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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 non-enzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger pro-inflammatory 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. Herein, 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 post-wounding 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 co-delivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.

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.

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.

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.

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., Graphical abstract

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 scaffold 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 endothelial networks. However, hiPSC-VSMC in the presence of fibronectin further substantiated the number and size of endothelial networks. A mechanistic study shows that blocking αvβ3 integrin signaling between hiPSC-VSMC and fibronectin impacts the endothelial network formation. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct.

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 β3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvβ3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin β3, 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.

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.

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.

To understand the diabetic wound healing microenvironment, we profiled 174,962 single cells from foot, forearm, and PBMCs using single-cell RNA sequencing (scRNASeq) approach. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes and M1 macrophage polarization in the DFU patients with healing wounds. Further, scRNASeq of spatially separated samples from same patient and spatial transcriptomics (ST) revealed preferential localization of these healing associated fibroblasts toward deep wound/ulcer bed as compared to wound edge or non-wounded skin. ST also validated our findings of higher enrichment 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.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) 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 function. In this study, our objective was to understand the effect of specific ECM ligand-integrin interaction on hiPSC- VSMC’s paracrine secretion, cell proliferation, and morphology. We here showed a precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin β3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin dependent via αvβ3 integrin interactions. Also, our data indicate the possible role of a positive feedback loop between integrin β3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC- VSMC’s morphology and cell proliferation. Finally, the secretome with improved proangiogenic activity shows potential for future regenerative applications.

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.

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.

Vascular smooth muscle cells (VSMCs) play a critical role during the development of blood vessels and cardiovascular disease progression. Although autologous VSMCs have been used in vascular tissue engineering, it would be desirable to have a renewable source of these cells. To this end, human-induced pluripotent stem cells (hiPSCs) have provided an attractive approach to generate patient-specific VSMCs in a large scale and have been spearheading the development of novel vascular regeneration strategies, disease modeling, and drug screening for the last decade. This book chapter concisely discusses the progress in the field of vascular regeneration strategies, reprogramming, and VSMC differentiation methods and examines current hiPSC-VSMC-based vascular constructs for regenerative therapy and disease modeling.

Buerger’s disease or Thromboangiitis Obliterans (TAO) is a nonatherosclerotic segmental vascular disease which affects small and medium arteries and veins in the upper and lower extremities. Based on pathological findings, TAO can be considered as a distinct form of vasculitis that is most prevalent in young male smokers. There is no definitive cure for this disease as therapeutic modalities are limited in number and efficacy. Surgical bypass has limited utility and 24% of patients will ultimately require amputation. Recently, studies have shown that therapeutic angiogenesis and immunomodulatory approaches through the delivery of stem cells to target tissues are potential options for ischemic lesion treatment. In this review, we summarize the current knowledge of TAO treatment and provide an overview of stem cell-based treatment modalities.

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 PEG-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. This article is protected by copyright. All rights reserved.

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&rsquo, 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.

Buerger's disease or Thromboangiitis Obliterans (TAO) is a nonatherosclerotic segmental vascular disease which affects small and medium arteries and veins in the upper and lower extremities. Based on pathological findings, TAO can be considered as a distinct form of vasculitis that is most prevalent in young male smokers. There is no definitive cure for this disease as therapeutic modalities are limited in number and efficacy. Surgical bypass has limited utility and 24% of patients will ultimately require amputation. Recently, studies have shown that therapeutic angiogenesis and immunomodulatory approaches through the delivery of cells to target tissues are potential options for ischemic lesion treatment. In this review, we summarize the current knowledge of TAO treatment and provide an overview of stem cell-based treatment modalities.

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 (MRTF/SRF) 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.

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.

Wound healing is the physiologic response to a disruption in normal skin architecture and requires both spatial and temporal coordination of multiple cell types and cytokines. This complex process is prone to dysregulation secondary to local and systemic factors such as ischemia and diabetes that frequently lead to chronic wounds. Chronic wounds such as diabetic foot ulcers are epidemic with great cost to the healthcare system as they heal poorly and recur frequently, creating an urgent need for new and advanced therapies. Stem cell therapy is emerging as a potential treatment for chronic wounds, and adult-derived stem cells are currently employed in several commercially available products; however, stem cell therapy is limited by the need for invasive harvesting techniques, immunogenicity, and limited cell survival in vivo. Induced pluripotent stem cells (iPSC) are an exciting cell type with enhanced therapeutic and translational potential. iPSC are derived from adult cells by in vitro induction of pluripotency, obviating the ethical dilemmas surrounding the use of embryonic stem cells; they are harvested non-invasively and can be transplanted autologously, reducing immune rejection; and iPSC are the only cell type capable of being differentiated into all of the cell types in healthy skin. This review focuses on the use of iPSC in animal models of wound healing including limb ischemia, as well as their limitations and methods aimed at improving iPSC safety profile in an effort to hasten translation to human studies.

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.

Advancements in the field of skin tissue engineering have been quite visible with the development of composite biomaterials. While natural polymers help increase the biocompatibility of the composite, synthetic polymers help optimize physical properties such as mechanical strength, degradation, controlled release, swelling capacity, and water retention. So far, a wide array of composite scaffolds has been fabricated matching the mechanical strength and stratified structure of skin and with an ability to deliver bioactive agents and stem cells. This book chapter reviews the use of various combinations of natural and synthetic polymers in engineering different kinds of composite matrices. Furthermore, we discuss their use in the delivery of a variety of bioactive agents and stem cells for skin tissue engineering applications.

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein, hiPSCs were differentiated into alpha-smooth muscle actin (α-SMA) and calponin-positive VSMCs, which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted, which contained cells largely positive for α-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover, when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts, they remained unruptured and patent with active vascular remodeling, and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs, and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease.

INTRODUCTION Fibroblasts produce extracellular matrix (ECM) molecules that regulate tissue strength and resilience. Imbalanced ECM maintenance leads to tissue dysfunction. Although multiple populations of fibroblasts support uninjured skin function, the extent of fibroblast diversity and the presence of functionally distinct subsets in adult fibrotic skin are poorly defined. The inability to find a single molecular marker that identifies all activated “myofibroblasts” during wound healing suggests the existence of multiple subsets of ECM-producing myofibroblasts. Furthermore, little is known about the cellular and molecular mechanisms that promote the expansion of individual myofibroblast subsets and support cellular heterogeneity. Although macrophages influence myofibroblast numbers and ECM deposition after injury, subpopulations of wound bed macrophages have only recently been defined, and specific interactions with fibroblasts have not been explored. RATIONALE Variation in healing and scarring rates in multiple tissues suggests that fibroblast diversity exists. To develop therapies targeting fibrotic responses, functionally distinct subsets of myofibroblasts and the mechanisms that support individual populations must be uncovered. We used a comprehensive, hierarchical fluorescence-activated cell sorting strategy to define myofibroblast subsets in skin wound beds from adult mice. This strategy revealed distinct subsets of wound bed myofibroblasts, including an abundant population that contains the cell surface marker profile of adipocyte precursor cells (APs). We examined myofibroblast subsets in different cutaneous fibrotic contexts and explored mechanisms that selectively promote the expansion of APs. RESULTS Genetic lineage tracing and flow cytometry revealed distinct subsets of wound bed myofibroblasts that express smooth muscle actin and collagen. The most abundant populations were CD26-expressing APs and a subset with high cell surface levels of CD29 (CD29 High ). Although transcriptomic analysis revealed that each myofibroblast subset has a distinct gene expression profile, functional analyses suggest that myofibroblast subsets make both overlapping and distinct contributions to repair. APs were significantly reduced and CD29 High cells were more abundant in wound beds from aged mice and skin from mice that underwent bleomycin-induced fibrosis, suggesting that the fibrotic environment influences myofibroblast composition. Injury and repair-related changes in AP transcription implicated macrophage signaling in the modulation of AP gene expression. Genetic ablation and cell transplantations of different myeloid cells revealed that macrophages expressing macrophage galactose N -acetylgalactosamine–specific lectin 2 ( Mgl2 /CD301b) directly stimulate proliferation in a subset of APs and not in other myofibroblast subsets. By combining in vitro cytokine stimulation with in vivo signaling pathway inhibition, we identified multiple CD301b + macrophage–secreted factors (platelet-derived growth factor C and insulin-like growth factor 1) that selectively stimulate AP proliferation, thus supporting the heterogeneity of wound bed myofibroblasts. CONCLUSION We identified multiple populations of skin myofibroblasts and observed that the composition of myofibroblasts is dependent upon the fibrotic environment. Distinct interactions allow CD301b + macrophage–derived signaling to selectively activate the proliferation of APs and not other myofibroblasts. These results have potential for the development of therapies that target multiple cellular populations or signaling pathways under conditions associated with excessive or deficient ECM deposition, such as wound healing and fibrosis.

The normal wound healing process involves a well-organized cascade of biological pathways and any failure in this process leads to wounds becoming chronic. Non-healing wounds are a burden on healthcare systems and set to increase with aging population and growing incidences of obesity and diabetes. Stem cell-based therapies have the potential to heal chronic wounds but have so far seen little success in the clinic. Current research has been focused on using polymeric biomaterial systems that can act as a niche for these stem cells to improve their survival and paracrine activity that would eventually promote wound healing. Furthermore, different modification strategies have been developed to improve stem cell survival and differentiation, ultimately promoting regenerative wound healing. This review focuses on advanced polymeric scaffolds that have been used to deliver stem cells and have been tested for their efficiency in preclinical animal models of wounds.

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.

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.

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Encapsulation of plasmid DNA (pDNA) in nanoparticulate gene delivery systems offers the possibility of control in dosing, enhanced pDNA uptake, increased resistance to nuclease degradation and sustained release of functionally active pDNA over time. Extracellular matrix based biomaterial i.e. hyaluronan (HA) was used to encapsulate pDNA (pCMV-GLuc, Gaussia Luciferase reporter plasmid DNA having CMV promoter) in submicron size particulate system. Nano size range (~400-600 nm) pDNA loaded hyaluronan nanoparticles were formulated by ionic gelation followed by the cross-linking method with high encapsulation efficiency (~75-85%). The particle preparation process was further optimized for molecular weight, cross-linking method, cross-linking time and plasmid/polymer ratio. The entrapped plasmid maintained its structural and functional integrity as revealed by agarose gel electrophoresis. The pDNA was released from the hyaluronan nanoparticles in a controlled manner over a period of one month. In vitro transfection by one-week released pDNA from nanoparticles with transfecting agent branched polyethyleneimine (bPEI) resulted in significantly higher expression levels than those in pDNA alone which demonstrated the functional bioactivity of released pDNA. For cellular localization studies, the hyaluronan nanoparticles encapsulated with FITC-dextran were incubated with adipose derived stem cells (ADSCs) and localization in the cellular environment were investigated. The results of this study illustrate that hyaluronan nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 24 h.

This study describes the potential use of silk gland sericin protein as a biocompatible natural biopolymer in its native form. The membranes were fabricated using native silk sericin protein extracted from middle silk gland of Antheraea mylitta, a non-mulberry tropical tasar silkworm without using any cross-linking agent. The fabricated membranes were biophysically characterized and optimized for cell culture. Silk sericin protein extracted from gland contained higher amount of beta-sheets, which increased upon treatment with ethanol as observed by FTIR and XRD. The membranes did show robustness, good mechanical strength and high temperature stability. Cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis using feline fibroblast cells. Morphology of growing cells was assessed by confocal microscopy that indicated normal spreading and proliferation on the silk sericin membranes. The membranes showed low inflammatory response as observed assaying TNF alpha release. This study reveals the potential of native silk sericin protein from silk gland as biocompatible biopolymer for potential biomedical applications.

Silk proteins consist of a fibrous core protein, fibroin, and glue proteins called sericins, which envelop the fibroin fiber with successive sticky layers that help in the formation of a cocoon by cementing together the silk fibers. This system is essentially a protein–fiber composite system, with fibers of the high-molecular-weight fibroin proteins surrounded by the glue-like continuous phase of the sericin. In mulberry silk produced by Bombyx mori, Ser1 and Ser2 are the two major genes encoding for sericin. Ser1 is the most abundant silk sericin of B. mori and consists of repeats of a 38-amino acid motif that contributes towards the hydrophilicity of sericin. The protein has partially been characterized from non-mulberry silkworms like the tropical tasar Antheraea mylitta, and studied for various potential applications because of its unique biochemical and biophysical properties. These applications include the skincare and food industries, as coating material for biomedical applications like anticoagulants, in anticancer drugs, for drug delivery, and in tissue engineering. Sericin promotes proliferation of cells when used as a constituent of cell culture in serum-free media. Sericin also supports cell adhesion and proliferation when used in pure form and/or blended in matrices. Sericin films, 3D scaffolds, nanoparticles, composites, conjugated drugs, and recombinant sericins offer potential future options for these needs.

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 rig) and IL-10 (10 mu g)/eNOS (20 mu g) and reduced inflammation with IL-10 (10 mu g) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELF injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. (C) 2015 Elsevier Ltd. All rights reserved. Science Foundation Ireland grant no. 07/SRC/B1163 peer-reviewed 2017-10-31

Journal article 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. peer-reviewed

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere's behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles.