a-full-skin-defect-model-to-evaluate-vascularization-biomaterials

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials <em>In Vivo</em>

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Technische Universität München

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factorscytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP) inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17beta-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smad1 physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type beta transforming growth factor family have important roles.

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk.

Optimizing seeding efficiency, reducing delayed culture periods and mimicking native tissue architecture are crucial requirements for the development of seeding procedures in tissue engineering. In vascular applications, the tubular geometry of the grafts further hampers the efficient delivery of cells onto the scaffold. To overcome these limitations, a novel technology based upon the use of magnetic fields is presented in this study: a radial magnetic force drives the cells immediately onto the luminal surface of a tubular scaffold and immobilizes the cells on the substrate's surface promoting cell attachment. Human smooth muscle cells (SMCs) labeled with CD44 magnetic Dynabeads were successively seeded onto the luminal surface of a tubular shaped collagen membrane. After 5 h, one additional layer of human umbilical vein endothelial cells (HUVECs) labeled with CD31 magnetic Dynabeads was seeded onto the luminal SMCs. The co-culture was incubated during 5 days prior to analysis. Cell viability and expression profiles were preserved during the entire seeding process. Histological examination of the constructs highlighted densely packed multilayers of SMCs covered by a monolayer of endothelial cells. SEM inspection confirmed a heterotypic multilayer assembly formed by multiple layers of elongated SMCs covered by a single layer of endothelial cells. Seeding kinetics of HUVECs and SMCs showed over 90% seeding efficiency after 20 and 40 min magnetic exposure respectively. Magnetically induced cell seeding provides a valuable tool for rapid seeding procedures of tubular scaffolds while complying with the histological architecture of tissue.

Direct magnetic tubular cell seeding: a novel approach for vascular tissue engineering.

Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP(+)) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with the scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.

The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration.

Pulsed electromagnetic fields (PEMFs) have been used extensively in bone fracture repairs and wound healing. It is accepted that the induced electric field is the dose metric. The mechanisms of interaction between weak magnetic fields and biological systems present more ambiguity than that of PEMFs since weak electric currents induced by PEMFs are believed to mediate the healing process, which are absent in magnetic fields. The present study examines the response of human umbilical vein endothelial cells to weak static magnetic fields. We investigated proliferation, viability, and the expression of functional parameters such as eNOS, NO, and also gene expression of VEGF under the influence of different doses of weak magnetic fields. Applications of weak magnetic fields in tissue engineering are also discussed. Static magnetic fields may open new venues of research in the field of vascular therapies by promoting endothelial cell growth and by enhancing the healing response of the endothelium.

Effects of weak static magnetic fields on endothelial cells.

The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo.

Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.

Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.

The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration.

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissue-derived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4 weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering.

Engineering of vascularized adipose constructs.

Vascularization is a key process in tissue engineering and regeneration and represents one of the most important issues in the field of regenerative medicine. Thus, several strategies to improve vascularization are currently under clinical evaluation. In this study, stem cells derived from human sweat glands were isolated, characterized, seeded in collagen scaffolds, and engrafted in a mouse full skin defect model for dermal regeneration. Results showed that these cells exhibit high proliferation rates and express stem cell and differentiation markers. Moreover, cells responded to angiogenic environments by increasing their migration (P<0.001) and proliferation (P<0.05) capacity and forming capillary-like structures. After seeding in the scaffolds, cells distributed homogeneously, interacting directly with the scaffold, and released bioactive molecules involved in angiogenesis, immune response, and tissue remodeling. In vivo, scaffolds containing cells were used to induce dermal regeneration. Here we have found that the presence of the cells significantly improved vascularization (P<0.001). As autologous sweat gland-derived stem cells are easy to obtain, exhibit a good proliferation capacity, and improve vascularization during dermal regeneration, we suggest that the combined use of sweat gland-derived stem cells and scaffolds for dermal regeneration might improve dermal regeneration in future clinical settings.

The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.

Article	Year
Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 12552124	2003
Direct magnetic tubular cell seeding: a novel approach for vascular tissue engineering. Cells, tissues, organs\| PubMed ID: 17108686	2006
The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration. Biomaterials\| PubMed ID: 19651436	2009
Effects of weak static magnetic fields on endothelial cells. Bioelectromagnetics\| PubMed ID: 20119972	2010
Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector. Biomaterials\| PubMed ID: 21159378	2011
The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration. Biomaterials\| PubMed ID: 21435711	2011
Engineering of vascularized adipose constructs. Cell and tissue research\| PubMed ID: 21850493	2012
The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration. The Journal of investigative dermatology\| PubMed ID: 22377762	2012
Development of photosynthetic biomaterials for in vitro tissue engineering. Acta biomaterialia\| PubMed ID: 24406198	2014

Ursula Hopfner

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