Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Unraveling Muscle Impairment Associated With COVID-19 and the Role of 3D Culture in Its Investigation.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been considered a public health emergency, extensively investigated by researchers. Accordingly, the respiratory tract has been the main research focus, with some other studies outlining the effects on the neurological, cardiovascular, and renal systems. However, concerning SARS-CoV-2 outcomes on skeletal muscle, scientific evidence is still not sufficiently strong to trace, treat and prevent possible muscle impairment due to the COVID-19. Simultaneously, there has been a considerable amount of studies reporting skeletal muscle damage in the context of COVID-19. Among the detrimental musculoskeletal conditions associated with the viral infection, the most commonly described are sarcopenia, cachexia, myalgia, myositis, rhabdomyolysis, atrophy, peripheral neuropathy, and Guillain-Barré Syndrome. Of note, the risk of developing sarcopenia during or after COVID-19 is relatively high, which poses special importance to the condition amid the SARS-CoV-2 infection. The yet uncovered mechanisms by which musculoskeletal injury takes place in COVID-19 and the lack of published methods tailored to study the correlation between COVID-19 and skeletal muscle hinder the ability of healthcare professionals to provide SARS-CoV-2 infected patients with an adequate treatment plan. The present review aims to minimize this burden by both thoroughly exploring the interaction between COVID-19 and the musculoskeletal system and examining the cutting-edge 3D cell culture techniques capable of revolutionizing the study of muscle dynamics.

Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

Tuning cytokines enriches dendritic cells and regulatory T cells in the periodontium.

BACKGROUND: Periodontal disease results from the pathogenic interactions between the tissue, immune system, and microbiota; however, standard therapy fails to address the cellular mechanism underlying the chronic inflammation. Dendritic cells (DC) are key regulators of T cell fate, and biomaterials that recruit and program DC locally can direct T cell effector responses. We hypothesized that a biomaterial that recruited and programmed DC toward a tolerogenic phenotype could enrich regulatory T cells within periodontal tissue, with the eventual goal of attenuating T cell mediated pathology. METHODS: The interaction of previously identified factors that could induce tolerance, granulocyte-macrophage colony stimulating factor (GM-CSF) and thymic stromal lymphopoietin (TSLP), with the periodontitis network was confirmed in silico. The effect of the cytokines on DC migration was explored in vitro using time-lapse imaging. Finally, regulatory T cell enrichment in the dermis and periodontal tissue in response to alginate hydrogels delivering TSLP and GM-CSF was examinedin vivo in mice using immunohistochemistry and live-animal imaging. RESULTS: The GM-CSF and TSLP interactome connects to the periodontitis network. GM-CSF enhances DC migration in vitro. An intradermal injection of an alginate hydrogel releasing GM-CSF enhanced DC numbers and the addition of TSLP enriched FOXP3+ regulatory T cells locally. Injection of a hydrogel with GM-CSF and TSLP into the periodontal tissue in mice increased DC and FOXP3+ cell numbers in the tissue, FOXP3+ cells in the lymph node, and IL-10 in the tissue. CONCLUSION: Local biomaterial-mediated delivery of GM-CSF and TSLP can enrich DC and FOXP3+ cells and holds promise for treating the pathologic inflammation of periodontal disease.

PRP and BMAC for Musculoskeletal Conditions via Biomaterial Carriers.

Platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) are orthobiologic therapies considered as an alternative to the current therapies for muscle, bone and cartilage. Different formulations of biomaterials have been used as carriers for PRP and BMAC in order to increase regenerative processes. The most common biomaterials utilized in conjunction with PRP and BMAC clinical trials are organic scaffolds and natural or synthetic polymers. This review will cover the combinatorial strategies of biomaterial carriers with PRP and BMAC for musculoskeletal conditions (MsCs) repair and regeneration in clinical trials. The main objective is to review the therapeutic use of PRP and BMAC as a treatment option for muscle, bone and cartilage injuries.

Characterizing the encapsulation and release of lentivectors and adeno-associated vectors from degradable alginate hydrogels.

Gene therapy using viral vectors has been licensed for clinical use both in the European Union and the United States. Lentivectors (LV) and adeno-associated vectors (AAV) are two promising and FDA approved gene-therapy viral vectors. Many future applications of these vectors will benefit from targeting specific regions of interest within the body. Therefore, building on the early success of these vectors may depend on finding effective delivery systems to localize therapeutic administration. Degradable alginate hydrogels have been tested as appealing delivery vehicles for the controlled delivery of vector payloads. In this study, we compare the ability of two different degradable alginate hydrogel formulations to efficiently deliver LV and AAV. We propose that release rates of viral vectors are dependent on the physical properties of both the hydrogels and vectors. Here, we demonstrate that the initial strength and degradation rate of alginate hydrogels provides levers of control for tuning LV release but do not provide control in the release of AAV. While both alginate formulations used showed sustained release of both LV and AAV, LV release was shown to be dependent on alginate hydrogel degradation, while AAV release was largely governed by diffusive mechanisms. Altogether, this study demonstrates alginate's use as a possible delivery platform for LV and, for the first time, AAV - highlighting the potential of injectable degradable alginate hydrogels to be used as a versatile delivery tool in gene therapy applications.

Heavy Metals of Santiago Island (Cape Verde) Alluvial Deposits: Baseline Value Maps and Human Health Risk Assessment.

The chemical composition of surface geological materials may cause metabolic changes and promote endemic diseases (e.g., oncological, gastrointestinal, neurological or cardiovascular diseases). The results of a geochemical survey is presented following the guidelines proposed by the International Project IGCP 259 performed on the alluvium of Santiago Island (Cape Verde) and focused on public health issues. Geochemical mapping is the base knowledge needed to determine critical contents of potential toxic elements and the potentially harmful regions in the planet. This work presents maps of baseline values of potentially toxic elements (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, V, and Zn) in Santiago alluvium and the assessment of their human health risks. According to the results the Cd, Co, Cr, Ni and V baseline values are above the Canadian guidelines for stream sediments (for any proposal use) and for soils (for agricultural and residential proposal uses) and also above the target values of Dutch guidelines. Hazard indexes (HI) were calculated for children and adults. For children (HI) are higher than 1 for Co, Cr and Mn, indicating potential non-carcinogenic risk. For the other elements and for adults there is no potential non-carcinogenic risk. Cancer risk was calculated for Cd, Cr and Ni exposures, for adults and children, and the results are only slightly higher than the carcinogenic target risk of 1 × 10-6 for adults exposed to Cr by inhalation. However, these results may be underestimated because alluvial contaminants may be indirectly ingested by groundwater and by crop and vegetables consumption.

Microgels produced using microfluidic on-chip polymer blending for controlled released of VEGF encoding lentivectors.

Alginate hydrogels are widely used as delivery vehicles due to their ability to encapsulate and release a wide range of cargos in a gentle and biocompatible manner. The release of encapsulated therapeutic cargos can be promoted or stunted by adjusting the hydrogel physiochemical properties. However, the release from such systems is often skewed towards burst-release or lengthy retention. To address this, we hypothesized that the overall magnitude of burst release could be adjusted by combining microgels with distinct properties and release behavior. Microgel suspensions were generated using a process we have termed on-chip polymer blending to yield composite suspensions of a range of microgel formulations. In this manner, we studied how alginate percentage and degradation relate to the release of lentivectors. Whereas changes in alginate percentage had a minimal impact on lentivector release, microgel degradation led to a 3-fold increase, and near complete release, over 10 days. Furthermore, by controlling the amount of degradable alginate present within microgels the relative rate of release can be adjusted. A degradable formulation of microgels was used to deliver vascular endothelial growth factor (VEGF)-encoding lentivectors in the chick chorioallantoic membrane (CAM) assay and yielded a proangiogenic response in comparison to the same lentivectors delivered in suspension. The utility of blended microgel suspensions may provide an especially appealing platform for the delivery of lentivectors or similarly sized therapeutics. STATEMENT OF SIGNIFICANCE: Genetic therapeutics hold considerable potential for the treatment of diseases and disorders including ischemic cardiovascular diseases. To realize this potential, genetic vectors must be precisely and efficiently delivered to targeted regions of the body. However, conventional methods of delivery do not provide sufficient spatial and temporal control. Here, we demonstrate how alginate microgels provide a basis for developing systems for controlled genetic vector release. We adjust the physiochemical properties of alginate for quicker or slower release, and we demonstrate how combining distinct formulations of microgels can tune the release of the overall composite microgel suspension. These composite suspensions are generated using a straightforward and powerful application of droplet microfluidics which allows for the real-time generation of a composite suspension.

VEGF and IGF Delivered from Alginate Hydrogels Promote Stable Perfusion Recovery in Ischemic Hind Limbs of Aged Mice and Young Rabbits.

Biomaterial-based delivery of angiogenic growth factors restores perfusion more effectively than bolus delivery methods in rodent models of peripheral vascular disease, but the same success has not yet been demonstrated in clinically relevant studies of aged or large animals. These studies explore, in clinically relevant models, a therapeutic angiogenesis strategy for the treatment of peripheral vascular disease that overcomes the challenges encountered in previous clinical trials. Alginate hydrogels providing sustained release of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF) were injected into ischemic hind limbs in middle-aged and old mice, and also in young rabbits, as a test of the scalability of this local growth factor treatment. Spontaneous perfusion recovery diminished with increasing age, and only the combination of VEGF and IGF delivery from gels significantly rescued perfusion in middle-aged (13 months) and old (20 months) mice. In rabbits, the delivery of VEGF alone or in combination with IGF from alginate hydrogels, at a dose 2 orders of magnitude lower than the typical doses used in past rabbit studies, enhanced perfusion recovery when given immediately after surgery, or as a treatment for chronic ischemia. Capillary density measurements and angiographic analysis demonstrated the benefit of gel delivery. These data together suggest that alginate hydrogels providing local delivery of low doses of VEGF and IGF constitute a safe and effective treatment for hind-limb ischemia in clinically relevant animal models, thereby supporting the potential clinical translation of this concept.

Alginate hydrogels allow for bioactive and sustained release of VEGF-C and VEGF-D for lymphangiogenic therapeutic applications.

Lymphatic dysfunction is associated with the progression of many cardiovascular disorders due to their role in maintaining tissue fluid homeostasis. Promoting new lymphatic vessels (lymphangiogenesis) is a promising strategy to reverse these cardiovascular disorders via restoring lymphatic function. Vascular endothelial growth factor (VEGF) members VEGF-C and VEGF-D are both potent candidates for stimulating lymphangiogenesis, though maintaining spatial and temporal control of these factors represents a challenge to developing efficient therapeutic lymphangiogenic applications. Injectable alginate hydrogels have been useful for the controlled delivery of many angiogenic factors, including VEGF-A, to stimulate new blood vasculature. However, the utility of these tunable hydrogels for delivering lymphangiogenic factors has never been closely examined. Thus, the objective of this study was to utilize ionically cross-linked alginate hydrogels to deliver VEGF-C and VEGF-D for potential lymphangiogenic applications. We demonstrated that lymphatic endothelial cells (LECs) are sensitive to temporal presentation of VEGF-C and VEGF-D but with different responses between the factors. The greatest LEC mitogenic and sprouting response was observed for constant concentrations of VEGF-C and a high initial concentration that gradually decreased over time for VEGF-D. Additionally, alginate hydrogels provided sustained release of radiolabeled VEGF-C and VEGF-D. Finally, VEGF-C and VEGF-D released from these hydrogels promoted a similar number of LEC sprouts as exogenously added growth factors and new vasculature in vivo via a chick chorioallantoic membrane (CAM) assay. Overall, these findings demonstrate that alginate hydrogels can provide sustained and bioactive release of VEGF-C and VEGF-D which could have applications for therapeutic lymphangiogenesis.

Comparison of Endothelial Differentiation Capacities of Human and Rat Adipose-Derived Stem Cells.

BACKGROUND: The authors compared the endothelial differentiation capacities of human and rat adipose-derived stem cells to determine whether human adipose-derived stem cells can be a source of endothelial cells clinically. METHODS: Human and rat adipose-derived stem cells were harvested and characterized with flow cytometry and trilineage differentiation. Cells from passages III through V were fed with endothelial cell differentiation medium for up to 3 weeks. Cells were harvested after 1, 2, and 3 weeks, and endothelial differentiation was evaluated with quantitative reverse-transcriptase polymerase chain reaction, flow cytometry, and angiogenic sprouting assays. RESULTS: Both human and rat adipose-derived stem cells were CD90, CD44, and CD31 before differentiation. The cells were successfully differentiated into adipogenic, osteogenic, and chondrogenic lineages. Expression of endothelial cell-specific genes peaked at the second week of differentiation in both human and rat cells. The fold changes in expression of CD31, vascular endothelial growth factor receptor-1, nitric oxide synthase, and von Willebrand factor genes at week 2 were 0.4 ± 0.1, 34.7 ± 0.3, 2.03 ± 0.25, and 12.5 ± 0.3 respectively, in human adipose-derived stem cells; and 1.5 ± 1.01, 21.6 ± 1.7, 17.9 ± 0.6, and 11.2 ± 1.3, respectively, in rat cells. The percentages of CD31 cells were 0.2, 0.64, and 1.6 in human cell populations and 0.5, 5.91, and 11.5 in rat cell populations at weeks 1, 2, and 3, respectively. Rat adipose-derived stem cell-derived endothelial cells displayed enhanced sprouting capability compared with the human cells. CONCLUSIONS: Human adipose-derived stem cells responded less strongly to EGM-2MV endothelial differentiation medium than did the rat cells. Still, the human cells have the potential to become a clinical source of endothelial cells with modifications in the differentiation conditions.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

Concomitant Benznidazole and Suramin Chemotherapy in Mice Infected with a Virulent Strain of Trypanosoma cruzi.

Although suramin (Sur) is suggested as a potential drug candidate in the management of Chagas disease, this issue has not been objectively tested. In this study, we examined the applicability of concomitant treatment with benznidazole (Bz) and suramin in mice infected with a virulent strain of Trypanosoma cruzi. Eighty 12-week-old male C57BL/6 mice were equally randomized in eight groups: (i) noninfected mice (negative control) and mice infected with T. cruzi Y strain receiving (ii) no treatment (positive control), (iii) Bz, 100 mg/kg of body weight per day, (iv) Sur, 20 mg/kg/day, and (v to viii) Sur, 20 mg/kg/day, combined with Bz, 100, 50, 25, or 5 mg/kg/day. Bz was administered by gavage, and Sur was administered intraperitoneally. Sur dramatically increased the parasitemia, cardiac content of parasite DNA, inflammation, oxidative tissue damage, and mortality. In response to high parasitic load in cardiac tissue, Sur stimulated the immune system in a manner typical of the acute phase of Chagas disease, increasing tissue levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and inducing a preferential IgG2a anti-T. cruzi serum pattern. When Sur and Bz were combined, the infection severity was attenuated, showing a dose-dependent Bz response. Sur therapy had a more harmful effect on the host than on the parasite and reduced the efficacy of Bz against T. cruzi infection. Considering that Sur drastically reinforced the infection evolution, potentiating the inflammatory process and the severity of cardiac lesions, the in vivo findings contradicted the in vitro anti-T. cruzi potential described for this drug.

The Role of Synthetic Extracellular Matrices in Endothelial Progenitor Cell Homing for Treatment of Vascular Disease.

Poor vascular homeostasis drives many clinical disorders including diabetes, arthritis, atherosclerosis, and peripheral artery disease. Local tissue ischemia resultant of insufficient blood flow is a potent stimulus for recruitment of endothelial progenitor cells (EPCs). This mobilization and homing is a multi-step process involving EPC detachment from their steady state bone marrow niches, entry into circulation, rolling along vessel endothelium, transmigration, and adhesion to denuded extracellular matrix (ECM) where they may participate in neovessel formation. However, these events are often interrupted in pathological conditions partly due to an imbalance in factor presentation at the tissue level. EPC number and function is impaired in patients with vascular diseases and this dysfunction has been proposed as a prominent contributor to disease pathogenesis. Research approaches aimed at providing therapeutic angiogenesis commonly involve the delivery of proangiogenic cells and/or soluble factors. Nevertheless, greater understanding of the mechanisms involved in EPC homing in both healthy and diseased states is critical for improving efficacy of such strategies. This review underscores the matrix-related signals necessary for enhancing EPC recruitment to ischemic tissue and provides an overview of the development of synthetic ECMs that aim to mimic functions of the local native microenvironment for use in therapeutic angiogenesis.

Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies.

Hypoxia augments outgrowth endothelial cell (OEC) sprouting and directed migration in response to sphingosine-1-phosphate (S1P).

Therapeutic angiogenesis provides a promising approach to treat ischemic cardiovascular diseases through the delivery of proangiogenic cells and/or molecules. Outgrowth endothelial cells (OECs) are vascular progenitor cells that are especially suited for therapeutic strategies given their ease of noninvasive isolation from umbilical cord or adult peripheral blood and their potent ability to enhance tissue neovascularization. These cells are recruited to sites of vascular injury or tissue ischemia and directly incorporate within native vascular endothelium to participate in neovessel formation. A better understanding of how OEC activity may be boosted under hypoxia with external stimulation by proangiogenic molecules remains a challenge to improving their therapeutic potential. While vascular endothelial growth factor (VEGF) is widely established as a critical factor for initiating angiogenesis, sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, has recently gained great enthusiasm as a potential mediator in neovascularization strategies. This study tests the hypothesis that hypoxia and the presence of VEGF impact the angiogenic response of OECs to S1P stimulation in vitro. We found that hypoxia altered the dynamically regulated S1P receptor 1 (S1PR1) expression on OECs in the presence of S1P (1.0 µM) and/or VEGF (1.3 nM). The combined stimuli of S1P and VEGF together promoted OEC angiogenic activity as assessed by proliferation, wound healing, 3D sprouting, and directed migration under both normoxia and hypoxia. Hypoxia substantially augmented the response to S1P alone, resulting in ~6.5-fold and ~25-fold increases in sprouting and directed migration, respectively. Overall, this report highlights the importance of establishing hypoxic conditions in vitro when studying ischemia-related angiogenic strategies employing vascular progenitor cells.

Refilling drug delivery depots through the blood.

Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that blood-borne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequence-targeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.

Injectable MMP-sensitive alginate hydrogels as hMSC delivery systems.

Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful tool for tissue-regeneration therapies. In this context, entrapped cells should be able to escape the matrix becoming more available to actively participate in the healing process. Here, we analyzed the performance of proteolytically degradable alginate hydrogels as vehicles for human mesenchymal stem cells (hMSC) transplantation. Alginate was modified with the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG), which did not promote dendritic cell maturation in vitro, neither free nor conjugated to alginate chains, indicating low immunogenicity. hMSC were entrapped within MMP-sensitive and MMP-insensitive alginate hydrogels, both containing cell-adhesion RGD peptides. Softer (2 wt % alginate) and stiffer (4 wt % alginate) matrices were tested. When embedded in a Matrigel layer, hMSC-laden MMP-sensitive alginate hydrogels promoted more extensive outward cell migration and invasion into the tissue mimic. In vivo, after 4 weeks of subcutaneous implantation in a xenograft mouse model, hMSC-laden MMP-sensitive alginate hydrogels showed higher degradation and host tissue invasion than their MMP-insensitive equivalents. In both cases, softer matrices degraded faster than stiffer ones. The transplanted hMSC were able to produce their own collagenous extracellular matrix, and were located not only inside the hydrogels, but also outside, integrated in the host tissue. In summary, injectable MMP-sensitive alginate hydrogels can act as localized depots of cells and confer protection to transplanted cells while facilitating tissue regeneration.

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Guided bone regeneration using injectable vascular endothelial growth factor delivery gel.

BACKGROUND: Vascularization underlies the success of guided bone regeneration (GBR) procedures. This study evaluates the regenerative potential of GBR in combination with vascular endothelial growth factor (VEGF) delivery via an injectable hydrogel system. METHODS: Critical-sized defects were created in rat calvariae, and GBR procedures were performed with a collagen membrane alone (control), or plus bolus delivery of VEGF, or plus application of VEGF-releasing hydrogels (VEGF-Alg). Four and 8 weeks after treatment, defect sites were evaluated with microcomputed tomographic and histomorphometric analyses for blood vessel and bone formation. RESULTS: At 4 weeks, relative to the control condition, the bolus addition of VEGF did not affect blood vessel density within the defect site, yet the application of VEGF-Alg significantly (P <0.05) increased blood vessel density. Although there was no difference in bone regeneration at 4 weeks, at 8 weeks there was a significant (P <0.05) increase in bone regeneration in the VEGF-Alg-treated defects. CONCLUSIONS: These data demonstrate that the application of VEGF-Alg enhanced early angiogenesis, whereas at a later time point, it enhanced bone regeneration. Controlled delivery approaches of angiogenic growth factors used adjunctively with GBR may be a promising strategy for enhancing outcomes of GBR.