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Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.
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Colágeno/química , Ácido Hialurônico/química , Hidrogéis/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologiaRESUMO
Fibrosis is a consequence of aberrant wound healing processes that can be debilitating for patients and often are associated with highly morbid disease processes. Myofibroblasts play an important role in determining an appropriate physiologic response to tissue injury or an excessive response leading to fibrosis. Specifically, "supermature" focal adhesions, α-smooth muscle actin, and the myocardin-related transcription factor/serum response factor pathway likely play a significant role in the differentiation and survival of myofibroblasts in fibrotic lesions. Thus, targeting each of these and disrupting their functioning could lead to the development of therapeutic options for patients suffering from fibrosis and other sequelae of dysregulated wound healing. In this paper, we review the current literature concerning the roles of these three constituents of fibrotic signaling pathways, work already done in attempting to regulate these processes, and discuss the potential of these biomolecular constituents as therapeutic targets in future translational research.
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Fibrose/genética , Regulação da Expressão Gênica , Transdução de Sinais , Cicatrização/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Feminino , Fibrose/patologia , Humanos , Masculino , Miofibroblastos/citologia , Pesquisa Translacional Biomédica , Cicatrização/fisiologiaRESUMO
BACKGROUND: A commonly used treatment for open wounds, negative pressure wound therapy (NPWT) has recently been used to optimize wound healing in the setting of surgically closed wounds; however, the specific mechanisms of action by which NPWT may benefit patients after surgery remain unknown. Using a swine wound healing model, the current study investigates angiogenesis as a candidate mechanism. METHODS: Multiple excisional wounds were created on the dorsa of 10 male Yorkshire pigs and closed by primary suture. The closed wounds underwent treatment with either NPWT dressing or control dressings in the absence of negative pressure. Dressings were maintained for 8 days followed by euthanasia of the animal. Scar evaluation of the wounds by photographic analysis was performed, and wounds were analyzed for angiogenesis markers by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Scar evaluation scores were observed to be significantly higher for the NPWT-treated sites compared with the control sites (P < 0.05). The enzyme-linked immunosorbent assay results demonstrated increases for vascular endothelial growth factor (VEGF) staining at the incision site treated with NPWT compared with other treatment groups (P < 0.05). In addition, an approximately 3-fold elevation in VEGF expression was observed at the NPWT-treated sites (2.8% vs. 1%, respectively; P < 0.0001).). However, there was no significant difference in immunohistochemistry staining. CONCLUSIONS: The use of NPWT improves the appearance of wounds and appears to increase VEGF expression after 8 days in the setting of a closed excisional wound model, suggesting that improved angiogenesis is one mechanism by which NPWT optimizes wound healing when applied to closed surgical wound sites.
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Tratamento de Ferimentos com Pressão Negativa , Neovascularização Fisiológica , Cicatrização/fisiologia , Animais , Masculino , Modelos Animais , Projetos Piloto , Fluxo Sanguíneo Regional , SuínosRESUMO
Vascular smooth muscle cells (VSMCs) play a major role in the pathophysiology of cardiovascular diseases. The advent of induced pluripotent stem cell (iPSC) technology and the capability of differentiating into virtually every cell type in the human body make this field a ray of hope for vascular regenerative therapy and understanding of the disease mechanism. In the present review, we first discuss the recent iPSC technology and vascular smooth muscle development from an embryo and then examine different methodologies to derive VSMCs from iPSCs, and their applications in regenerative therapy and disease modelling.
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Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Liso Vascular/metabolismo , Medicina Regenerativa/métodos , Doenças Vasculares/terapia , Animais , Embrião de Mamíferos/metabolismo , Humanos , Doenças Vasculares/metabolismoRESUMO
Sustained delivery of growth factors has emerged as an essential requirement for bone tissue engineering applications for the treatment of various kinds of bone defects. Chitosan (CH) has attracted particular attention for drug delivery and bone tissue engineering because of its favorable biocompatibility and biodegradability. In this study, a composite microsphere system containing CH and nanohydroxyapatite (nHA)-alendronate (AL) particles was fabricated by employing both emulsification and cross-linking strategies. The microspheres were characterized for their surface morphology, composition, size distribution, drug loading efficiency and release properties. The results showed that loading efficiency and sustained release of hydrophilic AL were significantly improved, which is ideal for locally sustained release in the bone microenvironment. In vitro osteogenic studies showed that the microspheres could enhance the osteogenic activity of rabbit adipose-derived stem cells. In conclusion, the CH/nHA-AL composite microspheres exhibit promising properties as a candidate for local treatment for bone defects.
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Alendronato/administração & dosagem , Quitosana/química , Preparações de Ação Retardada/síntese química , Durapatita/química , Nanopartículas/química , Osteoblastos/citologia , Células-Tronco/citologia , Alendronato/química , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/química , Cápsulas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Reagentes de Ligações Cruzadas/química , Preparações de Ação Retardada/administração & dosagem , Emulsões , Nanopartículas/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Coelhos , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
Objective: Chronic skin wounds are one of the most devastating complications in diabetic patients due to the formation of advanced glycation end-products (AGEs) resulting from nonenzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger proinflammatory signals that impair wound healing in diabetes and contribute to the pathology of chronic skin wounds. Approach: We previously developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that acts as a competitive inhibitor of AGEs, and another ELP fusion protein containing stromal cell-derived factor 1 (SDF1) that promotes revascularization. In this study, we report the effects of protein coacervates incorporating both vRAGE-ELP and SDF1-ELP on wound healing in an in vitro diabetes-mimicking cell culture system, and in in vivo in full-thickness wounds on diabetic mice. Results: The combination of vRAGE-ELP and SDF1-ELP increased cell metabolic activity in AGE-stimulated endothelial cells, promoted in vitro tube formation and accelerated healing in an in vitro cell migration assay. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure in the combination groups reached almost 100% on postwounding day 35, compared to 62% and 85% on the same days in animals treated with fibrin gel control and vehicle control consisting of ELP alone. Innovation: To our knowledge, this is the first study that attempts to reverse the AGE-RAGE-mediated signaling as well as to promote cell proliferation and vascularization in one single treatment. Conclusion: The codelivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.
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Diabetes Mellitus Experimental , Elastina , Camundongos , Animais , Elastina/química , Células Endoteliais/metabolismo , Peptídeos , Proteínas Recombinantes , Produtos Finais de Glicação AvançadaRESUMO
Introduction: hiPSC-VSMCs have been suggested as therapeutic agents for wound healing and revascularization through the secretion of proangiogenic factors. However, methods of increasing cell paracrine secretion and survivability have thus far yielded inconsistent results. This study investigates the effect of pre-conditioning of hiPSC-VSMCs with TNF-α and their integration into 3D collagen scaffolds on cellular viability and secretome. Methods: hiPSC-VSMCs were dual-plated in a 2D environment. TNF-α was introduced to one plate. Following incubation, cells from each plate were divided and added to type-I collagen scaffolds. TNF-α was introduced to two sets of scaffolds, one from each 2D plate. Following incubation, scaffolds were harvested for their media, tested for cell survivability, cytotoxicity, and imaged. Intra-media VEGF and bFGF levels were evaluated using ELISA testing. Results: hiPSC-VSMCs exposed to TNF-α during collagen scaffold proliferation and preconditioning showed an increase in cell viability and less cytotoxicity compared to non-exposed cells and solely-preconditioned cells. Significant increases in bFGF expression were found in pre-conditioned cell groups with further increases found in cells subsequently exposed during intra-scaffold conditioning. A significant increase in VEGF expression was found in cell groups exposed during both pre-conditioning and intra-scaffold conditioning. Fibroblasts treated with any conditioned media demonstrated increased migration potential. Conclusions: Conditioning hiPSC-VSMCs embedded in scaffolds with TNF-α improves cellular viability and increases the secretion of paracrine factors necessary for wound healing mechanisms such as migration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00764-0.
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Wound repair requires the coordination of multiple cell types including immune cells and tissue resident cells to coordinate healing and return of tissue function. Diabetic foot ulceration is a type of chronic wound that impacts over 4 million patients in the US and over 7 million worldwide (Edmonds et al., 2021). Yet, the cellular and molecular mechanisms that go awry in these wounds are not fully understood. Here, by profiling chronic foot ulcers from non-diabetic (NDFUs) and diabetic (DFUs) patients using single-cell RNA sequencing, we find that DFUs display transcription changes that implicate reduced keratinocyte differentiation, altered fibroblast function and lineages, and defects in macrophage metabolism, inflammation, and ECM production compared to NDFUs. Furthermore, analysis of cellular interactions reveals major alterations in several signaling pathways that are altered in DFUs. These data provide a view of the mechanisms by which diabetes alters healing of foot ulcers and may provide therapeutic avenues for DFU treatments.
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Nonviral gene delivery systems have a number of limitations including low transfection efficiency, specificity, and cytotoxicity, especially when the target cells are macrophages. To address these issues, the hypothesis tested in this study was that mannose functionalized nanohybrids composed of synthetic and natural polymers will improve transfection efficiency, cell viability, and cell specificity in macrophages. Robust nanohybrids were designed from hyaluronic acid (HA) and branched polyethyleneimine (bPEI) using carbodiimide chemistry. The reaction product, i.e., branched polyethyleneimine-hyaluronic acid (bPEI-HA) copolymer was subsequently functionalized with mannose at the terminal end of the copolymer to obtain mannosylated-bPEI-HA (Man-bPEI-HA) copolymer. UV spectroscopy and gel retardation studies confirmed the formation of polyplexes at polymer to DNA weight ratio ≥ 2. Alamar Blue and MTT assay revealed that the cytotoxicity of the developed nanohybrids were significantly (P < 0.05) lower than that of unmodified bPEI. Mannose functionalization of these nanohybrids showed specificity for both murine and human macrophage-like cell lines RAW 264.7 and human acute monocytic leukemia cell line (THP1), respectively, with a significant level (P < 0.05) of expression of gaussia luciferase (GLuc) and green fluorescent reporter plasmids. Internalization studies indicate that a mannose mediated endocytic pathway is responsible for this higher transfection rate. These results suggest that hyaluronan-based mannosylated nanohybrids could be used as efficient carriers for targeted gene delivery to macrophages.
Assuntos
DNA/administração & dosagem , Ácido Hialurônico/análogos & derivados , Manose/química , Nanoestruturas/química , Polietilenoimina/análogos & derivados , Transfecção , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Genes Reporter , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Manose/toxicidade , Camundongos , Nanoestruturas/toxicidade , Polietilenoimina/química , Polietilenoimina/toxicidadeRESUMO
iPSC technology is revolutionizing the field of regenerative medicine. The generation of patient-specific cells has huge potential for disease modeling as well as for clinical applications. iPSCs have been used as a renewable source of vascular cells, and in particular vascular smooth muscle cells. The use of these human iPSC-derived vascular smooth muscle cells is attractive for vascular tissue engineering. The cells are used in developing vascular grafts as well as in engineering disease models. Recent studies have shown the proangiogenic potentials of human iPSC-derived vascular smooth muscle cells in treating wounds. Here, we describe the VSMC differentiation protocol from human iPSCs and encapsulation methods in collagen scaffolds to promote proangiogenic potentials.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Humanos , Músculo Liso Vascular , Miócitos de Músculo Liso , Engenharia Tecidual/métodosRESUMO
Significance: Nonhealing wounds are a significant burden for the health care system all over the world. Existing treatment options are not enough to promote healing, highlighting the urgent need for improved therapies. In addition, the current advancements in tissue-engineered skin constructs and stem cell-based therapies are facing significant hurdles due to the absence of a renewable source of functional cells. Recent Advances: Induced pluripotent stem cell technology (iPSC) is emerging as a novel tool to develop the next generation of personalized medicine for the treatment of chronic wounds. The iPSC provides unlimited access to various skin cells to generate complex personalized three-dimensional skin constructs for disease modeling and autologous grafts. Furthermore, the iPSC-based therapies can target distinct wound healing phases and have shown accelerating wound closure by enhancing angiogenesis, cell migration, tissue regeneration, and modulating inflammation. Critical Issues: Since the last decade, iPSC has been revolutionizing the field of wound healing and skin tissue engineering. Despite the current progress, safety and heterogeneity among iPSC lines are still major hurdles in addition to the lack of large animal studies. These challenges need to be addressed before translating an iPSC-based therapy to the clinic. Future Directions: Future considerations should be given to performing large animal studies to check the safety and efficiency of iPSC-based therapy in a wound healing setup. Furthermore, strategies should be developed to overcome variation between hiPSC lines, develop an efficient manufacturing process for iPSC-derived products, and generate complex skin constructs with vasculature and skin appendages.
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Células-Tronco Pluripotentes Induzidas , Medicina Regenerativa , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Medicina Regenerativa/métodos , Pele , Engenharia Tecidual/métodos , Cicatrização/fisiologiaRESUMO
Human-induced pluripotent stem cells (hiPSC) and their differentiated vascular cells have been revolutionizing the field of regenerative wound healing. These cells are shown to be rejuvenated with immense potentials in secreting paracrine factors. Recently, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMC) have shown regenerative wound healing ability via their paracrine secretion. The quest to modulate the secretory function of these hiPSC-VSMC is an ongoing effort and involves the use of both biochemical and biophysical stimuli. This study explores the development and optimization of a reproducible, inexpensive protocol to form hiPSC-VSMC derived spheroids to investigate the implications of spheroid size on viability and paracrine secretion. Our data show the successful formation of different sizes of spheroids using various amount of hiPSC-VSMC. The hiPSC-VSMC spheroids formed with 10,000 cells strike an ideal balance between overall cell health and maximal paracrine secretion. The conditioned medium from these spheroids was found to be bioactive in enhancing human dermal fibroblast cell proliferation and migration. This research will inform future studies on the optimal spheroid size for regenerative wound healing applications.
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Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Meios de Cultivo Condicionados , Humanos , Músculo Liso Vascular , Miócitos de Músculo Liso , Esferoides CelularesRESUMO
Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.
Assuntos
Diabetes Mellitus/genética , Pé Diabético/genética , Fibroblastos/metabolismo , Macrófagos/metabolismo , Transcriptoma , Cicatrização/genética , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteína 1 Semelhante à Quitinase-3/genética , Proteína 1 Semelhante à Quitinase-3/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Pé Diabético/metabolismo , Pé Diabético/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Macrófagos/patologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 11 da Matriz/genética , Metaloproteinase 11 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Análise de Célula Única/métodos , Pele/metabolismo , Pele/patologia , Sequenciamento do ExomaRESUMO
Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) and their secretome have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC's functions. In this study, we investigated the effect of specific ECM ligand-integrin interaction on hiPSC-VSMC's paracrine secretion, cell viability, and morphology. Here, we show precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin ß3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvß3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin ß3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC-VSMC's morphology and cell viability. Finally, the secretome with enhanced bFGF shows potential for future wound healing applications.
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Células-Tronco Pluripotentes Induzidas , Materiais Biocompatíveis , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos , Humanos , Integrina beta3 , Metaloproteinase 2 da Matriz , Músculo Liso VascularRESUMO
Tissue-engineered constructs have immense potential as autologous grafts for wound healing. Despite the rapid advancement in fabrication technology, the major limitation is controlling angiogenesis within these constructs to form a vascular network. Here, we aimed to develop a 3D hydrogel that can regulate angiogenesis. We tested the effect of fibronectin and vascular smooth muscle cells derived from human induced pluripotent stem cells (hiPSC-VSMC) on the morphogenesis of endothelial cells. The results demonstrate that fibronectin increases the number of EC networks. However, hiPSC-VSMC in the hydrogel further substantiated the number and size of EC networks by vascular endothelial growth factor and basic fibroblast growth factor secretion. A mechanistic study shows that blocking αvß3 integrin signaling between hiPSC-VSMC and fibronectin impacts the EC network formation via reduced cell viability and proangiogenic growth factor secretion. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct.
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Significance: Chronic wounds are one of the major burdens of the U.S. health care system with an annual cost of $31.7 billion and affecting an estimated 2.4-4.5 million people. Several underlying molecular and cellular pathophysiological mechanisms, including poor vascularization, excessive extracellular matrix (ECM) degradation by proteases, decreased growth factor activity, and bacterial infection can lead to chronic wounds. More effective wound therapies need to address one or more of these mechanisms to significantly advance wound care. Recent Advances: Self-assembled nanomaterials may provide new therapeutic options for chronic wound healing applications as those materials generally exhibit excellent biocompatibility and can bear multiple functionalities, such as ECM-mimicking properties, drug delivery capabilities, and tunable mechanics. Furthermore, self-assembled nanomaterials can be produced at low cost, and owing to their ability to self-organize, generate complex multifunctional structures that can be tailored to the varying sizes and shapes of chronic wounds. Self-assembled nanomaterials have been engineered to serve as wound dressings, growth factor delivery systems, and antimicrobials. Critical Issues: As there are many different types of self-assembled nanomaterials, which in turn have different mechanisms of self-assembly and physiochemical properties, one type of self-assembled nanomaterials may not be sufficient to address all underlying mechanisms of chronic wounds. However, self-assembled nanomaterials can be easily tailored, and developing multifunctional self-assembled nanomaterials that can address various targets in chronic wounds will be needed. Future Directions: Future studies should investigate combinations of various self-assembled nanomaterials to take full advantage of their multifunctional properties.
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Nanoestruturas/química , Preparações Farmacêuticas , Cicatrização/efeitos dos fármacos , Animais , Doença Crônica , Sistemas de Liberação de Medicamentos , Humanos , Nanomedicina , Nanoestruturas/economia , Pele/patologiaRESUMO
Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. We developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates at around 30-31 °C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells, including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastase in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds.
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Diabetes Mellitus Experimental , Elastina , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Humanos , Camundongos , Peptídeos , Receptor para Produtos Finais de Glicação Avançada , Pele , CicatrizaçãoRESUMO
The application of human-induced pluripotent stem cells (hiPSCs) to generate vascular smooth muscle cells (hiPSC-VSMCs) in abundance is a promising strategy for vascular regeneration. While hiPSC-VSMCs have already been utilized for tissue-engineered vascular grafts and disease modeling, there is a lack of investigations exploring their therapeutic secretory factors. The objective of this manuscript was to understand how the biophysical property of a collagen-based scaffold dictates changes in the secretory function of hiPSC-VSMCs while developing hiPSC-VSMC-based therapy for durable regenerative wound healing. We investigated the effect of collagen fibrillar density (CFD) on hiPSC-VSMC's paracrine secretion and cytokines via the construction of varying density of collagen scaffolds. Our study demonstrated that CFD is a key scaffold property that modulates the secretory function of hiPSC-VSMCs. This study lays the foundation for developing collagen-based scaffold materials for the delivery of hiPSC-VSMCs to promote regenerative healing through guiding paracrine signaling pathways.
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Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Liso Vascular/metabolismo , Alicerces Teciduais/normas , Cicatrização/fisiologia , Animais , Diferenciação Celular , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
Aim: To assess the potential of human induced pluripotent stem cell-derived smooth muscle cells (hiPSC-SMC) to accelerate diabetic wound healing. Methods: hiPSC-SMC were embedded in 3D collagen scaffolds and cultured in vitro for 72 h; scaffolds were then applied to diabetic, nude mouse, splinted back wounds to assess in vivo healing. Cultured medium after scaffold incubation was collected and analyzed for expression of pro-angiogenic cytokines. Results: hiPSC-SMC secrete increased concentration of pro-angiogenic cytokines, compared with murine adipose derived stem cells. Delivery of hiPSC-SMC-containing collagen scaffolds accelerates diabetic wound healing and is associated with an increased number of total and M2 type macrophages. Conclusion: hiPSC-SMC promote angiogenesis and accelerate diabetic wound healing, making them a promising new candidate for treatment of diabetic wounds.
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Diabetes Mellitus Experimental/complicações , Pé Diabético/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/citologia , Neovascularização Fisiológica , Cicatrização , Animais , Pé Diabético/etiologia , Pé Diabético/patologia , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.