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Aerobic metabolism relies on external oxygen production through photosynthesis and its subsequent transport into each cell of the body via the cardiorespiratory system. This mechanism has successfully evolved over millions of years, enabling animals to inhabit most environments on Earth. However, the insufficient oxygen supply leads to several clinical problems, ranging from non-healing wounds to tumor resistance to therapy. Given that photosynthetic microorganisms are capable of producing oxygen and removing carbon dioxide from the environment, over the last decade, several groups worldwide have proposed their potential use as an alternative tissue oxygenation approach. While most studies have demonstrated safety and efficacy after local tissue administration, recent studies have also suggested that systemic administration could trigger intravascular photosynthesis. If successful, the development of a new generation of circulating cells, known as chlorocytes, may partially replace the role of erythrocytes in gas exchange within the body, without relying on external supply and vascular flow. This work reviews the existing literature on local and systemic administration of photosynthetic microorganisms, highlighting the main challenges in the field and potential solutions to unleash the enormous potential clinical impact of chlorocytes and intravascular photosynthesis. KEY POINTS: • Circulating photosynthetic microorganisms could deliver oxygen to tissues • Microalgae and cyanobacteria have shown safety and efficacy for oxygen delivery • Several key challenges need to be addressed for the clinical success of chlorocytes., (© 2024. The Author(s).)

Oxygen is essential for tissue regeneration, playing a crucial role in several processes, including cell metabolism and immune response. Therefore, the delivery of oxygen to wounds is an active field of research, and recent studies have highlighted the potential use of photosynthetic biomaterials as alternative oxygenation approach. However, while plants have traditionally been used to enhance tissue regeneration, their potential to produce and deliver local oxygen to wounds has not yet been explored. Hence, in this work we studied the oxygen-releasing capacity of Marchantia polymorpha explants, showing their capacity to release oxygen under different illumination settings and temperatures. Moreover, co-culture experiments revealed that the presence of these explants had no adverse effects on the viability and morphology of fibroblasts in vitro, nor on the viability of zebrafish larvae in vivo. Furthermore, oxygraphy assays demonstrate that these explants could fulfill the oxygen metabolic requirements of zebrafish larvae and freshly isolated skin biopsies ex vivo. Finally, the biocompatibility of explants was confirmed through a human skin irritation test conducted in healthy volunteers following the ISO-10993-10-2010. This proof-of-concept study provides valuable scientific insights, proposing the potential use of freshly isolated plants as biocompatible low-cost oxygen delivery systems for wound healing and tissue regeneration., (© 2024 Wiley Periodicals LLC.)

3D bioprinting technology has emerged as a tool that promises to revolutionize the biomedical field, including tissue engineering and regeneration. Despite major technological advancements, several challenges remain to be solved before 3D bioprinted tissues could be fully translated from the bench to the bedside. As oxygen plays a key role in aerobic metabolism, which allows energy production in the mitochondria; as a consequence, the lack of tissue oxygenation is one of the main limitations of current bioprinted tissues and organs. In order to improve tissue oxygenation, recent approaches have been established for a broad range of clinical applications, with some already applied using 3D bioprinting technologies. Among them, the incorporation of photosynthetic microorganisms, such as microalgae and cyanobacteria, is a promising approach that has been recently explored to generate chimerical plant-animal tissues where, upon light exposure, oxygen can be produced and released in a localized and controlled manner. This review will briefly summarize the state-of-the-art approaches to improve tissue oxygenation, as well as studies describing the use of photosynthetic microorganisms in 3D bioprinting technologies. STATEMENT OF SIGNIFICANCE: 3D bioprinting technology has emerged as a tool for the generation of viable and functional tissues for direct in vitro and in vivo applications, including disease modeling, drug discovery and regenerative medicine. Despite the latest advancements in this field, suboptimal oxygen delivery to cells before, during and after the bioprinting process limits their viability within 3D bioprinted tissues. This review article first highlights state-of-the-art approaches used to improve oxygen delivery in bioengineered tissues to overcome this challenge. Then, it focuses on the emerging roles played by photosynthetic organisms as novel biomaterials for bioink generation. Finally, it provides considerations around current challenges and novel potential opportunities for their use in bioinks, by comparing latest published studies using algae for 3D bioprinting., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

As animal cells cannot produce oxygen, erythrocytes are responsible for gas interchange, being able to capture and deliver oxygen upon tissue request. Interestingly, several other cells in nature produce oxygen by photosynthesis, raising the question of whether they could circulate within the vascular networks, acting as an alternative source for oxygen delivery. To address this long-term goal, here some physical and mechanical features of the photosynthetic microalga Chlamydomona reinhardtii were studied and compared with erythrocytes, revealing that both exhibit similar size and rheological properties. Moreover, key biocompatibility aspects of the microalgae were evaluated in vitro and in vivo, showing that C. reinhardtii can be co-cultured with endothelial cells, without affecting each other's morphology and viability. Moreover, short-term systemic perfusion of the microalgae showed a thoroughly intravascular distribution in mice. Finally, the systemic injection of high numbers of microalgae did not trigger deleterious responses in living mice. Altogether, this work provides key scientific insights to support the notion that photosynthetic oxygenation could be achieved by circulating microalgae, representing another important step towards human photosynthesis. KEY POINTS: • C. reinhardtii and endothelial cells are biocompatible in vitro. • C. reinhardtii distribute throughout the entire vasculature after mice perfusion. • C. reinhardtii do not trigger deleterious responses after injection in mice., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tissue regeneration capabilities vary significantly throughout an organism's lifespan. For example, mammals can fully regenerate until they reach specific developmental stages, after which they can only repair the tissue without restoring its original architecture and function. The high regenerative potential of fetal stages has been attributed to various factors, such as stem cells, the immune system, specific growth factors, and the presence of extracellular matrix molecules upon damage. To better understand the local differences between regenerative and reparative tissues, we conducted a comparative analysis of skin derived from mice at regenerative and reparative stages. Our findings show that both types of skin differ in their molecular composition, structure, and functionality. We observed a significant increase in cellular density, nucleic acid content, neutral lipid density, Collagen III, and glycosaminoglycans in regenerative skin compared with reparative skin. Additionally, regenerative skin had significantly higher porosity, metabolic activity, water absorption capacity, and elasticity than reparative skin. Finally, our results also revealed significant differences in lipid distribution, extracellular matrix pore size, and proteoglycans between the two groups. This study provides comprehensive data on the molecular and structural clues that enable full tissue regeneration in fetal stages, which could aid in developing new biomaterials and strategies for tissue engineering and regeneration.

The development of biomaterials to improve wound healing is a critical clinical challenge and an active field of research. As it is well described that oxygen plays a critical role in almost each step of the wound healing process, in this work, an oxygen producing photosynthetic biomaterial was generated, characterized, and further modified to additionally release other bioactive molecules. Here, alginate hydrogels were loaded with the photosynthetic microalgae Chlamydomonas reinhardtii, showing high integration as well as immediate oxygen release upon illumination. Moreover, the photosynthetic hydrogel showed high biocompatibility in vitro and in vivo, and the capacity to sustain the metabolic oxygen requirements of zebrafish larvae and skin explants. In addition, the photosynthetic dressings were evaluated in 20 healthy human volunteers following the ISO-10993-10-2010 showing no skin irritation, mechanical stability of the dressings, and survival of the photosynthetic microalgae. Finally, hydrogels were also loaded with genetically engineered microalgae to release human VEGF, or pre-loaded with antibiotics, showing sustained release of both bioactive molecules. Overall, this work shows that photosynthetic hydrogels represent a feasible approach for the local delivery of oxygen and other bioactive molecules to promote wound healing. STATEMENT OF SIGNIFICANCE: As oxygen plays a key role in almost every step of the tissue regeneration process, the development of oxygen delivering therapies represents an active field of research, where photosynthetic biomaterials have risen as a promising approach for wound healing. Therefore, in this work a photosynthetic alginate hydrogel-based wound dressing containing C. reinhardtii microalgae was developed and validated in healthy skin of human volunteers. Moreover, hydrogels were modified to additionally release other bioactive molecules such as recombinant VEGF or antibiotics. The present study provides key scientific data to support the use of photosynthetic hydrogels as customizable dressings to promote wound healing., Competing Interests: Declaration of Competing Interest JTE is CSO and co-founder of SymbiOx Inc., a start-up company that owns IP in the field of this work. During the conduct of this project, RC-O and CV were employees of SymbiOx Inc. All other authors declare that they have no competing interests., (Copyright © 2022 Acta Materialia Inc. All rights reserved.)

Chronic wounds cannot heal due to impairment of regeneration, mainly caused by the persistent infection of multispecies biofilms. Still, the effects of biofilm wound infection and its interaction with the host are not fully described. We aimed to study functional biofilms in physiological conditions in vitro, and their potential effects in health and regeneration in vivo. Therefore, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis were seeded in collagen-based scaffolds for dermal regeneration. After 24 h, scaffolds had bacterial loads depending on the initial inoculum, containing viable biofilms with antibiotic tolerance. Afterwards, scaffolds were implanted onto full skin wounds in mice, together with daily supervision and antibiotic treatment. Although all mice survived their health was affected, displaying fever and weight loss. After ten days, histomorphology of scaffolds showed high heterogeneity in samples and within groups. Wounds were strongly, mildly, or not infected according to colony forming units, and P. aeruginosa had higher identification frequency. Biofilm infection induced leucocyte infiltration and elevated interferon-γ and interleukin-10 in scaffolds, increase of size and weight of spleen and high systemic pro-calcitonin concentrations. This functional and implantable 3D biofilm model allows to study host response during infection, providing a useful tool for infected wounds therapy development., (© 2022. The Author(s).)

As hypoxic tumors show resistance to several clinical treatments, photosynthetic microorganisms have been recently suggested as a promising safe alternative for oxygenating the tumor microenvironment. The relationship between organisms and the effect microalgae have on tumors is still largely unknown, evidencing the need for a simple yet representative model for studying photosynthetic tumor oxygenation in a reproducible manner. Here, we present a 3D photosynthetic tumor model composed of human melanoma cells and the microalgae Chlamydomonas reinhardtii , both seeded into a collagen scaffold, which allows for the simultaneous study of both cell types. This work focuses on the biocompatibility and cellular interactions of the two cell types, as well as the study of photosynthetic oxygenation of the tumor cells. It is shown that both cell types are biocompatible with one another at cell culture conditions and that a 10:1 ratio of microalgae to cells meets the metabolic requirement of the tumor cells, producing over twice the required amount of oxygen. This 3D tumor model provides an easy-to-use in vitro resource for analyzing the effects of photosynthetically produced oxygen on a tumor microenvironment, thus opening various potential research avenues.

Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real-time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration in vitro and in vivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein (rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed in vitro and in vivo. Expression of 16S was significantly upregulated in scaffolds in vitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to in vitro (16S: in vivo Ct value 13.21 ± 0.32, in vitro 12.44 ± 0.42; rnpA in vivo Ct value 19.87 ± 0.41, in vitro 17.75 ± 1.41). Overall, the results demonstrate rnpA and 16S expression after 7 days of implantation in vitro and in vivo, proving the presence of living bacteria embedded in scaffolds using qPCR., (© 2022 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Oxygen is the key molecule for aerobic metabolism, but no animal cells can produce it, creating an extreme dependency on external supply. In contrast, microalgae are photosynthetic microorganisms, therefore, they are able to produce oxygen as plant cells do. As hypoxia is one of the main issues in organ transplantation, especially during preservation, the main goal of this work was to develop the first generation of perfusable photosynthetic solutions, exploring its feasibility for ex vivo organ preservation. Here, the microalgae Chlamydomonas reinhardtii was incorporated in a standard preservation solution, and key aspects such as alterations in cell size, oxygen production and survival were studied. Osmolarity and rheological features of the photosynthetic solution were comparable to human blood. In terms of functionality, the photosynthetic solution proved to be not harmful and to provide sufficient oxygen to support the metabolic requirement of zebrafish larvae and rat kidney slices. Thereafter, isolated porcine kidneys were perfused, and microalgae reached all renal vasculature, without inducing damage. After perfusion and flushing, no signs of tissue damage were detected, and recovered microalgae survived the process. Altogether, this work proposes the use of photosynthetic microorganisms as vascular oxygen factories to generate and deliver oxygen in isolated organs, representing a novel and promising strategy for organ preservation., Competing Interests: Author CG was employed by company Sky-Walker SpA. Competing Interests: JTE is CSO and co-founder of SymbiOx Inc., a start-up company that owns IP in the field of this work. Thanks to an R and D grant provided by the Chilean Ministry of Economics (CORFO), during the conduct of this project, DN, RE and RC-O were full-time employees of SymbiOx Inc. All other authors declare that they have no competing interests., (Copyright © 2021 Veloso-Giménez, Escamilla, Necuñir, Corrales-Orovio, Riveros, Marino, Ehrenfeld, Guzmán, Boric, Rebolledo and Egaña.)

Insufficient oxygen supply represents a relevant issue in several fields of human physiology and medicine. It has been suggested that the implantation of photosynthetic cells can provide oxygen to tissues in the absence of a vascular supply. This approach has been demonstrated to be successful in several in vitro and in vivo models; however, no data is available about their safety in human patients. Here, an early phase-1 clinical trial (ClinicalTrials.gov identifier: NCT03960164, https://clinicaltrials.gov/ct2/show/NCT03960164) is presented to evaluate the safety and feasibility of implanting photosynthetic scaffolds for dermal regeneration in eight patients with full-thickness skin wounds. Overall, this trial shows that the presence of the photosynthetic microalgae Chlamydomonas reinhardtii in the implanted scaffolds did not trigger any deleterious local or systemic immune responses in a 90 days follow-up, allowing full tissue regeneration in humans. The results presented here represent the first attempt to treat patients with photosynthetic cells, supporting the translation of photosynthetic therapies into clinics. Clinical Trial Registration: www.clinicaltrials.gov/ct2/show/NCT03960164, identifier: NCT03960164., Competing Interests: JTE is co-founder of SymbiOx Inc., a startup company that owns IP for the technology described here. During the conduct of the trial, MB, RC-O and FC were full-time employees of SymbiOx Inc., while AE-Z, XM and CW were part-time employees. All SymbiOx team members were financed with a R&D grant obtained from the Chilean Ministry of Economics (CORFO 18PIDE98887). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Obaíd, Camacho, Brenet, Corrales-Orovio, Carvajal, Martorell, Werner, Simón, Varas, Calderón, Guzmán, Bono, San Martín, Eblen-Zajjur and Egaña.)

Impaired wound healing represents an unsolved medical need with a high impact on patients´ quality of life and global health care. Even though its causes are diverse, ischemic-hypoxic conditions and exacerbated inflammation are shared pathological features responsible for obstructing tissue restoration. In line with this, it has been suggested that promoting a normoxic pro-regenerative environment and accelerating inflammation resolution, by reinstating the lymphatic fluid transport, could allow the wound healing process to be resumed. Our group was first to demonstrate the functional use of scaffolds seeded with photosynthetic microorganisms to supply tissues with oxygen. Moreover, we previously proposed a photosynthetic gene therapy strategy to create scaffolds that deliver other therapeutic molecules, such as recombinant human growth factors into the wound area. In the present work, we introduce the use of transgenic Synechococcus sp. PCC 7002 cyanobacteria (SynHA), which can produce oxygen and lymphangiogenic hyaluronic acid, in photosynthetic biomaterials. We show that the co-culture of lymphatic endothelial cells with SynHA promotes their survival and proliferation under hypoxic conditions. Also, hyaluronic acid secreted by the cyanobacteria enhanced their lymphangiogenic potential as shown by changes to their gene expression profile, the presence of lymphangiogenic protein markers and their capacity to build lymph vessel tubes. Finally, by seeding SynHA into collagen-based dermal regeneration materials, we developed a viable photosynthetic scaffold that promotes lymphangiogenesis in vitro under hypoxic conditions. The results obtained in this study lay the groundwork for future tissue engineering applications using transgenic cyanobacteria that could become a therapeutic alternative for chronic wound treatment. STATEMENT OF SIGNIFICANCE: In this study, we introduce the use of transgenic Synechococcus sp. PCC 7002 (SynHA) cyanobacteria, which were genetically engineered to produce hyaluronic acid, to create lymphangiogenic photosynthetic scaffolds for dermal regeneration. Our results confirmed that SynHA cyanobacteria maintain their photosynthetic capacity under standard human cell culture conditions and efficiently proliferate when seeded inside fibrin-collagen scaffolds. Moreover, we show that SynHA supported the viability of co-cultured lymphatic endothelial cells (LECs) under hypoxic conditions by providing them with photosynthetic-derived oxygen, while cyanobacteria-derived hyaluronic acid stimulated the lymphangiogenic capacity of LECs. Since tissue hypoxia and impaired lymphatic drainage are two key factors that directly affect wound healing, our results suggest that lymphangiogenic photosynthetic biomaterials could become a treatment option for chronic wound management., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Without the sustained provision of adequate levels of oxygen by the cardiovascular system, the tissues of higher animals are incapable of maintaining normal metabolic activity, and hence cannot survive. The consequence of this evolutionarily suboptimal design is that humans are dependent on cardiovascular perfusion, and therefore highly susceptible to alterations in its normal function. However, hope may be at hand. "Photosynthetic strategies," based on the recognition that photosynthesis is the source of all oxygen, offer a revolutionary and promising solution to pathologies related to tissue hypoxia. These approaches, which have been under development over the past 20 years, seek to harness photosynthetic microorganisms as a local and controllable source of oxygen to circumvent the need for blood perfusion to sustain tissue survival. To date, their applications extend from the in vitro creation of artificial human tissues to the photosynthetic maintenance of oxygen-deprived organs both in vivo and ex vivo , while their potential use in other medical approaches has just begun to be explored. This review provides an overview of the state of the art of photosynthetic technologies and its innovative applications, as well as an expert assessment of the major challenges and how they can be addressed., (Copyright © 2020 Chávez, Moellhoff, Schenck, Egaña and Nickelsen.)

After skin tissue injury or pathological removal, vascularization timing is paramount in graft survival. As full thickness skin grafts often fail to become perfused over larger surfaces, split-thickness grafts are preferred and can be used together with biomaterials, which themselves are non-angiogenic. One way of promoting vascular ingrowth is to "pre-vascularize" an engineered substitute by introducing endothelial cells (ECs). Since it has been previously demonstrated that surface structured biomaterials have an effect on wound healing, skin regeneration, and fibrosis reduction, we proposed that a microvascular-rich lipoconstruct with anisotropic topographical cues could be a clinically translatable vascularization approach. Murine lipofragments were formed with three polydimethylsiloxane molds (flat, 5 µm, and 50 µm parallel gratings) and implanted into the dorsal skinfold chamber of male C57BL/6 mice. Vascular ingrowth was observed through intravital microscopy over 21 days and further assessed by histology and protein identification. Our investigation revealed that topographical feature size influenced the commencement of neovascular ingrowth, with 5 µm gratings exhibiting early construct perfusion at 3 days post-operation, and 50 µm being delayed until day 5. We therefore postulate that surface structured lipoconstructs may serve as an easily obtained and produced construct suitable for providing soft tissue and ECs to tissue defects. STATEMENT OF SIGNIFICANCE: Skin graft failures due to inadequate or uneven perfusion frequently occur and can be even more complicated in deep, difficult to heal wounds, or bone coverage. In complex injuries, biomaterials are often used to cover bone structures with a standard split thickness graft; however, perfusion can take up to 3 weeks. Thus, any means to promote faster and uniform vascularization could significantly reduce healing time, as well as lower patient down-time. As pre-vascularized constructs have reported success in research, we created a cost-efficient, translatable method with no additional laboratory time as adipose tissue can be harvested and used immediately. We further used surface topography as an aspect to modulate construct perfusion, which has been reported for the first time here., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

The study of myocardial transmembrane ion currents is fundamental to understand frequent pathologies such as arrhythmias and ischemia. Conventional electrocardiography (ECG) is not able to record ion currents, while the use of intracellular microelectrodes in a beating heart has technical limitations. Myocardial monophasic action potentials (MAPs) recorded with suction electrodes allow the evaluation of ionic currents similar to those recorded by intracellular glass microelectrodes. The technique is based on the fact that suction, through a small diameter tube, on the myocardial cell, induces an opening at the membrane, connecting the intracellular media to the electrode by a saline bridge. The electrophysiology of zebrafish heart is remarkably similar to the human; however, in situ evaluation of MAPs has not been yet explored. In this study, we aimed to establish a myocardial MAP recording technique for adult zebrafish. Male adult wild-type zebrafish were anesthetized and 50% of the beating ventricle was exposed. A glass hematocrit capillary tube (1.1 mm inner diameter) was used as a suction electrode connected to a 3-way stopcock valve, which is also connected to a syringe containing a chloride-coated silver wire for signal recording. Gentle suction was exerted by a syringe filled with ringer and connected to the 3-way stopcock valve. Two needles were used for ground (tail) and indifferent (abdomen) electrodes. Without suction, the system can record conventional ECG, but applying suction MAPs are registered and show typical morphology with phase 0-4 sequence. MAP amplitude and duration values show low variability. Ischemia and/or lidocaine-induced Na + channel blocking dramatically reduced MAP amplitude. These results strongly suggest that the suction electrode technique is a promising method to record myocardial ion currents in situ in zebrafish.

The World Health Organization has estimated that, worldwide, cigarette smoking has caused more than 100 million deaths in the last century, a number that is expected to increase in the future. Understanding cigarette smoke toxicity is key for research and development of proper public health policies. The current challenge is to establish a reliable preclinical model to evaluate the effects of cigarette smoke. In this work, we describe a simple method that allows for quantifying the toxic effects of cigarette smoke using zebrafish. Here, viability of larvae and adult fish, as well as the effects of cigarette smoke extracts on vascular development and tissue regeneration, can be easily assayed. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

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., (Copyright © 2019 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier Ltd.)

Cigarette smoke is associated with several pathologies including chronic respiratory diseases and cancer. In addition, exposure to cigarette smoke is correlated with impaired wound healing, where a significant decrease in the regenerative capacity of smokers is well documented and broadly considered a negative risk factor after trauma or surgery. So far, some in vitro and in vivo models have been described to study how exposure to cigarette smoke diminishes the regenerative potential in different organisms. However, although useful, many of these models are difficult and expensive to implement and do not allow high-throughput screening approaches. In order to establish a reliable and accessible model, we have evaluated the effects of cigarette smoke extract (CSE) on zebrafish development and regeneration. In this work, zebrafish embryos and larvae were exposed to low doses of aqueous CSE showing severe developmental abnormalities in a dose-dependent manner. Furthermore, when adult zebrafish were subjected to caudal fin amputation, we observed a significant decrease in the regenerative capacity of animals exposed to CSE. The effect was exacerbated in male and aged fish compared to female or young organisms. The establishment of a zebrafish model to assess the consequences of cigarette smoke and its effects on animal physiology could provide a new tool to study the underlying mechanisms involved in impaired tissue regeneration, and aid the development of novel approaches to treat complications associated with cigarette smoke toxicity.

Mesenchymal stem cells (MSCs) are known to play a key role in tissue regeneration, while smoking cigarettes is described to impair it. This work focuses on the effect cigarette smoke extract (CSE) has on the migration, differentiation, and paracrine potential of human adipose derived MSCs (AdMSCs). To mimic native conditions in vitro, AdMSCs were cultured in either monolayer or three-dimensional pellet cultures. While constant exposure to high concentrations of CSE had lethal effects on AdMSCs, lower concentrations of CSE impaired cell migration when compared to control conditions. The secretion of key interleukins was downregulated when CSE was exposed to the cells at low concentrations. Moreover, in this work AdMSCs were exposed to CSE while simultaneously being induced to differentiate into adipocytes, osteoblasts, and chondrocytes to determine the effect of CSE on the cells potential to differentiate. While adipogenic differentiation showed no significant variation, AdMSCs exposed to osteogenic and chondrogenic supplements showed both early and late genetic level variation when acutely exposed to low concentrations of CSE. Our results indicate that even a small amount of cigarette smoke can have detrimental effects on the regenerative potential of MSCs.

Angiogenesis is the process through which new blood vessels are formed from preexisting ones and plays a critical role in several conditions including embryonic development, tissue repair and disease. Moreover, enhanced therapeutic angiogenesis is a major goal in the field of regenerative medicine and efficient vascularization of artificial tissues and organs is one of the main hindrances in the implementation of tissue engineering approaches, while, on the other hand, inhibition of angiogenesis is a key therapeutic target to inhibit for instance tumor growth. During the last decades, the understanding of cellular and molecular mechanisms involved in this process has been matter of intense research. In this regard, several in vitro and in vivo models have been established to visualize and study migration of endothelial progenitor cells, formation of endothelial tubules and the generation of new vascular networks, while assessing the conditions and treatments that either promote or inhibit such processes. In this review, we address and compare the most commonly used experimental models to study angiogenesis in vitro and in vivo. In particular, we focus on the implementation of the zebrafish (Danio rerio) as a model to study angiogenesis and discuss the advantages and not yet explored possibilities of its use as model organism.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many therapies using mesenchymal stem cells (MSC) rely on their ability to produce and release paracrine signals with chemotactic and pro-angiogenic activity. These characteristics, however, are mostly studied under standard in vitro culture conditions. In contrast, various novel cell-based therapies imply pre-seeding MSC into bio-artificial scaffolds. Here we describe human bone marrow-derived MSC seeded in Integra matrices, a common type of scaffold for dermal regeneration (SDR). We show and measured the distribution of MSC within the SDR, where cells clearly establish physical interactions with the scaffold, exhibiting constant metabolic activity for at least 15 days. In the SDR, MSC secrete VEGF and SDF-1α and induce transwell migration of CD34(+) hematopoietic/endothelial progenitor cells, which is inhibited in the presence of a CXCR4/SDF-1α antagonist. MSC in SDR respond to hypoxia by altering levels of angiogenic signals such as Angiogenin, Serpin-1, uPA, and IL-8. Finally, we show that MSC-containing SDR that have been pre-incubated in hypoxia show higher infiltration of endothelial cells after implantation into immune deficient mice. Our data show that MSC are fully functional ex vivo when implanted into SDR. In addition, our results strongly support the notion of hypoxic pre-conditioning MSC-containing SDR, in order to promote angiogenesis in the wounds.

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.

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., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

Disorders in skin wound healing are a major health problem that requires the development of innovative treatments. The use of biomaterials as an alternative of skin replacement has become relevant, but its use is still limited due to poor vascularization inside the scaffolds, resulting in insufficient oxygen and growth factors at the wound site. In this study, we have developed a cell-based wound therapy consisting of the application of collagen-based dermal scaffolds containing mesenchymal stem cells from Wharton's jelly (WJ-MSC) in an immunocompetent mouse model of angiogenesis. From our comparative study on the secretion profile between WJ-MSC and adipose tissue-derived MSC, we found a stronger expression of several well-characterized growth factors, such as VEGF-A, angiopoietin-1 and aFGF, which are directly linked to angiogenesis, in the culture supernatant of WJ-MSC, both on monolayer and 3D culture conditions. WJ-MSC proved to be angiogenic both in vitro and in vivo, through tubule formation and CAM assays, respectively. Moreover, WJ-MSC consistently improved the healing response in vivo in a mouse model of human-like dermal repair, by triggering angiogenesis and further providing a suitable matrix for wound repair, without altering the inflammatory response in the animals. Since these cells can be easily isolated, cultured with high expansion rates and cryopreserved, they represent an attractive stem cell source for their use in allogeneic cell transplant and tissue engineering.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one's own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

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., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Delayed wound healing and scar formation are among the most frequent complications after surgical interventions. Although biodegradable surgical sutures present an excellent drug delivery opportunity, their primary function is tissue fixation. Mesenchymal stem cells (MSC) act as trophic mediators and are successful in activating biomaterials. Here biodegradable sutures were filled with adipose-derived mesenchymal stem cells (ASC) to provide a pro-regenerative environment at the injured site. Results showed that after filling, ASCs attach to the suture material, distribute equally throughout the filaments, and remain viable in the suture. Among a broad panel of cytokines, cell-filled sutures constantly release vascular endothelial growth factor to supernatants. Such conditioned media was evaluated in an in vitro wound healing assay and showed a significant decrease in the open wound area compared to controls. After suturing in an ex vivo wound model, cells remained in the suture and maintained their metabolic activity. Furthermore, cell-filled sutures can be cryopreserved without losing their viability. This study presents an innovative approach to equip surgical sutures with pro-regenerative features and allows the treatment and fixation of wounds in one step, therefore representing a promising tool to promote wound healing after injury.

Objective: Recent findings have attested to EPO tissue-protective effects in ischemically challenged tissues. Therefore, the study aimed at elaborating the effect of systemic pre- and postconditioning using EPO in a mouse model of persistent ischemia of the skin., Methods: Three groups of nine C57Bl/6-mice each were analyzed. The experimental groups consisted of untreated controls, EPO preconditioning, and EPO postconditioning (500 IU EPO/kg bw/day for 10 days). Critically perfused skin flaps undergoing necrosis, if kept untreated, were mounted into dorsal skinfold chambers. Intravital epi-fluorescence microscopy was performed for 10 days to assess tissue necrosis, microcirculation, inflammation, and angiogenesis. Protein expression analysis of eNOS was performed. Hematocrit analyses were carried out separately in eight animals., Results: Only EPO preconditioning was able to significantly reduce necrosis, when compared with controls. This correlated with a significantly increased CD in the critically perfused tissue. Administration of EPO only slightly increased eNOS expression at day 10, when compared with controls. EPO induced angiogenesis and increased hematocrit. Finally, EPO significantly reduced leukocytic inflammation in arterioles in all EPO receiving mice., Conclusions: EPO preconditioning effectively reduces skin necrosis predominantly by capillary maintenance and reperfusion, as well as improved tissue regeneration. Thus, EPO preconditioning might represent a promising, non-invasive approach to reduce complications in ischemically challenged skin., (© 2013 John Wiley & Sons Ltd.)

The application of scaffolds in bone tissue engineering often comes along with side effects such as poor integrity, low regeneration rates of bone tissue with inadequate functionality, and, in case of non-degradable implants, the necessity of a second removal surgery after therapy. In this study, we coated a bioresorbable FDA-approved poly-(ε-caprolactone)-scaffold for bone regeneration with a poly-(D,L-lactide) layer containing copolymer-protected gene vectors to locally provide bone morphogenetic protein-2 (BMP-2). Results show that the presence of such gene vectors did not affect the distribution and attachment of seeded cells on gene-activated surfaces. BMP-2 was released into cell culture supernatants and furthermore detected in homogenised scaffolds. Increased amounts of osteoblastic markers, such as osteocalcin, osteopontin and the activity of alkaline phosphatase, in gene-activated scaffolds in vitro suggest a transdifferentiation of myoblastic C2C12 cells into the osteoblastic phenotype. With this study we present a new technology to bioactivate implant surfaces with non-viral gene vectors. This tool allows the stimulation of tissue regeneration by a local release of therapeutic proteins in vivo.

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.

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.

Purpose: Recent reports discuss the altered bone homeostasis in cigarette smokers, being a risk factor for osteoporosis and negatively influencing fracture healing. Cigarette smoke is known to induce oxidative stress in the body via an increased production of reactive oxygen species (ROS). These increases in ROS are thought to damage the bone-forming osteoblasts. Naturally occurring polyphenols contained in green tea extract (GTE), e.g., catechins, are known to have anti-oxidative properties. Therefore, the aim of this study was to investigate whether GTE and especially catechins protect primary human osteoblasts from cigarette smoke-induced damage and to identify the underlying mechanisms., Methods: Primary human osteoblasts were isolated from patients' femur heads. Cigarette smoke medium (CSM) was obtained using a gas-washing bottle and standardized by its optical density (OD(320)) at λ = 320 nm. ROS formation was measured using 2'7'dichlorofluorescein diacetate, and osteoblasts' viability was detected by resazurin conversion., Results: Co-, pre-, and post-incubation with GTE and catechins significantly reduced ROS formation and thus improved the viability of CSM-treated osteoblasts. Besides GTE's direct radical scavenging properties, pre-incubation with both GTE and catechins protected osteoblasts from CSM-induced damage. Inhibition of the anti-oxidative enzyme HO-1 significantly reduced the protective effect of GTE and catechins emphasizing the key role of this enzyme in GTE anti-oxidative effect., Conclusions: Our data suggest possible beneficial effects on bone homeostasis, fracture healing, and bone mineral density following a GTE-rich diet or supplementation.

The clinical utilization of resorbable bone substitutes has been growing rapidly during the last decade, creating a rising demand for new resorbable biomaterials. An ideal resorbable bone substitute should not only function as a load-bearing material but also integrate into the local bone remodeling process. This means that these bone substitutes need to undergo controlled resorption and then be replaced by newly formed bone structures. Thus the assessment of resorbability is an important first step in predicting the in vivo clinical function of bone substitute biomaterials. Compared with in vivo assays, cell-based assays are relatively easy, reproducible, inexpensive and do not involve the suffering of animals. Moreover, the discovery of RANKL and M-CSF for osteoclastic differentiation has made the differentiation and cultivation of human osteoclasts possible and, as a result, human cell-based bone substitute resorption assays have been developed. In addition, the evolution of microscopy technology allows advanced analyses of the resorption pits on biomaterials. The aim of the current review is to give a concise update on in vitro cell-based resorption assays for analyzing bone substitute resorption. For this purpose models using different cells from different species are compared. Several popular two-dimensional and three-dimensional optical methods used for resorption assays are described. The limitations and advantages of the current ISO degradation assay in comparison with cell-based assays are discussed., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy., Methods and Results: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed., Conclusions: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)