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Modified hyaluronic acid-collagen matrices trigger efficient gene transfer and prohealing behavior in fibroblasts for improved wound repair
Abstract
Growth factor therapy has demonstrated great promise for chronic wound repair, but controlling growth factor activity and cell phenotype over desired time frames remains a critical challenge. In this study, we developed a gene-activated hyaluronic acid-collagen matrix (GAHCM) comprising DNA/polyethylenimine (PEI) polyplexes retained on hyaluronic acid (HA)-collagen hydrogels using collagen mimetic peptides (CMPs). We hypothesized that manipulating both the number of CMP-collagen tethers and the ECM composition would provide a powerful strategy to control growth factor gene transfer kinetics while regulating cell behavior, resulting in enhanced growth factor activity for wound repair. We observed that polyplexes with 50% CMP-modified PEI (50 CP) showed enhanced retention of polyplexes in HCM hydrogels by 2.7-fold as compared to non-CMP modified polyplexes. Moreover, the incorporation of HA in the hydrogel promoted a significant increase in gene transfection efficiency based upon analysis of Gaussia luciferase (GLuc) reporter gene expression, and gene expression could be attenuated by blocking HA-CD44 signaling. Furthermore, when fibroblasts were exposed to vascular endothelial growth factor-A (VEGF-A)-GAHCM, the 50 CP matrix facilitated sustained VEGF-A production for up to 7 days, with maximal expression at day 5. Application of these VEGF-A-50 CP samples stimulated prolonged pro-healing responses, including the TGF-β1-induced myofibroblast-like phenotypes and enhanced closure of murine splinted wounds. Overall, these findings demonstrate the use of ECM-based materials to stimulate efficient gene transfer and regulate cellular phenotype, resulting in improved control of growth factor activity for wound repair. GAHCM have significant potential to overcome key challenges in growth factor therapy for regenerative medicine.
Statement of Significance
Despite great promise for growth factor therapies in wound treatment, controlling growth factor activity and providing a microenvironment for cells that maximizes growth factor signaling have continued to limit the success of existing formulations. Our GAHCM strategy, combining CMP gene delivery and hyaluronic acid-collagen matrix, enabled enhanced wound healing efficacy via the combination of controlled and localized growth factor expression and matrix-mediated regulation of cell behavior. Incorporation of CMPs and HA in the same matrix synergistically enhanced VEGF activity as compared with simpler matrices. Accordingly, GAHCM will advance our ability to leverage growth factor signaling for wound healing, resulting in new long-term treatments for recalcitrant wounds.
... 169 The major advantage of lipidoids over other lipid-based delivery materials is their straightforward such as collagen, gelatin, fibrin, and hyaluronic acid have demonstrated the ability to enhance GF stability, promote cell recruitment and differentiation, and facilitate GF delivery in a sustained and controllable manner during wound healing. 128,129,[176][177][178] Harnessing the potential of these biomaterials in hydrogels holds great promise for the development of innovative wound healing strategies which can effectively promote wound repair and regeneration. One key advantage of such hydrogels is their ability to provide a protective environment for gene-loaded nanoparticles, shielding them from protein-rich conditions commonly found in wound beds. ...
... 188 Although it is possible to manipulate mechanical properties of hydrogels through changes in density and chemical composition of cross-linkers, high levels of cross-linking can impede important cellular processes such as proliferation and migration. 189 The incorporation of nanoparticles, including dendrimers, polyplexes, lipoplexes, and carbon-and ceramic-based nanoparticles into hydrogels has enabled the development of threedimensional structures 128,129,184,190,191 that can mimic some of the functions of natural tissues, making them highly suitable for tissue engineering and gene delivery applications. ...
Chronic wounds are an unmet clinical need affecting millions of patients globally, and current standards of care fail to consistently promote complete wound closure and prevent recurrence. Disruptions in growth factor signaling, a hallmark of chronic wounds, have led researchers to pursue growth factor therapies as potential supplements to standards of care. Initial studies delivering growth factors in protein form showed promise, with a few formulations reaching clinical trials and one obtaining clinical approval. However, protein‐form growth factors are limited by instability and off‐target effects. Gene therapy offers an alternative approach to deliver growth factors to the chronic wound environment, but safety concerns surrounding gene therapy as well as efficacy challenges in the gene delivery process have prevented clinical translation. Current growth factor delivery and gene therapy approaches have primarily used single growth factor formulations, but recent efforts have aimed to develop multi‐growth factor approaches that are better suited to address growth factor insufficiencies in the chronic wound environment, and these strategies have demonstrated improved efficacy in preclinical studies. This review provides an overview of chronic wound healing, emphasizing the need and potential for growth factor therapies. It includes a summary of current standards of care, recent advances in growth factor, cell‐based, and gene therapy approaches, and future perspectives for multi‐growth factor therapeutics.
... This finding might be due to the antioxidant capabilities of HA, which binds to and inactivates ROS [9], favoring cell viability and, therefore, may increase the metabolic activity of cultures. In addition, HDF cells express CD44 receptors; which interact directly with HA, possibly influencing cell adhesion, migration, and proliferation via activation of the ERK/-MAPK signaling cascade [25]. Furthermore, HA-CD44 interactions may further signal to increase in Ca 2+ influx, essential for cell cycle progression and cell proliferation, as previously demonstrated for diverse cell types [25]. ...
... In addition, HDF cells express CD44 receptors; which interact directly with HA, possibly influencing cell adhesion, migration, and proliferation via activation of the ERK/-MAPK signaling cascade [25]. Furthermore, HA-CD44 interactions may further signal to increase in Ca 2+ influx, essential for cell cycle progression and cell proliferation, as previously demonstrated for diverse cell types [25]. Cell morphology was indistinguishable between the control and experimental groups ( Fig. 6-C). ...
Infections associated with the surfaces of medical devices represent a critical problem due to biofilm formation and the growing resistance towards antibacterial drugs. This is particularly relevant in commonly used invasive devices such as silicone-based ones where a demand for alternative antibiofilm surfaces is increasing. In this work, an antimicrobial chitosan-biosurfactant hydrogel mesh was produced by 3D-printing. The 3D structure was designed to coat polydimethylsiloxane-based medical devices for infection prevention. Additionally, the porous 3D structure allows the incorporation of customized bioactive components. For this purpose, two biosurfactants (surfactin and sophorolipids) were biosynthesized and tested for their antimicrobial activity. In addition, the printing of surfactant-chitosan-based coatings was optimized, and the resulting 3D structures were characterized (i.e., wettability, FTIR-ATR, antimicrobial activity, and biocompatibility). Compared with surfactin, the results showed a better yield and higher antibacterial activity against Gram-positive bacteria for sophorolipids (SLs). Thus, SLs were used to produce chitosan-based 3D-printed coatings. Overall, the SLs-impregnated coatings showed the best antibacterial activity against Staphylococcus aureus planktonic bacteria (61 % of growth inhibition) and antibiofilm activity (2 log units reduction) when compared to control. Furthermore, concerning biocompatibility, the coatings were cytocompatible towards human dermal fibroblasts. Finally, the coating presented a mesh suitable to be filled with a model bioactive compound (i.e., hyaluronic acid), paving the way to be used for customized therapeutics.
... This finding might be due to the antioxidant capabilities of HA, which binds to and inactivates ROS [9], favoring cell viability and, therefore, may increase the metabolic activity of cultures. In addition, HDF cells express CD44 receptors; which interact directly with HA, possibly influencing cell adhesion, migration, and proliferation via activation of the ERK/-MAPK signaling cascade [25]. Furthermore, HA-CD44 interactions may further signal to increase in Ca 2+ influx, essential for cell cycle progression and cell proliferation, as previously demonstrated for diverse cell types [25]. ...
... In addition, HDF cells express CD44 receptors; which interact directly with HA, possibly influencing cell adhesion, migration, and proliferation via activation of the ERK/-MAPK signaling cascade [25]. Furthermore, HA-CD44 interactions may further signal to increase in Ca 2+ influx, essential for cell cycle progression and cell proliferation, as previously demonstrated for diverse cell types [25]. Cell morphology was indistinguishable between the control and experimental groups ( Fig. 6-C). ...
The use of invasive medical devices is becoming more common nowadays, with catheters representing one of the most used medical devices. However, there is a risk of infection associated with the use of these devices, since they are made of materials that are prone to bacterial adhesion with biofilm formation, often requiring catheter removal as the only therapeutic option. Catheter-related urinary tract infections (CAUTIs) and central line-associated bloodstream infections (CLABSIs) are among the most common causes of healthcare-associated infections (HAIs) worldwide while endotracheal intubation is responsible for ventilator-associated pneumonia (VAP). Therefore, to avoid the use of biocides due to the potential risk of bacterial resistance development, antifouling strategies aiming at the prevention of bacterial adherence and colonization of catheter surfaces represent important alternative measures. This review is focused on the main strategies that are able to modify the physical or chemical properties of biomaterials, leading to the creation of antiadhesive surfaces. The most promising approaches include coating the surfaces with hydrophilic polymers, such as poly(ethylene glycol) (PEG), poly(acrylamide) and poly(acrylates), betaine-based zwitterionic polymers and amphiphilic polymers or the use of bulk-modified poly(urethanes). Natural polysaccharides and association with polysaccharides, namely with heparin, have also been used to improve hemocompatibility. Recently developed bioinspired techniques yielding very promising results in the prevention of bacterial adhesion and colonization of surfaces include slippery liquid-infused porous surfaces (SLIPS) based on the superhydrophilic rim of the pitcher plant and the Sharklet topography inspired by the shark skin, which are potential candidates as surface-modifying approaches for biomedical devices. Concerning the potential application of most of these strategies in catheters, more in vivo studies and clinical trials are needed to assure their efficacy and safety for possible future use.
... Fig. 7F shows that the SCE2 + NIR group had significantly more new blood vessel proliferation than the PBS group, as further detailed in Figs. S27A and S27B in the Supporting Information [55]. Consistent with results from the rat back skin wound experiments, the SCE2 + NIR set possessed the least CD86 expression and the highest CD206 expression, as evidenced in Fig. 7F and S28 in the Supporting Information. ...
Oral ulcers can be managed using a variety of biomaterials that deliver drugs or cytokines. However, many patients experience minimal benefits from certain medical treatments because of poor compliance, short retention times in the oral cavity, and inadequate drug efficacy. Herein, we present a novel hydrogel patch (SCE2) composed of a biopolymer matrix (featuring ultraviolet-triggered adhesion properties) loaded with cuttlefish ink nanoparticles (possessing pro-healing functions). Applying a straightforward local method initiates the formation of a hydrogel barrier that adheres to mucosal injuries under the influence of ultraviolet light. SCE2 then demonstrates exceptional capabilities for near-infrared photothermal sterilization and neutralization of reactive oxygen species. These properties contribute to the elimination of bacteria and the management of the oxidation process, thus accelerating the healing phase's progression from inflammation to proliferation. In studies involving diabetic rats with oral ulcers, the SCE2 adhesive patch significantly quickens recovery by altering the inflamed state of the injured area, facilitating rapid re-epithelialization, and fostering angiogenesis. In conclusion, this light-sensitive hydrogel patch offers a promising path to expedited wound healing, potentially transforming treatment strategies for clinical oral ulcers.
... [7,9,11,12] HA-CD44 interaction allows a better cell adhesion, migration, proliferation rates, and stimulates transforming growth factor β (TGF-β)-mediated differentiation which is crucial in the healing process. [7,13] Chen et al. conducted to see the effect of CD44 on mineralization by providing CD44 knockdown and the results showed a decrease in hDPSCs mineralization. This result concluded that CD44 plays a role in the mineralization process of hDPSCs. ...
Hyaluronic acid (HA) has the capability to influence dentin niche which is important in regenerative process. The CD44 as a specific receptor of HA was found to be related to dentin mineralization process. Meanwhile, transforming growth factor β1 (TGF-β1) has a vital role in the transition from proliferation into the differentiation of human dental pulp stem cell human dental pulp stem cells (hDPSCs) to become odontoblast cells and dentin mineralization. This study aims to analyzed HA's effect on dentin mineralization through CD44 and TGF-β1 expressions. Stem cells were cultured in four different supplemented conditioned media (control, +10 μg/mL, +20 μg/mL, and + 30 μg/mL of HA). Evaluation of CD44 expression was analyzed using flow cytometry and TGF-β1 was analyzed using enzyme-linked immunosorbent assay reader. Qualitative result using Alizarin red test after 21 days was done to confirm the formation of mineralization nodules. It was shown that HA expression of CD44 and TGF-β1 on hDPSCs were higher in AH groups compared to the control group and 30 μg/mL HA induced the highest TGF-β1 expression on hDPSCs. Alizarin red test also showed the highest mineralization nodules in the same group. Therefore, from this study, we found that supplemented 30 μg/mL of HA was proved in initiating hDPSCs differentiation process and promote dentin mineralization.
... [7,9,11,12] HA-CD44 interaction allows a better cell adhesion, migration, proliferation rates, and stimulates transforming growth factor β (TGF-β)-mediated differentiation which is crucial in the healing process. [7,13] Chen et al. conducted to see the effect of CD44 on mineralization by providing CD44 knockdown and the results showed a decrease in hDPSCs mineralization. This result concluded that CD44 plays a role in the mineralization process of hDPSCs. ...
Hyaluronic acid (HA) has the capability to influence dentin niche which is important in regenerative process. The CD44 as a specific receptor of HA was found to be related to dentin mineralization process. Meanwhile, transforming growth factor β1 (TGF-β1) has a vital role in the transition from proliferation into the differentiation of human dental pulp stem cell human dental pulp stem cells (hDPSCs) to become odontoblast cells and dentin mineralization. This study aims to analyzed HA's effect on dentin mineralization through CD44 and TGF-β1 expressions. Stem cells were cultured in four different supplemented conditioned media (control, +10 μg/mL, +20 μg/mL, and + 30 μg/mL of HA). Evaluation of CD44 expression was analyzed using flow cytometry and TGF-β1 was analyzed using enzyme-linked immunosorbent assay reader. Qualitative result using Alizarin red test after 21 days was done to confirm the formation of mineralization nodules. It was shown that HA expression of CD44 and TGF-β1 on hDPSCs were higher in AH groups compared to the control group and 30 μg/mL HA induced the highest TGF-β1 expression on hDPSCs. Alizarin red test also showed the highest mineralization nodules in the same group. Therefore, from this study, we found that supplemented 30 μg/mL of HA was proved in initiating hDPSCs differentiation process and promote dentin mineralization.
Skin wounds significantly impact the global health care system and represent a significant burden on the economy and society due to their complicated dynamic healing processes, wherein a series of immune events are required to coordinate normal and sequential healing phases, involving multiple immunoregulatory cells such as neutrophils, macrophages, keratinocytes, and fibroblasts, since dysfunction of these cells may impede skin wound healing presenting persisting inflammation, impaired vascularization, and excessive collagen deposition. Therefore, cellular target-based immunomodulation is promising to promote wound healing as cells are the smallest unit of life in immune response. Recently, immunomodulatory hydrogels have become an attractive avenue to promote skin wound healing. However, a detailed and comprehensive review of cellular targets and related hydrogel design strategies remains lacking. In this review, the roles of the main immunoregulatory cells participating in skin wound healing are first discussed, and then we highlight the cellular targets and state-of-the-art design strategies for immunomodulatory hydrogels based on immunoregulatory cells that cover defect, infected, diabetic, burn and tumor wounds and related scar healing. Finally, we discuss the barriers that need to be addressed and future prospects to boost the development and prosperity of immunomodulatory hydrogels.
Hyaluronic acid (HA), a non-sulfated glycosaminoglycan (GAG), is a significant component of the epidermal extracellular matrix (ECM). It plays multiple roles in the inflammatory response, cell adhesion, migration, proliferation, differentiation, angiogenesis, and tissue regeneration. Due to its inherent characteristics, including non-immunoreactivity, exceptional biocompatibility, biodegradability, native biofunctionality, hydrophilicity, and non-immunoreactivity, HA has found applications in the production of wound dressings. HA's synergistic role in enhancing deeper penetration into chronic wounds and its biofunctional properties in the healing process have been harnessed. HA-based wound dressings, often incorporating biomolecules or drugs to improve the dressing's biochemical performance during wound healing, have been developed. In this review, we explore the current state of knowledge regarding hydrogels based on HA, focusing on their biofunctional properties and delivery mechanisms. We present the latest developments in the research and development of HA-based hydrogels for the treatment of skin wounds."
Infections are severe complications associated with chronic wounds and tardy healing that should be timely treated to achieve rapid and proper tissue repair. To hinder such difficulties, a nanofibrous mat composed of polyvinyl alcohol and chitosan (PVA/CS) was developed by electrospinning method, containing thyme (Thymus vulgaris) and ginger (Zingiber officinale) extracts. The mat containing 10 wt% of the extracts (at the ratio of 50:50) exposed the nanofibers
(NFs) with the nanoscale diameter (average 382 ± 60 nm), smooth surface, and defect-free morphology. Likewise, the relevant analyses of the loaded mat displayed high wettability, porosity, and liquid absorption capacity without any adverse interaction. The obtained mat also provided a high antioxidant activity, and its release profile was continuous and sustained for nearly
72 hours. Besides, it inhibited the growth of both Gram-positive S.aureus and Gram-negative E.coli strains. Furthermore, the proposed mat significantly accelerated cutaneous wound healing in bacterial-infected rats by preventing bacteria growth at the wound site. At last, histopathology analysis confirmed the ample regeneration of skin structures, forming collagen fibers and appendages. Overall, the proposed mat containing ginger-thyme extracts provides multiple therapeutic capabilities with promising solutions for inhibiting wound infection and accelerating
the healing process.
Glycosaminoglycans (GAGs) are important for the occurrence of signaling molecules and maintenance of microenvironment within the extracellular matrix (ECM) in living tissues. GAGs and GAG‐based biomaterial approaches have been widely explored to promote in situ tissue regeneration and repair by regulating the wound microenvironment, accelerating re‐epithelialization, and controlling ECM remodeling. However, most approaches remain unacceptable for clinical applications. To improve insights into material design and clinical translational applications, this review highlights the innate roles and bioactive mechanisms of native GAGs during in situ wound healing and presents common GAG‐based biomaterials and the adaptability of application scenarios in facilitating wound healing. Furthermore, challenges before the widespread commercialization of GAG‐based biomaterials are shared, to ensure that future designed and constructed GAG‐based artificial biomaterials are more likely to recapitulate the unique and tissue‐specific profile of native GAG expression in human tissues. This review provides a more explicit and clear selection guide for researchers designing biomimetic materials, which will resemble or exceed their natural counterparts in certain functions, thereby suiting for specific environments or therapeutic goals.
Wound healing is a common occurrence. However, delayed healing and aberrant scarring result in pathological wound healing. Accordingly, a scarless wound healing remains a significant clinical challenge. In this study, we constructed hyaluronic acid (HA)-modified and verteporfin (VP)-loaded polylactic acid (PLA) nanogels (HA/VP-PLA) to promote scarless wound healing by accelerating wound re-epithelialization and controlling scar formation. Owing to the unique structure of HA incorporating and coating in VP-loaded PLA nanoparticles, HA/VP-PLA could be topically applied on wound to achieve targeted delivery to fibroblasts. Then, HA/VP-PLA released HA and lactic acid (LA) to stimulate the proliferation and migration of fibroblasts, as well as VP to inhibit Yes-associated protein (YAP) expression and nuclear localization to suppress fibrosis. In vitro (skin fibroblasts) and in vivo (rat and rabbit models) experiments strongly suggested that HA/VP-PLA promoted scarless wound healing by accelerating wound re-epithelialization and controlling scar formation. Therefore, our work provides a feasible strategy for scarless wound healing, and the sophisticated HA/VP-PLA exhibit a great potential for clinical applications.
The ECM (extracellular matrix) is a 3-dimensional network that supports cellular responses and maintains structural tissue integrity in healthy and pathological conditions. The interactions between ECM and cells trigger signaling cascades that lead to phenotypic changes and structural and compositional turnover of the ECM, which in turn regulates vascular cell behavior. Hydrogel biomaterials are a powerful platform for basic and translational studies and clinical applications due to their high swelling capacity and exceptional versatility in compositions and properties. This review highlights recent development and use of engineered natural hydrogel platforms that mimic the ECM and present defined biochemical and mechanical cues for vascularization. Specifically, we focus on modulating vascular cell stimulation and cell-ECM/cell-cell interactions in the microvasculature that are influenced by the established biomimetic microenvironment.
Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.
Bacterial infection and subsequent reactive oxygen species (ROS) damage are major factors that delay wound healing in infected skin. Recently, photothermal therapy (PTT), as a new antibacterial method, has shown great advantages in the treatment of infected skin wound. Antibacterial and antioxidant hydrogels can reduce bacterial colonization and infection, scavenge ROS, relieve inflammation, and accelerate wound healing. In this study, an enzyme-crosslinked hyaluronic acid-tyramine (HT) hydrogel loaded with antioxidant and photothermal silver nanoparticles (AgNPs), named HTA, was developed as functional wound dressing to promote the infected skin wound healing. Natural antioxidant tannic acids (TA) were used as both reducing and stabilizing agents to facilely synthesize the silver nanoparticles capped with TA (AgNPs@TA). The incorporation of AgNPs@TA significantly enhanced the antioxidant, antibacterial, photothermal antibacterial, adhesive, and hemostatic abilities of the resulted HTA hydrogel. Besides, HTA hydrogel has rapid gelation, well injection and biocompatibility. In vivo results on the Staphylococcus aureus and Escherichia coli co-infected mouse skin wound model showed that HTA0.4 (containing 0.4 mg/mL AgNPs@TA) hydrogel combined with near infrared ray radiation highly alleviated inflammation, promoted angiogenesis, and accelerated the healing process. Therefore, this nanocomposite hydrogel wound dressing with antibacterial and antioxidant capabilities has great application potential in the treatment of infected skin wounds.
The healing process of infected wounds was limited by bacterial infection, excessive reactive oxygen species (ROS) accumulation, and tissue hypoxia. In order to alleviate the above situations, herein, a copper-rich multifunctional ultra-small Prussian blue nanozymes ([email protected] NZs) was constructed for infected wound synergistic treatment. Firstly, hyaluronic acid was modified by branched polyethyleneimine which could form a complex with copper ions, to construct copper-rich Prussian blue nanozymes. Secondly, the [email protected] NZs have a uniform ultra-small nano size and excellent photothermal response performance, exhibition of multifunctional enzymatic activity and anti-inflammatory properties. Finally, the slow release of copper ions in the [email protected] NZs could effectively promote the formation of new blood vessels, thus giving it multifunctional properties. In vitro and in vivo experiments showed that it not only could effectively inhibit and kill bacteria under 808 nm near-infrared laser but also could remove excessive ROS, regulate oxygen levels, and anti-inflammation. More importantly, the release of copper ions could synergistically promote the healing of infected wounds as well as good biocompatibility. Overall, our studies provide a multifunctional strategy for infected wounds with synergistic treatment based on carrier construction.
Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-β1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-β1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.
The universal increase in the number of patients with nonhealing skin wounds imposes a huge social and economic burden on the patients and healthcare systems. Although, the application of traditional wound dressings contributes to an effective wound healing outcome, yet, the complexity of the healing process remains a major health challenge. Recent advances in materials and fabrication technologies have led to the fabrication of dressings that provide proper conditions for effective wound healing. The 3D‐printed wound dressings, biomolecule‐loaded dressings, as well as smart and flexible bandages are among the recent alternatives that have been developed to accelerate wound healing. Additionally, the new generation of wound dressings contains a variety of microelectronic sensors for real‐time monitoring of the wound environment and is able to apply required actions to support the healing progress. Moreover, advances in manufacturing flexible microelectronic sensors enable the development of the next generation of wound dressing substrates, known as electronic skin, for real‐time monitoring of the whole physiochemical markers in the wound environment in a single platform. The current study reviews the importance of smart wound dressings as an emerging strategy for wound care management and highlights different types of smart dressings for promoting the wound healing process.
Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
The use of drug delivery vehicles to improve the efficacy of drugs and to target their action at effective concentrations over desired periods of time has been an active topic of research and clinical investigation for decades. Both synthetic and natural drug delivery materials have facilitated locally controlled as well as targeted drug delivery. Extracellular matrix (ECM) molecules have generated widespread interest as drug delivery materials owing to the various biological functions of ECM. Hydrogels created using ECM molecules can provide not only biochemical and structural support to cells, but also spatial and temporal control over the release of therapeutic agents, including small molecules, biomacromolecules, and cells. In addition, the modification of drug delivery carriers with ECM fragments used as cell-binding ligands has facilitated cell-targeted delivery and improved the therapeutic efficiency of drugs through interaction with highly expressed cellular receptors for ECM. The combination of ECM-derived hydrogels and ECM-derived ligand approaches shows synergistic effects, leading to a great promise for the delivery of intracellular drugs, which require specific endocytic pathways for maximal effectiveness. In this review, we provide an overview of cellular receptors that interact with ECM molecules and discuss examples of selected ECM components that have been applied for drug delivery in both local and systemic platforms. Finally, we highlight the potential impacts of utilizing the interaction between ECM components and cellular receptors for intracellular delivery, particularly in tissue regeneration applications.
Non‐healing chronic wounds in the constantly growing elderly population represent a major public health problem with high socio‐economic burden. Yet the underlying mechanism of age‐related impairment of wound healing remains elusive. Here we show that the number of dermal cells expressing cyclin dependent kinase inhibitor p21 was elevated upon skin injury, particularly in aged population, in both man and mouse. The nuclear expression of p21 in activated wound fibroblasts delayed the onset of the proliferation phase of wound healing in a p53‐independent manner. Further, the local and transient inhibition of p21 expression by in vivo delivered p21‐targeting siRNA ameliorated the delayed wound healing in aged mice. Our results suggest that the increased number of p21+ wound fibroblasts enforces the age‐related compromised healing, and targeting p21 creates potential clinical avenues to promote wound healing in aged population. This article is protected by copyright. All rights reserved.
Macrophages are important for effective iron recycling and erythropoiesis, but they also play a crucial role in wound healing, orchestrating tissue repair. Recently, we demonstrated a significant accumulation of iron in healing wounds and a requirement of iron for effective repair. Herein, we sought to determine the influence of iron on macrophage function in the context of wound healing. Interestingly, wound macrophages extensively sequestered iron throughout healing, associated with a prohealing M2 phenotype. In delayed healing diabetic mouse wounds, both macrophage polarization and iron sequestration were impaired. In vitro studies revealed that iron promotes differentiation, while skewing macrophages toward a hypersecretory M2-like polarization state. These macrophages produced high levels of chemokine (C-C motif) ligands 17 and 22, promoting wound reepithelialization and extracellular matrix deposition in a human ex vivo wound healing model. Together, these findings reveal a novel, unappreciated role for iron in modulating macrophage behavior to promote subsequent wound repair. These findings support therapeutic evaluation of iron use to promote wound healing in the clinic.
Background: Hyaluronic acid (HA), a large glycosaminoglycan involved in proliferation, migration, and tissue repair, is suggested to be an important factor for keratinocyte activation and re-epithelialization. The experimental hypothesis of this study was that HA accelerates re-epithelialization, and we aimed to investigate the effect of exogenous intradermal HA during deep dermal, incisional wound healing in vivo in humans, the primary endpoint being re-epithelialization.
Methods: A total of 8 standardized deep dermal incisional wounds (depth 1.6 mm, width 1.8 mm) per subject were induced in 10 healthy volunteers. Two of the wound sites per subject were pretreated with injections of HA and 2 with saline solution. At 2 time points (24 hours and 14 days), 2 biopsies for each treatment group (one for histology and one for proteomics) were taken. Skin erythema was measured at 24-hour intervals for 14 days as a surrogate measurement of inflammation.
Results: At 24 hours, 8 of 9 wounds pretreated with HA showed complete re-epithelization, whereas none of the wounds pretreated with saline had re-epithelized. Wounds pretreated with HA also showed a 10-fold regulation of 8 identified proteins involved in wound healing compared to wounds treated with saline solution. No difference in inflammation, as measured as erythema, could be seen between any of the groups.
Conclusions: We conclude that HA accelerates re-epithelialization and stimulates an altered protein expression in vivo in human deep dermal incisional skin wounds, but has no effect on the inflammation process as measured by erythema.
Background
Human adipose stem cells (ASCs) have emerged as a promising treatment paradigm for skin wounds. Recent works demonstrate that the therapeutic effect of stem cells is partially mediated by extracellular vesicles, which comprise exosomes and microvesicles. In this study, we investigate the regenerative effects of isolated microvesicles from ASCs and evaluate the mechanisms how ASC microvesicles promote wound healing.
Methods
Adipose stem cell-derived microvesicles (ASC-MVs) were isolated by differential ultracentrifugation, stained by PKH26, and characterized by electron microscopy and dynamic light scattering (DLS). We examined ASC-MV effects on proliferation, migration, and angiogenesis of keratinocytes, fibroblasts, and endothelial cells both in vitro and in vivo. Next, we explored the underlying mechanisms by gene expression analysis and the activation levels of AKT and ERK signaling pathways in all three kinds of cells after ASC-MV stimulation. We then assessed the effect of ASC-MVs on collagen deposition, neovascularization, and re-epithelialization in an in vivo skin injury model.
Results
ASC-MVs could be readily internalized by human umbilical vein endothelial cells (HUVECs), HaCAT, and fibroblasts and significantly promoted the proliferation, migration, and angiogenesis of these cells both in vitro and in vivo. The gene expression of proliferative markers (cyclin D1, cyclin D2, cyclin A1, cyclin A2) and growth factors (VEGFA, PDGFA, EGF, FGF2) was significantly upregulated after ASC-MV treatment. Importantly, ASC-MVs stimulated the activation of AKT and ERK signaling pathways in those cells. The local injection of ASC-MVs at wound sites significantly increased the re-epithelialization, collagen deposition, and neovascularization and led to accelerated wound closure.
Conclusions
Our data suggest that ASC-MVs can stimulate HUVEC, HaCAT, and fibroblast functions. ASC-MV therapy significantly accelerates wound healing, and the benefits of ASC-MVs may due to the involvement of AKT and ERK signaling pathways. This illustrates the therapeutic potential of ASC-MVs which may become a novel treatment paradigm for cutaneous wound healing.
Electronic supplementary material
The online version of this article (10.1186/s13287-019-1152-x) contains supplementary material, which is available to authorized users.
Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale "cables" acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the non-enzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.
Three dimensional (3D) bioprinting has been proposed as a method for fabricating tissue engineered small diameter vascular prostheses. This technique not only involves constructing the structural features to obtain a desired pattern but the morphology of the pattern may also be used to influence the behavior of seeded cells. Herein, we 3D bioprinted a gelatin hydrogel microchannel construct to promote and preserve the contractile phenotype of vascular smooth muscle cells (vSMCs), which is crucial for vasoresponsiveness. The microchanneled surface of a gelatin hydrogel facilitated vSMC attachment and an elongated alignment along the microchannel direction. The cells displayed distinct F-actin anisotropy in the direction of the channel. The vSMC contractile phenotype was confirmed by the positive detection of contractile marker gene proteins (α-smooth muscle actin (α-SMA) and smooth muscle-myosin heavy chain (SM-MHC)). Having demonstrated the effectiveness of the hydrogel channels bioprinted on a film, the bioprinting was applied radially to the surface of a 3D tubular construct by integrating a rotating mandrel into the 3D bioprinter. The hydrogel microchannels printed on the 3D tubular vascular construct also orientated the vSMCs and strongly promoted the contractile phenotype. Together, our study demonstrated that microchannels bioprinted using a transglutaminase crosslinked gelatin hydrogel, could successfully promote and preserve vSMC contractile phenotype. Furthermore, the hydrogel bioink could be retained on the surface of a rotating polymer tube to print radial cell guiding channels onto a vascular graft construct.
Critical tissue defects frequently result from trauma, burns, chronic wounds and/or surgery. The ideal treatment for
such tissue loss is autografting, but donor sites are often limited. Tissue engineering (TE) is an inspiring alternative for
tissue repair and regeneration (TRR). One of the current state-of-the-art methods for TRR is gene therapy. Non-viral
gene delivery systems (nVGDS) have great potential for TE and have several advantages over viral delivery including
lower immunogenicity and toxicity, better cell specifcity, better modifability, and higher productivity. However, there
is no ideal nVGDS for TRR, hence, there is widespread research to improve their properties. This review introduces the
basic principles and key aspects of commonly-used nVGDSs. We focus on recent advances in their applications, current challenges, and future directions.
Oxygen deficiency after myocardial infarction (MI) leads to massive cardiac cell death. Protection of cardiac cells and promotion of cardiac repair are key therapeutic goals. These goals may be achieved by re-introducing oxygen into the infarcted area. Yet current systemic oxygen delivery approaches cannot efficiently diffuse oxygen into the infarcted area that has extremely low blood flow. In this work, we developed a new oxygen delivery system that can be delivered specifically to the infarcted tissue, and continuously release oxygen to protect the cardiac cells. The system was based on a thermosensitive, injectable and fast gelation hydrogel, and oxygen releasing microspheres. The fast gelation hydrogel was used to increase microsphere retention in the heart tissue. The system was able to continuously release oxygen for 4 weeks. The released oxygen significantly increased survival of cardiac cells under the hypoxic condition (1% O2) mimicking that of the infarcted hearts. It also reduced myofibroblast formation under hypoxic condition (1% O2). After implanting into infarcted hearts for 4 weeks, the released oxygen significantly augmented cell survival, decreased macrophage density, reduced collagen deposition and myofibroblast density, and stimulated tissue angiogenesis, leading to a significant increase in cardiac function.
The skin protects the body against harmful substances and microorganisms. When the skin is damaged, wound healing must be finely regulated to restore the normal function of skin tissue. Artocarpin (ARTO), a prenylated flavonoid purified from the plant Artocarpus communis, has been reported to have anti-inflammatory and anti-cancer properties. The aim of the present study was to evaluate the wound healing potential and therapeutic mechanism of ARTO. Immunohistochemical staining of neutrophils and macrophages and mouse cytokine array analysis demonstrated that ARTO accelerates inflammatory progression and subsequently decreases persistent inflammation. ARTO increases collagen production and increases human fibroblast proliferation and migration by activating the P38 and JNK pathways. Moreover, ARTO increases the proliferation and migration of human keratinocytes through the ERK and P38 pathways and augments human endothelial cell proliferation and tube formation through the Akt and P38 pathways. Together, our data suggested that ARTO enhances skin wound healing, possibly by accelerating the inflammatory phase and by increasing myofibroblast differentiation, proliferation and migration of fibroblasts and keratinocytes, collagen synthesis and maturation, re-epithelialization, and angiogenesis. These findings indicate that ARTO has potential as a potent therapeutic agent for the treatment of skin wounds.
Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a Design of Experiments (DOE) approach to determine the interaction between three input variables (number of cells/spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2 (PGE2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells/spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2 and VEGF production versus those formed with 10,000 cells/spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced 5-fold more PGE2 and 4-fold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model - demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. This article is protected by copyright. All rights reserved.
Arteries for bypass grafting are harvested either with neighboring tissue attached or as skeletonized vessels that are free of surrounding tissue. There are significant benefits to skeletonization, but reports suggest that skeletonized vessels may develop structural defects and are at risk for atherosclerosis. We investigated the specific short-term effects of skeletonization on carotid artery biomechanics and microanatomy in a rabbit model. Six carotid arteries were surgically skeletonized. To support healing, three of these received polyethylene glycol hydrogel injected along their exterior surfaces. M-mode ultrasonography was used to track circumferential cyclic strain in the skeletonized, hydrogel-treated, and contralateral vessels. On day 21, the arteries were harvested, and vessel structure was assessed by histology, immunofluorescence microscopy, two-photon elastin autofluorescence, and second harmonic generation (SHG) microscopy. Intimal-medial thickness appeared unaffected by skeletonization, but the SHG signals indicated significant changes in collagen turnover in the adventitia. Skeletonized arteries also exhibited significantly decreased radial compliance (circumferential cyclic strain dropped ∼30%) and decreased numbers of elastic laminae (9.1±0.4 to 2.3±0.4). Hydrogel treatment protected against these effects with treated vessels maintaining normal mechanical properties. These results indicate that arterial skeletonization triggers immediate effects on vessel remodeling and reduced vessel compliance resulting in specific tissue alterations within 21 days, but that these effects can be attenuated by the placement of hydrogel on the exterior surface of the skeletonized vessel. This article is protected by copyright. All rights reserved.
Growth factors (GFs) play vital roles in wound repair. Many GF therapies have reached clinical trials, but success has been hindered by safety concerns and a lack of efficacy. Previously, we presented an approach to produce protein factors in wound beds through localized gene delivery mediated by biomimetic peptides. Modification of polyethylenimine (PEI) DNA polyplexes with collagen-mimetic peptides (CMPs) enabled tailoring of polyplex release/retention and improved gene transfer activity in a cell-responsive manner. In this work, CMP-mediated delivery from collagen was shown to improve expression of platelet-derived growth factor?BB (PDGF-BB) and promote a diverse range of cellular processes associated with wound healing, including proliferation, extracellular matrix production, and chemotaxis. Collagens were pre-exposed to physiologically-simulating conditions (complete media, 37oC) for days to weeks prior to cell seeding to simulate the environment within typical wound dressings. In cell proliferation studies, significant increases in cell counts were demonstrated in collagen gels containing CMP-modified polyplex vs. unmodified polyplex, and these effects became most pronounced following prolonged pre-incubation periods of greater than a week. Collagen containing CMP-modified polyplexes also induced a 2-fold increase in gel contraction as well as enhanced directionality and migratory activity in response to cell-secreted PDGF-BB gradients. While these PDGF-BB-triggered behaviors were observed in collagens containing unmodified polyplexes, the responses withstood much longer preincubation periods in CMP-modified polyplex samples (10 d vs. <5 d). Furthermore, enhanced closure rates in an in vitro wound model suggested that CMP-based PDGF-BB delivery may have utility in actual wound repair and other regenerative medicine applications. This article is protected by copyright. All rights reserved.
From the moment we are born, every injury to the skin has the potential to form a scar, many of which can impair form and/or function. As such, scar management constitutes a billion-dollar industry. However, effectively promoting scarless wound healing remains an elusive goal. The complex interactions of wound healing contribute to our inability to recapitulate scarless wound repair as it occurs in nature, such as in fetal skin and the oral mucosa. However, many new advances have occurred in recent years, some of which have translated scientific findings from bench to bedside. In vivo lineage tracing has helped establish a variety of novel cellular culprits that may act as key drivers of the fibrotic response. These newly characterized cell populations present further targets for therapeutic intervention, some of which have previously demonstrated promising results in animal models. Here, we discuss several recent studies that identify exciting approaches for diminishing scar formation. Particular attention will also be paid to the canonical Wnt/β-catenin signaling pathway, which plays an important role in both embryogenesis and tissue repair. New insights into the differential effects of Wnt signaling on heterogeneous fibroblast and keratinocyte populations within the skin further demonstrate methods by which wound healing can be re-directed to a more fetal scarless phenotype.
Graphical abstractRecent approaches to reducing scar formation. Representation showing novel scientific approaches for decreasing scar formation, including the targeting of pro-fibrotic cell populations based on surface molecule expression (e.g. DPP4⁺ fibroblasts, ADAM12⁺ pericytes). Modulation of cellular mechanotransduction pathways are another means to reduce scar formation, both at the molecular level or, macroscopically with dressings designed to offload tension, at cutaneous wound sites (ADAM12 a disintegrin and metalloprotease 12, DPP4 dipeptidyl peptidase-4, FAK focal adhesion kinase)
Hyaluronic acid (HA), the main component of extracellular matrix, is considered one of the key players in the tissue regeneration process. It has been proven to modulate via specific HA receptors, inflammation, cellular migration, and angiogenesis, which are the main phases of wound healing. Studies have revealed that most HA properties depend on its molecular size. High molecular weight HA displays anti-inflammatory and immunosuppressive properties, whereas low molecular weight HA is a potent proinflammatory molecule. In this review, the authors summarize the role of HA polymers of different molecular weight in tissue regeneration and provide a short overview of main cellular receptors involved in HA signaling. In addition, the role of HA in 2 major steps of wound healing is examined: inflammation and the angiogenesis process. Finally, the antioxidative properties of HA are discussed and its possible clinical implication presented.
The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenviron-mental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.
Polyethylenimine (PEI) has been used as a vehicle to deliver genes to cancer cells and somatic cells. In this study, cationic polymers of PEI were shielded with anionic polymers of hyaluronic acid (HA) to safely and effectively deliver genes into human mesenchymal stem cells (hMSCs). HA interacted with CD44 in the plasma membranes of hMSCs to facilitate the internalization of HA-shielded PEI/pDNA complexes. The HA-shielded PEI/pDNA nanogels were confirmed by size changes, ζ-potential, and gel retardation assays. HA-shielded nanogels were easily internalized by hMSCs, and this was reduced by pretreatment with a specific monoclonal antibody that blocked CD44. By shielding PEI/pDNA complexes with HA, nanogels were easily internalized to hMSCs when it did not blocked by anti-CD44. These shielded nanogels were also easily internalized by HeLa cells, and this was reduced by pretreatment with an anti-CD44 monoclonal antibody. Following internalization of the SOX9 gene, chondrogenesis of hMSCs was increased, as determined by RT-PCR, real-time quantitative PCR, and histological analyses.
Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ∼75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.
A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.
Repair following injury involves a range of processes - such as re-epithelialization, scar formation, angiogenesis, inflammation, and more - that function, often together, to restore tissue architecture. MMPs carry out diverse roles in all of these activities. In this article, we discuss how specific MMPs act on ECM during two critical repair processes: re-epithelialization and resolution of scar tissue. For wound closure, we discuss how two MMPs - MMP1 in human epidermis and MMP7 in mucosal epithelia - facilitate re-epithelialization by cleaving different ECM or ECM-associated proteins to affect similar integrin:matrix adhesion. In scars and fibrotic tissues, we discuss that a variety of MMPs carry out a diverse range of activities that can either promote or limit ECM deposition. However, few of these MMP-driven activities have been demonstrated to be due a direct action on ECM.
Copyright © 2015. Published by Elsevier B.V.
Significance: When a cutaneous injury occurs, the wound heals via a dynamic series of physiological events, including coagulation, granulation tissue formation, re-epithelialization, and extracellular matrix (ECM) remodeling. The final stage can take many months, yet the new ECM forms a scar that never achieves the flexibility or strength of the original tissue. In certain circumstances, the normal scar is replaced by pathological fibrotic tissue, which results in hypertrophic or keloid scars. These scars cause significant morbidity through physical dysfunction and psychological stress. Recent Advances and Critical Issues: The cutaneous ECM comprises a complex assortment of proteins that was traditionally thought to simply provide structural integrity and scaffolding characteristics. However, recent findings show that the ECM has multiple functions, including, storage and delivery of growth factors and cytokines, tissue repair and various physiological functions. Abnormal ECM reconstruction during wound healing contributes to the formation of hypertrophic and keloid scars. Whereas adult wounds heal with scarring, the developing foetus has the ability to heal wounds in a scarless fashion by regenerating skin and restoring the normal ECM architecture, strength, and function. Recent studies show that the lack of inflammation in fetal wounds contributes to this perfect healing. Future Directions: Better understanding of the exact roles of ECM components in scarring will allow us to produce therapeutic agents to prevent hypertrophic and keloid scars. This review will focus on the components of the ECM and their role in both physiological and pathological (hypertrophic and keloid) cutaneous scar formation.
(Myo)fibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myo)fibroblasts are embedded in a sophisticated extracellular matrix (ECM) that they secrete, and a complex and interactive dialogue exists between (myo)fibroblasts and their microenvironment. In addition to the secretion of the ECM, (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myo)fibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myo)fibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis), or during aging, this dialogue between the (myo)fibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myo)fibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment.
Significance
Glycan-based interactions can organize the plasma membrane into specialized domains that perform unique functions. One of their major roles is to regulate the turnover of receptors on the cell membrane. However, there is no clear picture on how this occurs. In this work we visualize cell membrane micropatterning mediated by glycans using a combination of superresolution imaging techniques and dual-color single-particle tracking. We find that this micropatterning corrals the receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) into clathrin active regions, thereby increasing clathrin–receptor interactions, and potentially influencing clathrin-mediated endocytosis of DC-SIGN-bound ligands. We also establish that clathrin–receptor encounters do not occur in a random fashion and further substantiate the dynamic and transient behavior of clathrin interactions with their cargo before successful internalization.
Wound healing involves a series of carefully modulated steps, from initial injury and blood clot to the final reconstituted tissue or scar. A dynamic reciprocity exists throughout between the wound, blood elements, extracellular matrix, and the cells that participate in healing. Multiple cytokines and signal transduction pathways regulate these reactions.A major component throughout most of the process is hyaluronan, a straight-chain carbohydrate extracellular matrix polymer.Hyaluronan occurs in multiple forms, chain length being the only distinguishing difference between these sugar chains.Levels of hyaluronan in its high molecular weight form are prominent in the earliest stages of wound repair.Progressively more fragmented forms occur in a manner not previously appreciated.We outline here steps in the wound-healing cascade in which hyaluronan participates, as well as provide a review of its metabolism. Although described by necessity in a series of quantum steps, the healing process is constituted by a smooth continuum of overlapping reactions. The prevalence of hyaluronan in the wound, particularly in its early stages, termed initially as hexosamine-containing mucopolysaccharides, was pointed out over half a century ago by the Harvard surgeon, J. Engelbert Dunphy. It appears we are now returning to where we started.
Significance
Inducing the growth of new blood vessels by specific factors is an attractive strategy to restore blood flow in ischemic tissues. Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, yet clinical trials of VEGF gene delivery failed. Major challenges include the need to control the tissue distribution of factor dose and the duration of expression. Here, we developed a highly tunable fibrin-based platform to precisely control the dose and duration of VEGF protein delivery in tissues. Optimized delivery of fibrin-bound VEGF ensured normal, stable, and functional angiogenesis and improved perfusion of ischemic tissues, without genetic modification and with limited duration of VEGF delivery. These findings suggest a strategy to improve both safety and efficacy of therapeutic angiogenesis.
The generation of functional microvascular networks is critical for the development of advanced in vitro models to replicate pathophysiological conditions. Mural cells provide structural support to blood vessels and secrete biomolecules contributing to vessel stability and functionality. We investigated the role played by two endothelium-related molecules, angiopoietin (Ang-1) and transforming growth factor (TGF-β1), on bone marrow-derived human mesenchymal stem cell (BM-hMSC) phenotypic transition toward a mural cell lineage, both in monoculture and in direct contact with human endothelial cells (ECs), within 3D fibrin gels in microfluidic devices. We demonstrated that the effect of these molecules is dependent on direct heterotypic cell-cell contact. Moreover, we found a significant increase in the amount of α-smooth muscle actin in microvascular networks with added VEGF and TGF-β1 or VEGF and Ang-1 compared to networks with added VEGF alone. However, the addition of TGF-β1 generated a non-interconnected microvasculature, while Ang-1 promoted functional networks, confirmed by microsphere perfusion and permeability measurements. The presence of mural cell-like BM-hMSCs coupled with the addition of Ang-1 increased the number of network branches and reduced mean vessel diameter compared to EC only vasculature. This system has promising applications in the development of advanced in vitro models to study complex biological phenomena involving functional and perfusable microvascular networks.
Immune modulators play critical roles in the progression of wounds to normal or conversely delayed healing, through the regulation of normal tissue regrowth, scarring, inflammation, and growth factor expression. Many immune modulator recombinants are under active preclinical study or in clinical trial to promote improved acute or chronic wound healing and to reduce scarring. Viruses have evolved highly efficient immune modulators for the evasion of host-defensive immune responses that target and kill invasive viruses. Recent studies have proven that some of these virus-derived immune modulators can be used to promote wound healing with significantly improved speed and reduced scarring in rodent models. Mouse full-thickness excisional wound model is one of the most commonly used animal models used to study wound healing for its similarity to humans in the healing phases and associated cellular and molecular mechanisms. This chapter introduces this mouse dermal wound healing model in detail for application in studying viral immune modulators as new treatments to promote wound healing. Details of hydrogel, protein construction, and topical application methods for these therapeutic proteins are provided in this chapter.
Wound healing is a complex biological process that requires coordinated cell proliferation, migration, and extracellular matrix production/remodeling, all of which are inhibited/delayed in chronic wounds. In this study, a formulation was developed that marries a fibrin-based, provisional-like matrix with collagen mimetic peptide (CMP)/PDGF gene-modified collagens, leading to the formation of robust gels that supported temporally controlled PDGF expression and facile application within the wound bed. Analysis employing in vitro co-gel scaffolds confirmed sustained and temporally controlled gene release based on matrix metalloproteinase (MMP) activity, with ~30% higher PDGF expression in MMP producing fibroblasts as-compared with non-MMP-expressing cells. The integration of fibrin with the gene-modified collagens resulted in co-gels that strongly supported both fibroblast cell recruitment/invasion as well as multiple aspects of the longer-term healing process. The excisional wound healing studies in mice established faster wound closure using CMP-modified PDGF polyplex-loaded co-gels, which exhibited up to 24% more wound closure (achieved with ~2 orders of magnitude lower growth factor dosing) after 9 days as compared to PDGF-loaded co-gels, and 19% more wound closure after 9 days as compared to CMP-free polyplex loaded co-gels. Moreover, minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation was observed following CMP-modified PDGF polyplex-loaded co-gel application on wounds. Taken together, the combined properties of the co-gels, including their stability and capacity to control both cell recruitment and cell phenotype within the murine wound bed, strongly supports the potential of the co-gel scaffolds for improved treatment of chronic non-healing wounds.
Hyaluronic acid (HA), a non-sulfated glycosaminoglycan (GAG), is a major component of skin extracellular matrix (ECM) and it is involved in the inflammatory response, angiogenesis, and tissue regeneration process. Due to the intrinsic properties of HA (such as biocompatibility, biodegradability and hydrophilic character), it has been used to produce different wound dressings, namely sponges, films, hydrogels, and electrospun membranes. Herein, an overview of the different HA-based wound dressings that have been produced so far is provided as well as the future directions regarding the strategies aimed to improve the mechanical stability of HA-based wound dressings, along with the incorporation of biomolecules intended to ameliorate their biological performance during the healing process.
Growth factors play a crucial role in tissue engineering by directing the fate of cells and allowing the formation of tissues. Understanding the key requirements for growth factor delivery can point the way towards novel tissue engineering platforms. Growth factor suffer from a tendency to lose their bioactivity upon environmental and physical changes. The need for biomaterials that can preserve the native form of growth factor has directed interest towards hydrogels (e.g. gelatin, alginate) and hydrophobic polymers (e.g. polycaprolactone). In recent advances, the use of composite materials such as PCL-chitosan (hydrogel-hydrophobic polymer) and gelatin-alginate (hydrogel-hydrogel polymers) has opened the possibility of fine tuning the delivery systems. Other current research is exploring new delivery strategies for growth factors such as the use of bioreactors and delivery by molecule-recognition. In particular, systems that allow the delivery of growth factors in a 3-dimensional manner are promising compared to conventional methods. This review analyses the biological considerations, material selection, and delivery strategies that have been established as the pivotal components for growth factor delivery and will support the next generation of tissue engineering platforms by providing a comprehensive landscape for the growth factor delivery field. The last part of the review discusses the current challenges and promising future directions for delivering growth factors.
Hyaluronic acid (HA), also termed hyaluronan, is a key extracellular matrix glycosaminoglycan that not only provides tissue hydration, but also shows intrinsic bioactivity towards cellular proliferation, differentiation and inflammatory response. These properties make HA attractive both as a potential target for the treatment of several diseases and as an effective biomaterial to control cell behavior. This concise review aims at emphasizing the increasing role of HA as a promising biomaterial for advanced medical applications that include, among others, the development of targeted therapies for cancer treatment and the development of scaffolds for regenerative medicine. Although the bioactive mechanisms of HA are not fully understood, its molecular weight and purity are well known to affect the cellular and immunological responses to HA-based products. Furthermore, in spite of the complex translation of novel HA-based medical devices from the lab bench to the clinic, a rapid growth of the HA market is foreseen in the next future, also due to the growing aging of the population and the increasing demand for anti-aging products. In this perspective, the industrial HA production is briefly addressed, with reference to the technological issues that are currently undergoing extensive research to improve the HA yield and quality, in an attempt to meet the quickly rising HA demand.
Significance: A 2018 retrospective analysis of Medicare beneficiaries identified that ∼8.2 million people had wounds with or without infections. Medicare cost estimates for acute and chronic wound treatments ranged from 96.8 billion. Highest expenses were for surgical wounds followed by diabetic foot ulcers, with a higher trend toward costs associated with outpatient wound care compared with inpatient. Increasing costs of health care, an aging population, recognition of difficult-to-treat infection threats such as biofilms, and the continued threat of diabetes and obesity worldwide make chronic wounds a substantial clinical, social, and economic challenge. Recent Advances: Chronic wounds are not a problem in an otherwise healthy population. Underlying conditions ranging from malnutrition, to stress, to metabolic syndrome, predispose patients to chronic, nonhealing wounds. From an economic point of view, the annual wound care products market is expected to reach $15-22 billion by 2024. The National Institutes of Health's (NIH) Research Portfolio Online Reporting Tool (RePORT) now lists wounds as a category. Future Directions: A continued rise in the economic, clinical, and social impact of wounds warrants a more structured approach and proportionate investment in wound care, education, and related research.
Growth factors (GFs) are versatile signalling molecules that orchestrate the dynamic, multi-stage process of wound healing. Delivery of exogenous GFs to the wound milieu to mediate healing in an active, physiologically-relevant manner has shown great promise in laboratories; however, the inherent instability of GFs, accompanied with numerous safety, efficacy and cost concerns, has hindered the clinical success of GF delivery. In this article, we highlight that the key to overcoming these challenges is to enhance the control of the activities of GFs throughout the delivering process. We summarise the recent strategies based on biomaterials matrices and molecular engineering, which aim to improve the conditions of GFs for delivery (at the 'supply' end of the delivery), increase the stability and functions of GFs in extracellular matrix (in transportation to target cells), as well as enhance the GFs/receptor interaction on the cell membrane (at the 'destination' end of the delivery). Many of these investigations have led to encouraging outcomes in various in vitro and in vivo regenerative models with considerable translational potential.
The contribution of epithelial-to-mesenchymal transition (EMT) to the pro-fibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on pro-fibrotic conditions including stiff substrata and TGF-β1, and analyzed in terms of morphology, stiffness and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype upon TGF-β1 stimulation. Yet, IPF-myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAK(Y397) activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAK(Y397) hyperactivation may underlie the aberrant mechanobiology of IPF-fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF-myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from non-activated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAK(Y397) may rescue normal mechanobiology in IPF.
Statement of significance:
In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release over nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy.
Adventitial fibroblasts (AFs) are key determinants of arterial function and critical mediators of arterial disease progression. The effects of altered stiffness, particularly those observed across individuals during normal vascular function, and the mechanisms by which AFs respond to altered stiffness, are not well understood. To study the effects of matrix stiffness on AF phenotype, cytokine production, and the regulatory pathways utilized to interpret basic cell-matrix interactions, human aortic AFs were grown in 5%, 7.5%, and 10% (w/v%) PEG-based hydrogels with Young's moduli of 1.2, 3.3, and 9.6 kPa, respectively. In 5% gels, AFs had higher proliferation rates, elevated monocyte chemoattractant protein-1 secretion, and enhanced monocyte recruitment. Significantly more AFs were α-smooth muscle actin positive in 7.5% gels, indicating myofibroblast development. AFs in 10% gels had low proliferation rates but produced high levels of interleukin-6 and vascular endothelial growth factor-A. Importantly, these modulus-dependent changes in AF phenotype were accompanied by alterations in the mitogen-activated protein kinase (MAPK) pathways contributing to the production of cytokines. These data indicate that complex cell regulatory changes occur with altered tissue stiffness and suggest that therapeutics affecting MAPK pathways may have altered effects on AFs depending on substrate stiffness.
The proteolytic processing of collagen (collagenolysis) is critical in development and homeostasis, but also contributes to numerous pathologies. Mammalian interstitial collagenolytic enzymes include members of the matrix metalloproteinase (MMP) family and cathepsin K. While MMPs have long been recognized for their ability to catalyze the hydrolysis of collagen, the roles of individual MMPs in physiological and pathological collagenolysis are less defined. The use of knockout and mutant animal models, which reflect human diseases, has revealed distinct collagenolytic roles for MT1-MMP and MMP-13. A better understanding of temporal and spatial collagen processing, along with the knowledge of the specific MMP involved, will ultimately lead to more effective treatments for cancer, arthritis, cardiovascular conditions, and infectious diseases. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.
The extracellular matrix (ECM) is a network of secreted proteins that, beyond providing support for tissues and organs, is involved in the regulation of a variety of cell functions, including cell proliferation, polarity, migration and oncogenic transformation. ECM homeostasis is maintained through a tightly controlled balance between synthesis, deposition and degradation. While the role of metalloproteases in ECM degradation is widely recognised, the contribution of ECM internalisation and intracellular degradation to ECM maintenance has been mostly overlooked. In this review, I will summarise what is known about the molecular mechanisms mediating ECM endocytosis and how this process impacts on diseases, such as fibrosis and cancer.
There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels.
Gene delivery is an important tool to study and manipulate human pluripotent stem cells for regenerative medicine purposes. Yet current methods of transient gene delivery to stem cells are still inefficient. Through the combination of biologically based concepts and material design, we aim to develop new methods to enhance the efficiency of gene delivery to stem cells. Specifically, we use poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PVBLG-8), a membrane-active helical, cationic polypeptide, to condense plasmid DNA to form stable nanocomplexes, which are further coated with hyaluronic acid (HA). HA not only shields the positive charges of PVBLG-8 to reduce toxicity, but also acts as a targeting moiety for cell surface receptor CD44, which binds HA and facilitates the internalization of the nanocomplexes. Upon entering cells, HA is degraded by hyaluronidase in endosomes and PVBLG-8 is exposed, facilitating the endosomal escape of DNA/polypeptide complex. Our studies show that the coating of HA significantly increases gene transfection efficiency of DNA/PVBLG-8 nanocomplexes from about 28 to 36% with largely reduced toxicity.
Data from registries can be especially useful in the evaluation of healthcare effectiveness. Thus, the goal of this study was to report on use of the US Wound Registry to investigate the outcomes of a broad population of patients undergoing treatment. Using a 5-year slice of de-identified data from electronic health records originating from 59 hospital-based outpatient wound centers in 18 states, outcomes, pa- tient and wound variables, and costs for facility and physician fees and procedures were analyzed for 5240 patients with 7099 wounds. Mean patient age was 61.7 years with 52.3% being male and the majority Caucasian (73.1%) and Medicare beneficiaries (52.6%). The mean number of serious comorbid conditions per patient was 1.8, with the most common being diabetes (46.8%), obese or overweight (71.3%), and having cardiovascular or peripheral vascular disease (51.3%). More than 1.6% of patients died in service or within 4 weeks of the last visit. Almost two thirds of wounds healed (65.8%) with an aver- age time to heal of 15 weeks and 10% of wounds taking 33 weeks or more to heal. The average wound surface area was 19.5 cm2. Half of wounds that healed did so with only the use of moist wound care (50.8%) and without the need for advanced therapeutics. Mean cost to heal per wound was 9358). This Registry would seem ideal for comparative effectiveness research in wound care, as it includes patients often ex- cluded from randomized controlled trials and reflects actual practice.
Treatment with vascular endothelial growth factor (VEGF) to reduce ischemia and enhance both endogenous muscle repair and regenerative cell therapy in Duchenne muscular dystrophy (DMD) has been widely proposed in recent years. However, the interaction between angiogenesis and fibrosis, a hallmark feature of DMD, remains unclear. To date, it has not been determined whether VEGF exerts a pro-fibrotic effect on DMD-derived fibroblasts, which may contribute to further disease progression. Thus, the purpose of this study was to investigate the effect of exogenous VEGF on fibroblast cultures established from a murine model of DMD. Primary fibroblast cultures were established from gastrocnemius and diaphragm muscles of 10 week-old mdx/utrn+/- mice. Quantitative polymerase chain reaction (qPCR) was employed to assess changes in transcript expression of alpha-smooth muscle actin (Acta2), type-1 collagen (Col1a1), connective tissue growth factor (Ctgf/ccn2) and fibronectin (Fn1). Immunofluorescence and Western blot analysis was further employed to visualize changes in protein expression of alpha-smooth muscle actin (α-SMA), CTGF/CCN2 and fibronectin. mRNA levels of Col1a1, Ctgf/ccn2, and FN did not increase following treatment with VEGF in fibroblasts derived from either diaphragm or gastrocnemius muscles. Acta2 expression increased significantly in diaphragm-derived fibroblasts following treatment with VEGF. Morphological assessment revealed increased stress fiber formation in VEGF-treated fibroblasts compared to the untreated control fibroblasts. The findings from this study suggest that further investigation into the effect of VEGF on fibroblast function is required prior to the utilization of the growth factor as a treatment for DMD.
The treatment of impaired wounds requires the use of biomaterials that can provide mechanical and biological queues to the surrounding environment to promote angiogenesis, granulation tissue formation, and wound closure. Porous hydrogels show promotion of angiogenesis, even in the absence of proangiogenic factors. It is hypothesized that the added delivery of nonviral DNA encoding for proangiogenic growth factors can further enhance this effect. Here, 100 and 60 μm porous and nonporous (n-pore) hyaluronic acid-MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used to investigate scaffold-mediated gene delivery for local gene therapy in a diabetic wound healing mouse model. Porous hydrogels allow for significantly faster wound closure compared with n-pore hydrogels, which do not degrade and essentially provide a mechanical barrier to closure. Interestingly, the delivery of pDNA/PEI polyplexes positively promotes granulation tissue formation even when the DNA does not encode for an angiogenic protein. And although transfected cells are present throughout the granulation tissue surrounding, all hydrogels at 2 weeks, pVEGF delivery does not further enhance the angiogenic response. Despite this, the presence of transfected cells shows promise for the use of polyplex-loaded porous hydrogels for local gene delivery in the treatment of diabetic wounds.
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The use of collagen-based biomaterials in regenerative medicine has rapidly increased over the past decade. The unique structural and biochemical properties of collagen make it a particularly promising material for delivering both protein and DNA-based therapeutics. Although many collagen modification techniques have been developed, the majority of them require multi-step chemical treatments that can modify the natural favourable properties of collagen. We have developed a promising biomimetic modification technique employing collagen-mimetic peptides (CMP)s to control the retention and delivery of DNA polyplexes from collagen structures, including both monomeric 2-D collagen films and fibrous, 3-D gels. Variations in the concentration of CMPs displayed on polyplexes enabled tuning of polyplex retention vs. release over periods of at least 2 weeks on films and a month on gels. Retention of CMP-modified polyplexes (20 days) was substantially improved compared to non-modified polyplexes, which were retained for only 2 days. The activity of bound polyplex in collagen gels was shown, through a series of transfection studies, to be maintained in the presence of serum for a minimum of 2 weeks. Only matrix metalloproteinase (MMP)-stimulated cells exhibited significant levels of transfection suggesting that cell mobility within the gel was vital and that collagen remodelling played a role in stimulating gene release and expression. To our knowledge, this study is the first to deliver genes with CMP-modified polyplexes and to examine the effects of CMP display on DNA release. The results suggest that this technique may be used more broadly to create tuneable, collagen-based delivery systems.
Significance: Angiogenesis, the growth of new blood vessels from existing vessels, is an important aspect of the repair process. Restoration of blood flow to damaged tissues provides oxygen and nutrients required to support the growth and function of reparative cells. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic growth factors in the skin, and the amount of VEGF present in a wound can significantly impact healing. Recent Advances: The activity of VEGF was once considered to be specific for endothelial cells lining the inside of blood vessels, partly because VEGF receptor (VEGFR) expression was believed to be restricted to endothelial cells. It is now known, however, that VEGFRs can be expressed by a variety of other cell types involved in wound repair. For example, keratinocytes and macrophages, which both carry out important functions during wound healing, express VEGFRs and are capable of responding directly to VEGF. Critical Issues: The mechanisms by which VEGF promotes angiogenesis are well established. Recent studies, however, indicate that VEGF can directly affect the activity of several nonendothelial cell types present in the skin. The implications of these extra-angiogenic effects of VEGF on wound repair are not yet known, but they suggest that this growth factor may play a more complex role during wound healing than previously believed. Future Directions: Despite the large number of studies focusing on VEGF and wound healing, it is clear that the current knowledge of how VEGF contributes to the repair of skin wounds is incomplete. Further research is needed to obtain a more comprehensive understanding of VEGF activities during the wound healing process.