The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation.

BACKGROUND: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. METHODS: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. RESULTS: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. CONCLUSION: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs.

Enoxaparin does not affect network formation of adipose tissue-derived microvascular fragments.

Dermal substitutes are frequently used for the initial coverage of extensive skin defects. The seeding of these implants with adipose tissue-derived microvascular fragments (ad-MVF) has recently been shown to accelerate their vascularization and incorporation. In the present study we analyzed whether these processes are affected by a thromboprophylactic therapy with the low molecular weight heparin (LMWH) enoxaparin (enox). Green fluorescent protein (GFP)+ ad-MVF were isolated from enox- (8 mg/kg s.c.) and vehicle-treated (0.9% NaCl s.c.) (C57BL/6-Tg(CAG-EGFP)1Osb/J mice and seeded onto Integra matrices. Subsequently, these were implanted into full-thickness skin defects within dorsal skinfold chambers of enox- and vehicle-treated C57BL/6 wild-type mice. Repetitive stereomicroscopy and intravital fluorescence microscopy over 2 weeks as well as histological and immunohistochemical analyses on day 14 revealed that enox does not inhibit the reassembly of ad-MVF into new microvascular networks. In addition, treatment with the anticoagulative compound did not promote implant-induced hemorrhage formation. Accordingly, Integra matrices in enox- and vehicle-treated animals exhibited a comparable final microvessel density, fraction of GFP+ blood vessels originating from seeded ad-MVF, collagen fiber content, and epithelialization. These novel findings demonstrate that the seeding of dermal substitutes with ad-MVF may be applied also during thromboprophylactic therapy without affecting implant vascularization and bleeding risk.

Dual inhibition of PI3K and mTOR by VS-5584 suppresses thrombus formation.

VS-5584 is a highly selective dual kinase inhibitor which suppresses phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) activity. Because these kinases are crucially involved in primary hemostasis, we herein investigated the effect of this compound on thrombus formation in vitro and in vivo. Pretreatment of washed platelets (WP) or platelet-rich plasma (PRP) with VS-5584 inhibited the agonist-induced activation of surface glycoprotein complex (GP)IIb/IIIa and the upregulation of P-selectin. This was associated with a significantly reduced formation of platelet-leukocyte aggregates (PLA). VS-5584 further attenuated platelet aggregation and adhesion after agonist stimulation. In contrast, endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and secretion of von Willebrand Factor (vWF) were not affected by the dual kinase inhibitor. In vivo, VS-5584 inhibited photochemically induced thrombus formation as shown by a significantly prolonged time to complete vessel occlusion when compared to vehicle-treated controls. This was associated with an elevated tail vein bleeding time, indicating a potential hemorrhagic risk in VS-5584-treated mice. Taken together, these novel findings demonstrate that VS-5584 is a potent inhibitor of primary hemostasis targeting multiple platelet functions.

The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity.

BACKGROUND: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. METHODS: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. RESULTS: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. CONCLUSION: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

Retention of Human iPSC-Derived or Primary Cells Following Xenotransplantation into Rat Immune-Privileged Sites.

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites.

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.

Establishment of a Reliable Model to Study the Failure of Fracture Healing in Aged Mice.

The failure of fracture healing represents a substantial clinical problem. Moreover, aged patients demonstrate an elevated risk for failed bone healing. However, murine models to study the failure of fracture healing are established only in young adult animals. Therefore, the aim of this study was to develop a reliable model to study failed fracture healing in aged mice. After creation of a 1.8-mm segmental defect and periosteal resection, femora of aged mice (18-20 months) and young adult control mice (3-4 months) were stabilized by pin-clip fixation. Segmental defects were analyzed by means of biomechanics, x-ray, and micro-computed tomography, as well as histomorphometric, immunohistochemical, and Western blot analysis. After 10 weeks, all animals showed a complete lack of osseous bridging, resulting in fracture healing failure. Segmental defects in aged mice revealed a reduced bone formation and vascularization when compared to young adult mice. This was associated with a decreased expression of bone formation markers. In addition, we detected a reduced number of tartrate-resistant acid phosphatase-positive osteoclasts and an elevated osteoprotegerin/receptor activator of NF-ĸB ligand ratio in aged animals, indicating a reduced osteoclast activity. Moreover, aged animals showed also an enhanced inflammatory response, characterized by an increased infiltration of macrophages within the callus tissue. Taken together, we herein report for the first time a reliable model to study fracture healing failure in aged mice. In the future, the use of this model enables us to study novel therapeutic strategies and molecular mechanics of failed fracture healing during aging.

Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis.

Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cultures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the therapeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches.

Vascularization Strategies for Porous Polyethylene Implants.

Porous polyethylene (pPE) is a frequently implanted biomaterial in craniofacial reconstructive surgery. Its rapid vascularization and tissue incorporation are major prerequisites to prevent complications, such as material infection, migration, and extrusion. To achieve this, several sophisticated strategies have been introduced and evaluated during the last 20 years. These include (i) the angiogenic stimulation of the host tissue with epidermal growth factor, basic fibroblast growth factor or macrophage-activating lipopeptide-2, (ii) material modifications, such as increase of surface roughness and incorporation of bioactive glass particles, (iii) surface coatings with growth factors, glycoproteins, acrylic acid, arginine/glycine/aspartic acid peptide as well as components of the plasminogen activation system and autologous clotted blood or serum, and (iv) the seeding with fibroblasts, chondrocytes, stem cells, or adipose-tissue-derived microvascular fragments. The majority of these approaches showed promising results in experimental studies and, thus, may be capable of improving the success rates after pPE implantation in future clinical practice.

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.

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 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.

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels.

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the ß-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.

Macrophages promote network formation and maturation of transplanted adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments rapidly reassemble into microvascular networks within implanted scaffolds. Herein, we analyzed the contribution of macrophages to this process. C57BL/6 mice received clodronate (clo)-containing liposomes for macrophage depletion, whereas animals treated with phosphate-buffered-saline-containing liposomes served as controls. Microvascular fragments were isolated from clo- and phosphate-buffered-saline-treated donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of clo- and phosphate-buffered-saline-treated recipient mice. The implants' vascularization and incorporation were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. Compared to controls, matrices within clo-treated animals exhibited a significantly reduced functional microvessel density. Moreover, they contained a lower fraction of microvessels with an α-smooth muscle actin (SMA)+ cell layer, indicating impaired vessel maturation. This was associated with a deteriorated implant incorporation. These findings demonstrate that macrophages not only promote the reassembly of microvascular fragments into microvascular networks, but also improve their maturation during this process.

Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine.

Cell-derived extracellular matrices (CD-ECMs) captured increasing attention since the first studies in the 1980s. The biological resemblance of CD-ECMs to their in vivo counterparts and natural complexity provide them with a prevailing bioactivity. CD-ECMs offer the opportunity to produce microenvironments with costumizable biological and biophysical properties in a controlled setting. As a result, CD-ECMs can improve cellular functions such as stemness or be employed as a platform to study cellular niches in health and disease. Either on their own or integrated with other materials, CD-ECMs can also be utilized as biomaterials to engineer tissues de novo or facilitate endogenous healing and regeneration. This review provides a brief overview over the methodologies used to facilitate CD-ECM deposition and manufacturing. It explores the versatile uses of CD-ECM in fundamental research and therapeutic approaches, while highlighting innovative strategies. Furthermore, current challenges are identified and it is accentuated that advancements in methodologies, as well as innovative interdisciplinary approaches are needed to take CD-ECM-based research to the next level.

Biological coating with platelet-rich plasma and adipose tissue-derived microvascular fragments improves the vascularization, biocompatibility and tissue incorporation of porous polyethylene.

Porous polyethylene (pPE) is a commonly used biomaterial in craniofacial reconstructive surgery. However, implant failure due to insufficient vascularization represents a major issue. To overcome this problem, we herein introduce an effective strategy to improve the vascularization and incorporation of pPE. Adipose tissue-derived microvascular fragments (MVF) from transgenic green fluorescent protein (GFP)+ mice were suspended in platelet-rich plasma (PRP) for the coating of pPE. PRP/MVF-coated pPE as well as PRP-coated and uncoated controls were subsequently implanted into the dorsal skinfold chamber and the flanks of GFP- wild-type mice to analyze their in vivo performance throughout 2, 4 and 8 weeks by means of intravital fluorescence microscopy, histology and immunohistochemistry. The GFP+/GFP- cross-over design allowed the identification of GFP+ MVF within the implants. Shortly after implantation, they rapidly reassembled into new blood-perfused microvascular networks, resulting in a significantly accelerated vascularization of PRP/MVF-coated pPE when compared to both controls. The overall numbers of rolling and adherent leukocytes within the microcirculation as well as macrophages, multi-nucleated giant cells and mast cells around the implants did not differ between the three groups. However, in contrast to uncoated controls, PRP/MVF-coated and PRP-coated pPE promoted pro-angiogenic M2 macrophage polarization at the implantation site. These findings demonstrate that PRP/MVF-coating represents a highly effective strategy to enhance the vascularization, biocompatibility and tissue incorporation of pPE. STATEMENT OF SIGNIFICANCE: The clinical in vivo performance of implanted biomaterials is crucially dependent on their adequate incorporation into the body. To achieve this, we herein introduce an effective biological coating strategy. Our results demonstrate that coating with PRP and MVF accelerates and enhances the vascularization, biocompatibility and tissue incorporation of porous polyethylene. Because this type of biological coating is easily applicable on any type of biomaterial, our approach may rapidly be translated into clinical practice to improve the outcome of various regenerative approaches.

Pantoprazole impairs fracture healing in aged mice.

Proton pump inhibitors (PPIs) belong to the most common medication in geriatric medicine. They are known to reduce osteoclast activity and to delay fracture healing in young adult mice. Because differentiation and proliferation in fracture healing as well as pharmacologic actions of drugs markedly differ in the elderly compared to the young, we herein studied the effect of the PPI pantoprazole on bone healing in aged mice using a murine fracture model. Bone healing was analyzed by biomechanical, histomorphometric, radiological and protein biochemical analyses. The biomechanical analysis revealed a significantly reduced bending stiffness in pantoprazole-treated animals when compared to controls. This was associated with a decreased amount of bone tissue within the callus, a reduced trabecular thickness and a higher amount of fibrous tissue. Furthermore, the number of osteoclasts in pantoprazole-treated animals was significantly increased at 2 weeks and decreased at 5 weeks after fracture, indicating an acceleration of bone turnover. Western blot analysis showed a lower expression of the bone morphogenetic protein-4 (BMP-4), whereas the expression of the pro-angiogenic parameters was higher when compared to controls. Thus, pantoprazole impairs fracture healing in aged mice by affecting angiogenic and osteogenic growth factor expression, osteoclast activity and bone formation.

In Vitro and in Vivo Analysis of Adhesive, Anti-Inflammatory, and Proangiogenic Properties of Novel 3D Printed Hyaluronic Acid Glycidyl Methacrylate Hydrogel Scaffolds for Tissue Engineering.

In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusion three-dimensional (3D) printing with photocured (λ = 445 nm) hyaluronic acid glycidyl methacrylate (HAGM). The developed product was compared with the polylactic-co-glycolic acid (PLGA) scaffolds generated by means of the original antisolvent 3D printing methodology. The cytotoxicity and cytocompatibility of the scaffolds were analyzed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests, flow cytometry, and scanning electron microscopy. Anti-inflammatory and proangiogenic properties of the scaffolds were evaluated in the dorsal skinfold chamber mouse model by means of intravital fluorescence microscopy, histology, and immunohistochemistry throughout an observation period of 14 days. In vitro, none of the scaffolds revealed cytotoxicity on days 1, 2, and 5 after seeding with umbilical cord-derived multipotent stromal cells, and the primary cell adhesion to the surface of HAGM scaffolds was low. In vivo, implanted HAGM scaffolds showed enhanced vascularization and host tissue ingrowth, and the inflammatory response to them was less pronounced compared with PLGA scaffolds. The results indicate excellent biocompatibility and vascularization capacity of the developed 3D printed HAGM scaffolds and position them as strong candidates for advanced tissue engineering applications.

In vivo biocompatibility, vascularization, and incorporation of Integra® dermal regenerative template and flowable wound matrix.

Integra® matrix wound dressing (MWD) is used for the reconstruction of full-thickness skin defects. For the treatment of complex wounds, this dermal substitute is available as a flowable wound matrix (FWM) of identical composition. To clarify whether variations in sample preparation and consistency affect the biocompatibility and tissue incorporation, we herein compared MWD and FWM. The matrices were characterized using scanning electron microscopy and histology. Moreover, they were implanted in mouse dorsal skinfold chambers to analyze their in vivo performance over 2 weeks. Scanning electron microscopy showed a planar surface of MWD whereas FWM presented an irregular, fissured morphology. However, histology of the two matrices revealed an identical fiber thickness, fiber length, and interfiber distance. Repetitive stereo-microscopy and immunohistochemical analyses of MWD and FWM showed a comparable epithelialization of the implants in the dorsal skinfold chamber model. At day 14, both matrices exhibited a low collagen content and microvessel density. Moreover, they were infiltrated by a high number of myeloperoxidase (MPO)-positive neutrophilic granulocytes and a lower number of MAC387-positive macrophages and CD3-positive lymphocytes. These findings demonstrate that differences in preparation and consistency do not affect the tissue response to MWD and FWM, indicating a comparable regenerative capacity in wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 52-60, 2018.

Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments.

BACKGROUND: The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach. RESULTS: SVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)+ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP- recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31+/GFP+ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time. CONCLUSIONS: The present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.