Wound fluid under occlusive dressings from diabetic patients show an increased angiogenic response and fibroblast migration.

INTRODUCTION: Metabolic diseases like diabetes mellitus often show prolonged healing and chronic wounds. Occlusive wound dressings are known to support wound closure by creating a moist environment which supports collagen synthesis, epithelialization and angiogenesis. We aimed to assess the effect of occlusion on diabetic wound fluid on the cellular level regarding fibroblast activity and angiogenetic response. MATERIAL AND METHODS: 22 split skin donor sites from 22 patients (11 patients with diabetes mellitus) were treated with occlusive dressings intraoperatively. On day 3, fluid and blood serum samples were harvested while changing the dressings. The influence of wound fluid on fibroblasts was assessed by measuring metabolic activity (Alamar Blue assay, Casey Counter), cell stress/death (LDH assay) and migration (in vitro wound healing assay) of fibroblasts. Angiogenesis of endothelial cells (HUVEC) was analyzed with the tube formation assay. Furthermore, a Magnetic Luminex Assay for multi-cytokines detection was performed focusing on inflammatory and pro-angiogenetic cytokines. RESULTS: The influence of wound fluid under occlusive dressings from diabetic patients showed a significantly increased angiogenic response and fibroblast migration compared to the non-diabetic patient group. Additionally, cell stress was increased in the diabetic group. Cytokine analysis showed an increase in VEGF-A in the diabetic group. CONCLUSION: Occlusive dressings may stimulate regenerative effects in diabetic wounds. Our in-vitro study shows the influence of wound fluid under occlusive dressings from diabetic patients on angiogenesis, migration and proliferation of fibroblasts, which are essential modulators of wound healing and scar modulation.

Deferiprone Stimulates Aged Dermal Fibroblasts via HIF-1α Modulation.

BACKGROUND: Hypoxia-inducible factor 1α (HIF-1α), a transcription factor responsible for tissue homeostasis and regeneration, presents reduced functionality in advanced age. In addition to absence of oxygen, sequestration of iron also stimulates HIF-1α. Therefore, we analyzed the efficacy of the iron-chelator deferiprone (DFP) at stimulating dermal fibroblasts. OBJECTIVES: The main objective of this study was to quantify the DFP concentrations capable of stimulating dermal fibroblasts in vitro and to correlate the effective DFP concentrations with the ability of DFP to penetrate the epidermis, reach the dermis, and activate HIF-1α in vivo. METHODS: We measured cell proliferation, metabolic activity, HIF-1α expression, and lactate dehydrogenase levels of both young and aged fibroblasts after a 24-hour in vitro preconditioning with DFP. In addition, we evaluated cell survival rates and morphology with different cellular stainings. Finally, we performed a transdermal permeation study with a 1% DFP topical formulation to quantify the concentration required to reach the dermis. RESULTS: In vitro administration of iron-chelation therapy (156-312.5 µg/mL DFP ) on aged fibroblasts resulted in activation of various antiaging processes. The concentration required to reach the dermis within 24 hours was 1.5% (0.15 mg/mL), which corresponds well with the effective doses of our laboratory analyses. CONCLUSIONS: The activation of HIF-1α by DFP enhances cell metabolism, proliferation, and survival of fibroblasts while reducing lactate dehydrogenase levels. Modulation of HIF-1α is linked to activation of key regeneration enzymes and proteins, and by proxy, antiaging. Therefore, the antiaging properties of DFP and its satisfactory dermal penetration make it a promising regenerative agent.

Osteoidosis leads to altered differentiation and function of osteoclasts.

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone.

Single-Cell Transcriptomics of Human Mesenchymal Stem Cells Reveal Age-Related Cellular Subpopulation Depletion and Impaired Regenerative Function.

Although bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely recognized as promising therapeutic agents, the age-related impacts on cellular function remain largely uncharacterized. In this study, we found that BM-MSCs from young donors healed wounds in a xenograft model faster compared with their aged counterparts (p < .001). Given this significant healing advantage, we then used single-cell transcriptomic analysis to provide potential molecular insights into these observations. We found that the young cells contained a higher proportion of cells characterized by a higher expression of genes involved in tissue regeneration. In addition, we identified a unique, quiescent subpopulation that was exclusively present in young donor cells. Together, these findings may explain a novel mechanism for the enhanced healing capacity of young stem cells and may have implications for autologous cell therapy in the extremes of age. Stem Cells 2019;37:240-246.

Towards a biotechnological platform for the production of human pro-angiogenic growth factors in the green alga Chlamydomonas reinhardtii.

The recent use of photosynthetic organisms such as Chlamydomonas reinhardtii in biomedical applications has demonstrated their potential for the treatment of acute and chronic tissue hypoxia. Moreover, transgenic microalgae have been suggested as an alternative in situ drug delivery system. In this study, we set out to identify the best available combination of strains and expression vectors to establish a robust platform for the expression of human pro-angiogenic growth factors, i.e., hVEGF-165, hPDGF-B, and hSDF-1, in biomedical settings. As a case study, combinations of two expression vectors (pOpt and pBC1) and two C. reinhardtii strains (UVM4 and UVM11) were compared with respect to hVEGF-165 transgene expression by determination of steady-state levels of transgenic transcripts and immunological detection of recombinant proteins produced and secreted by the generated strains. The results revealed the combination of the UVM11 strain with the pBC1 vector to be the most efficient one for high-level hVEGF-165 production. To assess the robustness of this finding, the selected combination was used to create hPDGF-B and hSDF-1 transgenic strains for optimized recombinant protein expression. Furthermore, biological activity and functionality of algal-produced recombinant pro-angiogenic growth factors were assessed by receptor phosphorylation and in vitro angiogenesis assays. The results obtained revealed a potentiating effect in the combinatorial application of transgenic strains expressing either of the three growth factors on endothelial cell tube formation ability, and thus support the idea of using transgenic algae expressing pro-angiogenic growth factors in wound healing approaches.

High Efficiency Low Cost Fibroblast Nucleofection for GMP Compatible Cell-based Gene Therapy.

Background: Dermal fibroblast is a powerful tool for the study of ex vivo DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. Results: the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Conclusion: Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research.

Engineering of vascularized adipose constructs.

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.

Effects of weak static magnetic fields on endothelial cells.

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.

Deferoxamine enhances the regenerative potential of diabetic Adipose Derived Stem Cells.

INTRODUCTION: Diabetes mellitus remains a significant public health problem, consuming over $400 billion every year. While Diabetes itself can be controlled effectively, impaired wound healing still occurs frequently in diabetic patients. Adipose-derived mesenchymal stem cells (ASCs) provide an especially appealing source for diabetic wound cell therapy. With autologous approaches, the functionality of ASCs largely underlie patient-dependent factors. Diabetes is a significant diminishing factor of MSC functionality. Here, we explore a novel strategy to enhance diabetic ASC functionality through deferoxamine (DFO) preconditioning. MATERIAL AND METHODS: Human diabetic ASCs have been preconditioned with 150 µM and 300 µM DFO in vitro and analyzed for regenerative cytokine expression. Murine diabetic ASCs have been preconditioned with 150 µM DFO examined for their in vitro and in vivo vasculogenic capacity in Matrigel assays. Additionally, a diabetic murine wound healing model has been performed to assess the regenerative capacity of preconditioned cells. RESULTS: DFO preconditioning enhances the VEGF expression of human diabetic ASCs through hypoxia-inducible factor upregulation. The use of 150 µM of DFO was an optimal concentration to induce regenerative effects. The vasculogenic potential of preconditioned diabetic ASCs is significantly greater in vitro and in vivo. The enhanced regenerative functionality of DFO preconditioned ASCs was further confirmed in a model of diabetic murine wound healing. CONCLUSION: These results demonstrate that DFO significantly induced the upregulation of hypoxia-inducible factor-1 alpha and VEGF in diabetic ASCs and showed efficacy in the treatment of diabetes-associated deficits of wound healing. The favorable status of DFO as a small molecule drug approved since decades for multiple indications makes this approach highly translatable.

Long-term pre- and postconditioning with low doses of erythropoietin protects critically perfused musculocutaneous tissue from necrosis.

It has been shown that pre- and postconditioning of ischemically challenged tissue with erythropoietin (EPO) is able to reduce necrosis in a dose-dependent manner. The aim of this study was to determine the tissue-protective effects of different EPO dosages and administration regimes. Three groups of six C57Bl/6-mice each were analyzed: (1) pre- and postconditioning with initial high doses of EPO (starting at 2500 I.U./kg bw i.p.) followed by low doses of EPO (125 I.U./kg bw i.p.) (EPO-high-dose); (2) pre- and postconditioning with low doses of EPO (125 I.U./kg bw i.p.) (EPO-low-dose); and (3) untreated control group. Randomly perfused musculocutaneous flaps were mounted on dorsal skinfold chambers undergoing acute persistent ischemia and developing ∼50% necrosis without treatment. Intravital epifluorescence microscopy was performed at days 1, 3, 5, 7, and 10 after surgery, assessing flap necrosis, microcirculation, and angiogenesis. The hematocrit was measured at days 0, 3, 7, and 10. Only the EPO-low-dose regimen was associated with a significant reduction of necrosis when compared to untreated controls. EPO-low-dose showed a higher increase in both arteriolar diameter and velocity, thereby resulting in a significantly increased arteriolar blood flow and a hence higher functional capillary density (FCD) of the critically perfused zone. EPO-induced angiogenesis was significantly increased in EPO-low-dose at days 7 and 10. Only EPO-high-dose reached a significant hematocrit increase by day 10. Tissue pre- and postconditioning with low doses of EPO protects the critically perfused musculocutaneous tissue by maintaining capillary perfusion because of increased arteriolar blood flow mediated by nitric oxide (NO) expression.

Adipose Stem Cells from Lipedema and Control Adipose Tissue Respond Differently to Adipogenic Stimulation In Vitro.

BACKGROUND: Lipedema is characterized by localized accumulation of fat in the extremities, which is typically unresponsive to dietary regimens or physical activity. Although the disease is well described and has a high incidence, little is known regarding the molecular and cellular mechanisms underlying its pathogenesis. The aim of this study was to investigate the pathophysiology of lipedema adipose cells in vitro. METHODS: Adipose-derived stem cells were isolated from lipoaspirates derived from lipedema and nonlipedema patients undergoing tumescent liposuction. In vitro differentiation studies were performed for up to 14 days using adipogenic or regular culture medium. Supernatants and cell lysates were tested for adiponectin, leptin, insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 content at days 7 and 14, using enzyme-linked immunosorbent assays. Adipogenesis was evaluated by visualizing and measuring cytoplasmic lipid accumulation. RESULTS: Lipedema adipose-derived stem cells showed impeded adipogenesis already at early stages of in vitro differentiation. Concomitant with a strongly reduced cytoplasmic lipid accumulation, significantly lower amounts of adiponectin and leptin were detectable in supernatants from lipedema adipose-derived stem cells and adipocytes compared with control cells. In addition, lipedema and nonlipedema cells differed in their expression of insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 and in their proliferative activity. CONCLUSIONS: The authors' findings indicate that in vitro adipogenesis of lipedema adipose-derived stem cells is severely hampered compared with nonlipedema adipose-derived stem cells. Lipedema adipose cells differ not only in their lipid storage capacity but also in their adipokine expression pattern. This might serve as a valuable marker for diagnosis of lipedema, probably from an early stage on.

In Vitro Characterization of Hypoxia Preconditioned Serum (HPS)-Fibrin Hydrogels: Basis for an Injectable Biomimetic Tissue Regeneration Therapy.

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS-fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS-fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS-fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration.

Fibrin Glue Enhances Adipose-Derived Stromal Cell Cytokine Secretion and Survival Conferring Accelerated Diabetic Wound Healing.

INTRODUCTION: Although chronic wounds are a major personal and economic burden, treatment options are still limited. Among those options, adipose-derived stromal cell- (ASC-) based therapies rank as a promising approach but are restricted by the harsh wound environment. Here we use a commercially available fibrin glue to provide a deliverable niche for ASCs in chronic wounds. MATERIAL AND METHODS: To investigate the in vitro effect of fibrin glue, cultivation experiments were performed and key cytokines for regeneration were quantified. By using an established murine chronic diabetic wound-healing model, we evaluated the influence of fibrin glue spray seeding on cell survival (In Vivo Imaging System, IVIS), wound healing (wound closure kinetics), and neovascularization of healed wounds (CD31 immunohistochemistry). RESULTS: Fibrin glue seeding leads to a significantly enhanced secretion of key cytokines (SDF-1, bFGF, and MMP-2) of human ASCs in vitro. IVIS imaging showed a significantly prolonged murine ASC survival in diabetic wounds and significantly accelerated complete wound closure in the fibrin glue seeded group. CD31 immunohistochemistry revealed significantly more neovascularization in healed wounds treated with ASCs spray seeded in fibrin glue vs. ASC injected into the wound bed. CONCLUSION: Although several vehicles have shown to successfully act as cell carrier systems in preclinical trials, regulatory issues have prohibited clinical usage for chronic wounds. By demonstrating the ability of fibrin glue to act as a carrier vehicle for ASCs, while simultaneously enhancing cellular regenerative function and viability, this study is a proponent of clinical translation for ASC-based therapies.

Occlusive dressing-induced secretomes influence the migration and proliferation of mesenchymal stem cells and fibroblasts differently.

BACKGROUND: Fingertip injuries treated with occlusive dressings (ODs) lead to nearly scar-free, functionally, and aesthetically pleasing results. We hypothesized that paracrine factors in the wound fluid (secretome) may influence migration and proliferation of mesenchymal stem cells (MSCs) and fibroblasts and modulate the wound-healing process. METHODS: We could collect wound fluid samples from 4 fingertip injuries and 7 split skin donor sites at the 5th day during dressing change. Blood serum samples served as controls. The proliferation rate of MSCs and fibroblasts (HS27) was continuously measured through impedance analysis for 60 h and by Alamarblue analysis after 72 h. Cell migration was evaluated continuously for 15 h and confirmed by the in vitro wound-healing assay. RESULTS: Migration of MSCs under the influence of both wound fluids was significantly faster than controls from 4 to 6 h after incubation and reversed after 9 h. MSC proliferation in wound fluid groups showed a significant increase at 5 and 10 h and was significantly decreased after 45 h. Fibroblasts in wound fluid groups showed overall a significant increase in migration and a significant decrease in proliferation compared to controls. CONCLUSION: OD-induced secretomes influence MSCs and fibroblasts and thereby possibly modulate wound healing and scar tissue formation.

Development of photosynthetic sutures for the local delivery of oxygen and recombinant growth factors in wounds.

Surgical sutures represent the gold standard for wound closure, however, their main purpose is still limited to a mechanical function rather than playing a bioactive role. Since oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors (VEGF, PDGF-BB, or SDF-1α) at the wound site. Here, photosynthetic genetically modified microalgae were seeded in commercially available sutures and their distribution and proliferation capacity was evaluated. Additionally, the mechanical properties of seeded sutures were compared to unseeded controls that showed no significant differences. Oxygen production, as well as recombinant growth factor release was quantified in vitro over time, and confirmed that photosynthetic sutures are indeed a feasible approach for the local delivery of bioactive molecules. Finally, photosynthetic sutures were tested in order to evaluate their resistance to mechanical stress and freezing. Significant stability was observed in both conditions, and the feasibility of their use in the clinical practice was therefore confirmed. Our results suggest that photosynthetic gene therapy could be used to produce a new generation of bioactive sutures with improved healing capacities. STATEMENT OF SIGNIFICANCE: Disruption of the vascular network is intrinsic to trauma and surgery, and consequently, wound healing is characterized by diminished levels of blood perfusion. Among all the blood components, oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process. Therefore, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors at the wound site. This novel concept has never been explored before for this type of material and represents the first attempt to create a new generation of bioactive sutures with improved regenerative capabilities.

Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for genetically-modified fibroblasts.

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.

Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.

The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

In Vitro Evaluation of Scaffolds for the Delivery of Mesenchymal Stem Cells to Wounds.

Mesenchymal stem cells (MSCs) have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs) on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis. In vitro results demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCs in vivo. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.

Generation of Viable Plant-Vertebrate Chimeras.

The extreme dependence on external oxygen supply observed in animals causes major clinical problems and several diseases are related to low oxygen tension in tissues. The vast majority of the animals do not produce oxygen but a few exceptions have shown that photosynthetic capacity is physiologically compatible with animal life. Such symbiotic photosynthetic relationships are restricted to a few aquatic invertebrates. In this work we aimed to explore if we could create a chimerical organism by incorporating photosynthetic eukaryotic cells into a vertebrate animal model. Here, the microalgae Chlamydomonas reinhardtii was injected into zebrafish eggs and the interaction and viability of both organisms were studied. Results show that microalgae were distributed into different tissues, forming a fish-alga chimera organism for a prolonged period of time. In addition, microscopic observation of injected algae, in vivo expression of their mRNA and re-growth of the algae ex vivo suggests that they survived to the developmental process, living for several days after injection. Moreover microalgae did not trigger a significant inflammatory response in the fish. This work provides additional evidence to support the possibility that photosynthetic vertebrates can be engineered.

Photosynthetic biomaterials: a pathway towards autotrophic tissue engineering.

Engineered tissues are highly limited by poor vascularization in vivo, leading to hypoxia. In order to overcome this challenge, we propose the use of photosynthetic biomaterials to provide oxygen. Since photosynthesis is the original source of oxygen for living organisms, we suggest that this could be a novel approach to provide a constant source of oxygen supply independently of blood perfusion. In this study we demonstrate that bioartificial scaffolds can be loaded with a solution containing the photosynthetic microalgae Chlamydomonas reinhardtii, showing high biocompatibility and photosynthetic activity in vitro. Furthermore, when photosynthetic biomaterials were engrafted in a mouse full skin defect, we observed that the presence of the microalgae did not trigger a native immune response in the host. Moreover, the analyses showed that the algae survived for at least 5 days in vivo, generating chimeric tissues comprised of algae and murine cells. The results of this study represent a crucial step towards the establishment of autotrophic tissue engineering approaches and suggest the use of photosynthetic cells to treat a broad spectrum of hypoxic conditions.