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.

Combining "waste utilization" and "tissue to tissue" strategies to accelerate vascularization for bone repair.

Background: A pivotal determinant for the success of tissue regeneration lies in the establishment of sufficient vasculature. Utilizing autologous tissue grafts from donors offers the dual advantage of mitigating the risk of disease transmission and circumventing the necessity for post-transplant immunosuppression, rendering it an exemplary vascularization strategy. Among the various potential autologous donors, adipose tissue emerges as a particularly auspicious source, being both widely available and compositionally rich. Notably, adipose-derived microvascular fragments (ad-MVFs) are a promising candidate for vascularization. ad-MVFs can be isolated from adipose tissue in a short period of time and show high vascularized capacity. In this study, we extracted ad-MVFs from adipose tissue and utilized their strong angiogenic ability to accelerate bone repair by promoting vascularization. Methods: ad-MVFs were extracted from the rat epididymis using enzymatic hydrolysis. To preserve the integrity of the blood vessels, gelatin methacryloyl (GelMA) hydrogel was chosen as the carrier for ad-MVFs in three-dimensional (3D) culture. The ad-MVFs were cultured directly on the well plates for two-dimensional (2D) culture as a control. The morphology of ad-MVFs was observed under both 2D and 3D cultures, and the release levels of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) were assessed under both culture conditions. In vitro studies investigated the impact of ad-MVFs/GelMA hydrogel on the toxicity, osteoblastic activity, and mineralization of rat bone marrow mesenchymal stem cells (rBMSCs), along with the examination of osteogenic gene and protein expression. In vivo experiments involved implanting the ad-MVFs/GelMA hydrogel into critical-size skull defects in rats, and its osteogenic ability was evaluated through radiographic and histological methods. Results: ad-MVFs were successfully isolated from rat adipose tissue. When cultured under 2D conditions, ad-MVFs exhibited a gradual disintegration and loss of their original vascular morphology. Compared with 2D culture, ad-MVFs can not only maintain the original vascular morphology, but also connect into a network in hydrogel under 3D culture condition. Moreover, the release levels of VEGF and BMP-2 were significantly higher than those in 2D culture. Moreover, the ad-MVFs/GelMA hydrogel exhibited superior osteoinductive activity. After implanting into the skull defect of rats, the ad-MVFs/GelMA hydrogel showed obvious effects for angiogenesis and osteogenesis. The translational potential of this article: The utilization of autologous adipose tissue as a donor presents a more direct route toward clinical translation. Anticipated future clinical applications envision the transformation of discarded adipose tissue into a valuable resource for personalized tissue repair, thereby realizing a paradigm shift in the utilization of this abundant biological material.

Human Adipose-Derived Microvessel Fragments: A Natural Vascularization Units for Ischemic Diseases.

BACKGROUND: In plastic surgery tissue transplantation, tissue ischemia limits transplanted tissue survival. Adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) show potential for promoting angiogenesis and rescuing ischemic conditions. However, when SVF and ASC suspensions are utilized without the protection of extracellular matrix, the retention rate of transplanted cells tends to be diminished, leading to an unsatisfactory therapeutic outcome. To overcome this, adipose tissue-derived microvascular fragments (ad-MVFs) have emerged as a promising solution. METHODS: We conducted enzymatic digestion on human adipose tissue to generate ad-MVFs. These fragments underwent a thorough characterization process, utilizing electron microscopy to assess their structural attributes and enabling a detailed analysis of their intricate morphology. Furthermore, our team investigated the cellular composition of these microvascular fragments, subsequently confirming their ability to enhance the viability of ischemic skin flaps. RESULTS: The resulting product primarily comprised fragments with sizes ranging from 20 to 50 µm, and some exhibited a sophisticated network-like structure. Electron microscopy examination revealed the presence of collagen components in the product. Additionally, flow cytometry analysis indicated a substantial abundance of adipose-derived stem cells and endothelial cells within these microvascular fragments. Significantly, when tested in treating an ischemic skin flap in a nude mouse model, the product exhibited superior therapeutic efficacy compared to SVF cell suspension. CONCLUSION: We have successfully generated human ad-MVFs and established standardized procedures. Compared with SVF, Ad-MVFs have a better effect in the treatment of ischemic diseases. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

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.

Bioprinted dermis with human adipose tissue-derived microvascular fragments promotes wound healing.

Tissue-engineered skin is an effective material for treating large skin defects in a clinical setting. However, its use is limited owing to vascular complications. Human adipose tissue-derived microvascular fragments (HaMVFs) are vascularized units that form vascular networks by rapid reassembly. In this study, we designed a vascularized bionic skin tissue using a three-dimensional (3D) bioprinter of HaMVFs and human fibroblasts encapsulated in a hybrid hydrogel composed of GelMA, HAMA, and fibrinogen. Tissues incorporating HaMVFs showed good in vitro vascularization and mechanical properties after UV crosslinking and thrombin exposure. Thus, the tissue could be sutured appropriately to the wound. In vivo, the vascularized 3D bioprinted skin promoted epidermal regeneration, collagen maturation in the dermal tissue, and vascularization of the skin tissue to accelerate wound healing. Overall, vascularized 3D bioprinted skin with HaMVFs is an effective material for treating skin defects and may be clinically applicable to reduce the necrosis rate of skin grafts.

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.

Transplantation of in vitro prefabricated adipose organoids attenuates skin fibrosis by restoring subcutaneous fat and inducing dermal adipogenesis.

Localized scleroderma is a complex autoimmune disease characterized by dermal fibrosis and loss of cutaneous fat. While cytotherapy offers a promising treatment option, stem cell transplantation results in low survival rates and fails in target cell differentiation. In this study, we aimed to prefabricate syngeneic adipose organoids (ad-organoids) using microvascular fragments (MVFs) via three-dimensional (3D) culturing and transplant them beneath the fibrotic skin to restore subcutaneous fat and reverse the pathological manifestation of localized scleroderma. We employed 3D culturing of syngeneic MVFs with stepwise angiogenic and adipogenic induction to produce ad-organoids and evaluated their microstructure and paracrine function in vitro. C57/BL6 mice with induced skin scleroderma were treated with adipose-derived stem cells (ASCs), adipocytes, ad-organoids, and Matrigel, and the therapeutic effect was assessed histologically. Our results showed that ad-organoids derived from MVF contained mature adipocytes and a well-established vessel network, secreted multiple adipokines, promoted adipogenic differentiation of ASCs, and suppressed proliferation and migration of scleroderma fibroblasts. Subcutaneous transplantation of ad-organoids reconstructed the subcutaneous fat layer and stimulated dermal adipocyte regeneration in bleomycin-induced scleroderma skin. It reduced collagen deposition and dermal thickness, attenuating dermal fibrosis. Moreover, ad-organoids suppressed macrophage infiltration and promoted angiogenesis in the skin lesion. In conclusion, 3D culturing of MVFs with stepwise angiogenic and adipogenic induction is an effective strategy for the fabrication of ad-organoids, and the transplantation of prefabricated ad-organoids can improve skin sclerosis by restoring cutaneous fat and attenuating skin fibrosis. These findings offer a promising therapeutic approach for the treatment of localized scleroderma.

Microvascular Fragments Protect Ischemic Musculocutaneous Flap Tissue from Necrosis by Improving Nutritive Tissue Perfusion and Suppressing Apoptosis.

Microvascular fragments (MVF) derived from enzymatically digested adipose tissue are functional vessel segments that have been shown to increase the survival rate of surgical flaps. However, the underlying mechanisms have not been clarified so far. To achieve this, we raised random-pattern musculocutaneous flaps on the back of wild-type mice and mounted them into dorsal skinfold chambers. The flaps were injected with MVF that were freshly isolated from green fluorescent protein-positive (GFP+) donor mice or saline solution (control). On days 1, 3, 5, 7, and 10 after surgery, intravital fluorescence microscopy was performed for the quantitative assessment of angiogenesis, nutritive blood perfusion, and flap necrosis. Subsequently, the flaps were analyzed by histology and immunohistochemistry. The injection of MVF reduced necrosis of the ischemic flap tissue by ~20%. When compared to controls, MVF-injected flaps also displayed a significantly higher functional capillary density and number of newly formed microvessels in the transition zone, where vital tissue bordered on necrotic tissue. Immunohistochemical analyses revealed a markedly lower number of cleaved caspase-3+ apoptotic cells in the transition zone of MVF-injected flaps and a significantly increased number of CD31+ microvessels in both the flaps' base and transition zone. Up to ~10% of these microvessels were GFP+, proving their origin from injected MVF. These findings demonstrate that MVF reduce flap necrosis by increasing angiogenesis, improving nutritive tissue perfusion, and suppressing apoptosis. Hence, the injection of MVF may represent a promising strategy to reduce ischemia-induced flap necrosis in future clinical practice.

Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model.

Chronic lymphedema after cancer treatment is common and there is still no cure for this disease. We herein investigated the lymphangiogenic capacity of adipose tissue-derived microvascular fragments (MVF), which contain stem cells and lymphatic vessel fragments. Secondary lymphedema was induced in the hindlimbs of C57BL/6J mice. Green fluorescence protein (GFP)+ MVF were isolated from transgenic C57BL/6Tg (CAG-EGFP)1Osb/J mice, suspended in collagen hydrogel, and injected in the lymphadenectomy defect of wild-type animals. This crossover model allowed the detection of MVF-derived blood and lymphatic vessels after transplantation. The MVF group was compared with animals receiving collagen hydrogel only or a sham intervention. Lymphangiogenic effects were analyzed using volumetry, magnetic resonance (MR) lymphography, histology, and immunohistochemistry. MVF injection resulted in reduced hindlimb volumes when compared to non-treated controls. MR lymphography revealed lymphatic regeneration with reduced dermal backflow after MVF treatment. Finally, MVF transplantation promoted popliteal angiogenesis and lymphangiogenesis associated with a significantly increased microvessel and lymphatic vessel density. These findings indicate that MVF transplantation represents a promising approach to induce therapeutic lymphangiogenesis.

Engineering Functional Vascularized Beige Adipose Tissue from Microvascular Fragments of Models of Healthy and Type II Diabetes Conditions.

Engineered beige adipose tissues could be used for screening therapeutic strategies or as a direct treatment for obesity and metabolic disease. Microvascular fragments are vessel structures that can be directly isolated from adipose tissue and may contain cells capable of differentiation into thermogenic, or beige, adipocytes. In this study, culture conditions were investigated to engineer three-dimensional, vascularized functional beige adipose tissue using microvascular fragments isolated from both healthy animals and a model of type II diabetes (T2D). Vascularized beige adipose tissues were engineered and exhibited increased expression of beige adipose markers, enhanced function, and improved cellular respiration. While microvascular fragments isolated from both lean and diabetic models were able to generate functional tissues, differences were observed in regard to vessel assembly and tissue function. This study introduces an approach that could be employed to engineer vascularized beige adipose tissues from a single, potentially autologous source of cells.

Engineering Human Beige Adipose Tissue.

In this study, we described a method for generating functional, beige (thermogenic) adipose microtissues from human microvascular fragments (MVFs). The MVFs were isolated from adipose tissue acquired from adults over 50 years of age. The tissues express thermogenic gene markers and reproduce functions essential for the potential therapeutic impact of beige adipose tissues such as enhanced lipid metabolism and increased mitochondrial respiration. MVFs serve as a potential single, autologous source of cells that can be isolated from adult patients, induced to recreate functional aspects of beige adipose tissue and enable rapid vascularization post-transplantation. This approach has the potential to be used as an autologous therapy for metabolic diseases or as a model for the development of a personalized approach to high-throughput drug development/screening for adipose tissue.

Vascularization of Microvascular Fragment Isolates from Visceral and Subcutaneous Adipose Tissue of Mice.

BACKGROUND: Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies in rodents exclusively use epididymal adipose tissue as a visceral fat source for MVF isolation. However, in future clinical practice, MVF may be rather isolated from liposuctioned subcutaneous fat tissue of patients. Therefore, we herein compared the vascularization characteristics of MVF isolates from visceral and subcutaneous fat tissue of murine origin. METHODS: MVF isolates were generated from visceral and subcutaneous fat tissue of donor mice using two different enzymatic procedures. For in vivo analyses, the MVF isolates were seeded onto collagen-glycosaminoglycan scaffolds and implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice. RESULTS: By means of the two isolation procedures, we isolated a higher number of MVF from visceral fat tissue when compared to subcutaneous fat tissue, while their length distribution, viability and cellular composition were comparable in both groups. Intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a significantly reduced vascularization of implanted scaffolds seeded with subcutaneous MVF isolates when compared to implants seeded with visceral MVF isolates. Light and scanning electron microscopy showed that this was due to high amounts of undigested connective tissue within the subcutaneous MVF isolates, which clogged the scaffold pores and prevented the interconnection of individual MVF into new microvascular networks. CONCLUSION: These findings indicate the need for improved protocols to generate connective tissue-free MVF isolates from subcutaneous fat tissue for future translational studies.

The simpler, the better: tissue vascularization using the body's own resources.

Tissue regeneration is crucially dependent on sufficient vascularization. In regenerative medicine, this can be effectively achieved by autologous vascularization strategies using the body's own resources. These strategies include the administration of blood-derived factor preparations, adipose tissue-based vascularization, and the in situ engineering of vascularized tissue. Due to their simplicity, the translation of these strategies into clinical practice is easier in terms of feasibility, safety requirements, and regulatory hurdles compared with complex and time-consuming procedures involving intensive cell manipulation. Hence, they are close to clinical application or are already being used to successfully treat patients by distinct personalized medicine concepts.

Microvascular Fragments in Microcirculation Research and Regenerative Medicine.

Adipose tissue-derived microvascular fragments (MVF) are functional vessel segments, which rapidly reassemble into new microvasculatures under experimental in vitro and in vivo conditions. Accordingly, they have been used for many years in microcirculation research to study basic mechanisms of endothelial cell function, angiogenesis, and microvascular network formation in two- and three-dimensional environments. Moreover, they serve as vascularization units for musculoskeletal regeneration and implanted biomaterials as well as for the treatment of myocardial infarction and the generation of prevascularized tissue organoids. Besides, multiple factors determining the vascularization capacity of MVF have been identified, including their tissue origin and cellular composition, the conditions for their short- and long-term storage, as well as their implantation site and the general health status and medication of the recipient. The next challenging step is now the successful translation of all these promising experimental findings into clinical practice. If this succeeds, a multitude of future therapeutic applications may significantly benefit from the remarkable properties of MVF. Impact Statement The present review provides a complete overview of the broad application spectrum of adipose tissue-derived microvascular fragments (MVF) in angiogenesis research and regenerative medicine. Moreover, it systematically describes factors determining their vascularization capacity. These factors may be used to further improve the remarkable properties of MVF in personalized medicine and to promote their rapid introduction as vascularization units into clinical practice.

Adipose Tissue-Derived Microvascular Fragments From Male and Female Fat Donors Exhibit a Comparable Vascularization Capacity.

Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies exclusively use epididymal fat tissue of male donor mice as a source for MVF isolation. However, in future clinical practice, MVF-based approaches may be applied in both male and female patients. Therefore, we herein compared the vascularization capacity of MVF isolated from the epididymal and peri-ovarian fat tissue of male and female donor mice. Freshly isolated MVF from male and female donors did not differ in their number, length distribution, viability and cellular composition. After their assembly into spheroids, they also exhibited a comparable in vitro sprouting activity. Moreover, they could be seeded onto collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects within mouse dorsal skinfold chambers. Repetitive intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a comparable vascularization and incorporation of implants seeded with MVF of male and female origin. Taken together, these findings demonstrate that the vascularization capacity of MVF is not gender-specific.

Microvascular fragment spheroids: Three-dimensional vascularization units for tissue engineering and regeneration.

Adipose tissue-derived microvascular fragments (MVF) serve as vascularization units in tissue engineering and regenerative medicine. Because a three-dimensional cellular arrangement has been shown to improve cell function, we herein generated for the first time MVF spheroids to investigate whether this further increases their vascularization potential. These spheroids exhibited a morphology, size, and viability comparable to that of previously introduced stromal vascular fraction (SVF) spheroids. However, MVF spheroids contained a significantly higher number of CD31-positive endothelial cells and α-smooth muscle actin (SMA)-positive perivascular cells, resulting in an enhanced angiogenic sprouting activity. Accordingly, they also exhibited an improved in vivo vascularization and engraftment after transplantation into mouse dorsal skinfold chambers. These findings indicate that MVF spheroids are superior to SVF spheroids and, thus, may be highly suitable to improve the vascularization of tissue defects and implanted tissue constructs.

3D Collagen Hydrogel Promotes In Vitro Langerhans Islets Vascularization through ad-MVFs Angiogenic Activity.

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33-955 µm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF-islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.

Systemic low-dose erythropoietin administration improves the vascularization of collagen-glycosaminoglycan matrices seeded with adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (MVF) are used as vascularization units in tissue engineering. In this study, we investigated whether the vascularization capacity of MVF can be improved by systemic low-dose erythropoietin (EPO) administration. MVF were isolated from the epididymal fat of donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice. Both donor and recipient mice were treated daily with either EPO (500 IU/kg) or vehicle (0.9% NaCl). The implants were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. EPO-treated MVF contained a comparable number of proliferating Ki67+ but less apoptotic cleaved caspase-3+ endothelial cells when compared to vehicle-treated controls. Moreover, EPO treatment accelerated and improved the in vivo vascularization, blood vessel maturation, and epithelialization of MVF-seeded matrices. These findings indicate that systemic low-dose EPO treatment is suitable to enhance the viability and network-forming capacity of MVF.

Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Dental Pulp Regeneration.

INTRODUCTION: The transplantation of dental pulp stem cells (DPSCs) has emerged as a novel strategy for the regeneration of lost dental pulp after pulpitis and trauma. Dental pulp regeneration of the young permanent tooth with a wide tooth apical foramen has achieved significant progress in the clinical trials. However, because of the narrow apical foramen, dental pulp regeneration in adult teeth using stem cells remains difficult in the clinic. Finding out how to promote vascular reconstitution is essential for the survival of stem cells and the regeneration of dental pulp after transplantation into the adult tooth. METHODS: Adipose tissue-derived microvascular fragments (ad-MVFs) were isolated from human adipose tissues. The apoptosis and senescence of DPSCs cultured in conditioned media were evaluated to explore the effects of ad-MVFs on DPSCs. DPSCs combined with ad-MVFs were inserted into the human tooth root segments and implanted subcutaneously into immunodeficient mice. Regenerated pulplike tissues were analyzed by hematoxylin and eosin and immunohistochemistry. The vessels in regenerated tissues were analyzed by Micro-CT and immunofluorescence. RESULTS: The isolated ad-MVFs contained endothelial cells and pericytes. ad-MVFs effectively prevented the apoptosis and senescence of the transplanted DPSCs both in vivo and in vitro. Combined with DPSCs, ad-MVFs obviously facilitated the formation of vascular networks in the transplants. DPSCs combined with ad-MVFs formed dental pulp-like tissues with abundant cells and matrix after 4 weeks of implantation. The supplementation of ad-MVFs led to more odontoblastlike cells and increased the formation of mineralized substance around the root canal. CONCLUSIONS: Cotransplantation with ad-MVFs promotes the angiogenesis and revascularization of transplanted DPSC aggregates, leading to robust regeneration of dental pulp.

Microvascular Fragments: More Than Just Natural Vascularization Units.

Adipose tissue-derived microvascular fragments serve as natural vascularization units in angiogenesis research and tissue engineering due to their ability to rapidly reassemble into microvascular networks. Recent studies indicate that they exhibit additional unique properties that may be beneficial for a wide range of future biomedical applications. Their angiogenic activity can be increased during short-term cultivation as a means of adapting their vascularization capacity to patient-specific needs. Moreover, they are a source of endothelial progenitor cells, multipotent mesenchymal stromal cells, and lymphatic vessel fragments. Finally, they exert immunomodulatory effects, determining the tissue integration of implanted biomaterials. Hence, microvascular fragments represent versatile building blocks for the improvement of vascularization, organotypic tissue formation, lymphatic regeneration, and implant integration.