Adipose tissue-derived microvascular fragments: natural vascularization units for regenerative medicine (original) (raw)
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Journal of Visualized Experiments, 2017
A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.
Short-term post-implantation dynamics of in vitro engineered human microvascularized adipose tissues
Biomedical materials (Bristol, England), 2018
Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adi...
European Cells and Materials, 2015
Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFPpositive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, noncultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.
2019
The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31 + , smooth muscle actin +). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.
The guidance of endothelial cell organization into a capillary network has been a long-standing challenge in tissue engineering. Some research efforts have been made to develop methods to promote capillary networks inside engineered tissue constructs. Capillary and vascular networks that would mimic blood microvessel function can be used to subsequently facilitate oxygen and nutrient transfer as well as waste removal. Vascularization of engineering tissue construct is one of the most favorable strategies to overpass nutrient and oxygen supply limitation, which is often the major hurdle in developing thick and complex tissue and artificial organ. This paper addresses recent advances and future challenges in developing three-dimensional culture systems to promote tissue construct vascularization allowing mimicking blood microvessel development and function encountered in vivo. Bioreactors systems that have been used to create fully vascularized functional tissue constructs will also be outlined.
Tissue engineering, 2007
Vascularization is critical to the survival of engineered tissues. This study combined biophysical and bioactive approaches to induce neovascularization in vivo. Further, we tested the effects of engineered vascularization on adipose tissue grafts. Hydrogel cylinders were fabricated from poly(ethylene glycol) diacrylate (PEG) in four configurations: PEG alone, PEG with basic fibroblast growth factor (bFGF), microchanneled PEG, or both bFGF-adsorbed and microchanneled PEG. In vivo implantation revealed no neovascularization in PEG, but substantial angiogenesis in bFGF-adsorbed and/or microchanneled PEG. The infiltrating host tissue consisted of erythrocyte-filled blood vessels lined by endothelial cells, and immunolocalized to vascular endothelial growth factor (VEGF). Human mesenchymal stem cells were differentiated into adipogenic cells, and encapsulated in PEG with both microchanneled and adsorbed bFGF. Upon in vivo implantation subcutaneously in immunodeficient mice, oil red O po...
Tissue engineering of blood vessels
British Journal of Surgery, 2006
Background: Tissue engineering techniques have been employed successfully in the management of wounds, burns and cartilage repair. Current prosthetic alternatives to autologous vascular bypass grafts remain poor in terms of patency and infection risk. Growing biological blood vessels has been proposed as an alternative.
In tissue engineering, the generation of tissue constructs comprising preformed microvessels is a promising strategy to guarantee their adequate vascularisation after implantation. Herein, we analysed whether this may be achieved by seeding porous scaffolds with adipose tissue-derived microvascular fragments. Green fluorescent protein (GFP)-positive microvascular fragments were isolated by enzymatic digestion from epididymal fat pads of male C57BL/6-TgN(ACTB-EGFP)1Osb/J mice. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were seeded with these fragments and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study inosculation and vascularisation of the implants by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Empty scaffolds served as controls. Vital microvascular fragments could be isolated from adipose tissue and seeded onto the scaffolds under dynamic pressure conditions. In the dorsal skinfold chamber, the fragments survived and exhibited a high angiogenic activity, resulting in the formation of GFP-positive microvascular networks within the implants. These networks developed interconnections to the host microvasculature, resulting in a significantly increased functional microvessel density at day 10 and 14 after implantation when compared to controls. Immunohistochemical analyses of vessel-seeded scaffolds revealed that >90 % of the microvessels in the implants' centre and ~60 % of microvessels in the surrounding host tissue were GFP-positive. This indicates that the scaffolds primarily vascularised by external inosculation. These novel findings demonstrate that the vascularisation of implanted porous scaffolds can be improved by incorporation of microvascular fragments. Accordingly, this approach may markedly contribute to the success of future tissue engineering applications in clinical practice.
Stem Cell Sources for Vascular Tissue Engineering and Regeneration
Tissue Engineering Part B: Reviews, 2012
This review focuses on the stem cell sources with the potential to be used in vascular tissue engineering and to promote vascular regeneration. The first clinical studies using tissue-engineered vascular grafts are already under way, supporting the potential of this technology in the treatment of cardiovascular and other diseases. Despite progress in engineering biomaterials with the appropriate mechanical properties and biological cues as well as bioreactors for generating the correct tissue microenvironment, the source of cells that make up the vascular tissues remains a major challenge for tissue engineers and physicians. Mature cells from the tissue of origin may be difficult to obtain and suffer from limited proliferative capacity, which may further decline as a function of donor age. On the other hand, multipotent and pluripotent stem cells have great potential to provide large numbers of autologous cells with a great differentiation capacity. Here, we discuss the adult multipotent as well as embryonic and induced pluripotent stem cells, their differentiation potential toward vascular lineages, and their use in engineering functional and implantable vascular tissues. We also discuss the associated challenges that need to be addressed in order to facilitate the transition of this technology from the bench to the bedside. Vascular Tissue Engineering: Unmet Clinical Need C ardiovascular disease, and coronary artery disease (CAD) in particular, is the leading cause of mortality in the United States, necessitating *500,000 coronary artery bypass graft (CABG) surgeries annually. 1 Surgically harvested autologous grafts, such as the left internal mammary and radial arteries or the greater saphenous vein, from patients are considered the gold standard for CABG procedures. 2-5 Other autologous arterial/venous grafts, cryopreserved cadaveric grafts, umbilical vein grafts, and arterial allografts have also been tried but with limited success because of associated complications. 6-11 Although autologous vessels from patients remain the grafts of choice, in many cases, previous harvest, morbidity at the donor site, or disease progression limit the availability of native grafts. 12,13 Clinical studies suggest that only a limited number of patients undergoing CABG surgeries have suitable arterial grafts and up to 30% of patients requiring venous grafts for peripheral vascular diseases lack transplantable veins. 14,15 While synthetic vascular prostheses such as expanded polytetrafluoroethylene (ePTFE) and Dacron are available alternatives for high-flow, low-resistance, large peripheral vessel pathologies, their clinical outcome for small-diameter (< 6 mm) vessel replacement has been grim. 16-20 Prosthetic graft failure has been attributed to intimal hyperplasia, thrombogenicity, compliance mismatch, and diameter mismatch between the graft and native artery. 21-24 Despite decades of effort, the successful fabrication of an ideal vascular graft still remains a challenge. Ideally, a vascular graft should be strong, biocompatible, nontoxic, nonimmunogenic, antithrombotic, compliant, vasoactive, and amenable to postimplantation remodeling by the host tissue. To this end, tissue-engineered vessels (TEVs) that can withstand the challenging arterial hemodynamic microenvironment and are amenable to physiological remodeling represent an attractive alternative. Vascular Tissue Engineering Approaches Three major approaches have been proposed for the tissue engineering of vascular grafts: (1) decellularized matrices; (2) cell-sheet engineering; and (3) biodegradable scaffolds from natural or synthetic polymers. Decellularized blood vessels as well as small intestinal submucosa (SIS) have been used to fabricate vascular grafts. The main advantage of using decellularized tissue is that the native three-dimensional (3D) architecture of matrix molecules-mainly type 1 collagen and elastin-is preserved 25 and might be helpful in guiding tissue repair and remodeling postimplantation. Decellularized blood vessels
Journal of Vascular Surgery, 2007
The burgeoning field of vascular tissue engineering holds promise for the creation of a practical and successful small-diameter arterial bypass graft. Many creative combinations of autologous cells and scaffolds exist along with an equally long list of microenvironmental cues used to create a functional arterial conduit. This review outlines our work using abdominal wall fat as a source of autologous stem cells for vascular tissue engineering, focusing specifically on this stem cell's availability and potency to differentiate into endothelial-like cells. In a series of 49 patients undergoing elective peripheral vascular surgery, an abundant quantity of adult stem cells was harvested from fat obtained via liposuction. The efficacy of the isolation did not appear influenced by advanced age, obesity, renal failure or vascular disease, although fat from diabetic patients yielded significantly less stem cells. Additionally, these adipose-derived stem cells acquired several morphological and molecular endothelial phenotypes when exposed to growth factors (ECGS, VEGF) and physiologic shear stress in vitro. Taken together, these studies suggest that fat appears to be a viable source of autologous stem cells for use in vascular tissue engineering.