidase with urokinase intravenously at different postobstruction time points (10, 20, 30, 40, 50, and 60 minutes). Saline was given to the control group. Fundus vascular (retinal and choroid artery) reperfusion status and the effectiveness of the solution on the obstruction of each group were observed for 5 days. RESULTS: The animal model closely imitated actual hyaluronic acid ophthalmic/retinal artery obstructions. Three vascular conditions were observed after hyaluronidase with urokinase injection: total, partial, and no reperfusion. Groups A, B, and C showed a significantly higher overall solution effectiveness rate (total/partial reperfusion) compared with the control group (p = 0.001, p = 0.001, and p = 0.005, respectively). Solution effectiveness in groups D, E, and F showed no difference compared with the control group (p = 0.628, p = 1.000, and p = 1.000, respectively). The effectiveness of the solution drops dramatically if given after 30 minutes of obstruction. CONCLUSIONS: The authors' method can indeed help resolve retinal artery hyaluronic acid obstruction. Intravenous hyaluronidase with urokinase technique shows possible potential to become a standardized treatment protocol for intraretinal artery hyaluronic acid embolism with further clinical tests.