Microvascular Fragment-Loaded Platelet-Rich Plasma Dressing Promotes Cutaneous Wound Healing.

Objective: Chronic wounds represent a considerable burden for the affected patients and the health care system. To overcome this problem, effective treatment strategies are urgently required. In this study, we tested a novel approach by combining platelet-rich plasma (PRP) and microvascular fragments (MVF) to create a prevascularized gel dressing. Approach: MVF were enzymatically isolated from the epididymal fat pads of transgenic green fluorescent protein (GFP)+ C57BL/6J donor mice. Subsequently, 5,000 MVF were suspended in 10 µL murine PRP as carrier and transferred into full-thickness skin wounds within dorsal skinfold chambers of C57BL/6J wild-type mice (PRP+MVF). Wound healing in comparison to empty wounds (control) and wounds filled with PRP alone was repeatedly analyzed throughout 14 days by means of stereomicroscopy, histology, and immunohistochemistry. Results: Planimetric assessment of the wound size over time revealed a significantly accelerated and improved healing of PRP+MVF-treated wounds when compared with PRP-treated and empty control wounds. These wounds also exhibited a significantly higher density of blood and lymph vessels, which originated from the GFP+ MVF isolates and effectively promoted granulation tissue formation inside the skin defects. Innovation: This study is the first to combine PRP and MVF for the improvement of wound healing. Conclusion: The combination of PRP and MVF represents a promising approach for the future treatment of wounds that do not heal spontaneously due to poor wound-healing conditions.

Biofabrication of prevascularized spheroids for bone tissue engineering by fusion of microvascular fragments with osteoblasts.

Introduction: Spheroids are promising building blocks for scaffold-free bone tissue engineering. Their rapid vascularization is of major importance to guarantee their survival after transplantation. To achieve this, we herein introduce the biofabrication of prevascularized spheroids by fusion of adipose tissue-derived microvascular fragments (MVF) with osteoblasts (OB). Methods: For this purpose, 200 MVF from donor mice and 5,000, 10,000 or 20,000 murine OB (MC3T3-E1) were co-cultured in a liquid overlay system for 3 days to generate OB + MVF spheroids. OB mono-culture spheroids served as controls. Results and discussion: During the generation process, the diameters of all spheroids progressively decreased, resulting in compact, viable spheroids of homogeneous sizes. MVF promoted the maturation of spheroids containing 5,000 OB, as shown by an accelerated decline of cell proliferation due to contact inhibition. Moreover, MVF most effectively reassembled into new microvascular networks within these small spheroids when compared to the other spheroid types, indicating the most beneficial MVF to OB ratio. Accordingly, these spheroids also showed a high angiogenic sprouting activity in vitro. In contrast to OB spheroids, they further rapidly vascularized in vivo after transplantation into dorsal skinfold chambers. This was caused by the interconnection of incorporated MVF with surrounding blood vessels. These findings indicate that OB + MVF spheroids may be suitable for bone tissue engineering, which should be next tested in appropriate in vivo bone defect models.

Generation of Connective Tissue-Free Microvascular Fragment Isolates from Subcutaneous Fat Tissue of Obese Mice.

BACKGROUND: Microvascular fragment (MVF) isolates are generated by short-term enzymatic digestion of adipose tissue and contain numerous vessel segments for the vascularization of tissue defects. Recent findings indicate that the functionality of these isolates is determined by the quality of the fat source. Therefore, we compared MVF isolates from subcutaneous adipose tissue of obese and lean mice. METHODS: MVF isolates were generated from subcutaneous adipose tissue of donor mice, which received a high fat or control diet for 12 weeks. The isolates were analyzed in vitro and in vivo. RESULTS: Feeding of mice with a high fat diet induced obesity with adipocyte hypertrophy, resulting in a significantly lower collagen fraction and microvessel density within the subcutaneous fat depots when compared to lean controls. Accordingly, MVF isolates from obese mice also contained a reduced number of MVF per mL adipose tissue. However, these MVF tended to be longer and, in contrast to MVF from lean mice, were not contaminated with collagen fibers. Hence, they could be freely seeded onto collagen-glycosaminoglycan scaffolds, whereas MVF from lean controls were trapped in between large amounts of collagen fibers that clogged the pores of the scaffolds. In line with these results, scaffolds seeded with MVF isolates from obese mice exhibited a significantly improved in vivo vascularization after implantation into full-thickness skin defects. CONCLUSION: Subcutaneous adipose tissue from obese mice facilitates the generation of connective tissue-free MVF isolates. Translated to clinical conditions, these findings suggest that particularly obese patients may benefit from MVF-based vascularization strategies.

Absent in Melanoma (AIM)2 Promotes the Outcome of Islet Transplantation by Repressing Ischemia-Induced Interferon (IFN) Signaling.

Clinical islet transplantation is limited by ischemia-induced islet cell death. Recently, it has been reported that the absent in melanoma (AIM)2 inflammasome is upregulated by ischemic cell death due to recognition of aberrant cytoplasmic self-dsDNA. However, it is unknown whether AIM2 determines the outcome of islet transplantation. To investigate this, isolated wild type (WT) and AIM2-deficient (AIM2-/-) islets were exposed to oxygen-glucose deprivation to mimic ischemia, and their viability, endocrine function, and interferon (IFN) signaling were assessed. Moreover, the revascularization and endocrine function of grafted WT and AIM2-/- islets were analyzed in the mouse dorsal skinfold chamber model and the diabetic kidney capsule model. Ischemic WT and AIM2-/- islets did not differ in their viability. However, AIM2-/- islets exhibited a higher protein level of p202, a transcriptional regulator of IFN-ß and IFN-γ gene expression. Accordingly, these cytokines were upregulated in AIM2-/- islets, resulting in a suppressed gene expression and secretion of insulin. Moreover, the revascularization of AIM2-/- islet grafts was deteriorated when compared to WT controls. Furthermore, transplantation of AIM2-/- islets in diabetic mice failed to restore physiological blood glucose levels. These findings indicate that AIM2 crucially determines the engraftment and endocrine function of transplanted islets by repressing IFN signaling.