Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns.

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase's ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

Regenerative Effects of Hypoxia Primed Flowable Placental Formulation in Muscle and Dermal Injury.

The placental tissue, due to its angiogenic, anti-inflammatory, antioxidative, antimicrobial, and anti-fibrotic properties, has become a compelling source towards a solution for several indications in regenerative medicine. However, methods to enhance and capture the therapeutic properties with formulations that can further the applications of viable placental tissue have not been explored. In this study, we investigated the regenerative effects of a hypoxia primed flowable placental formulation (FPF), composed of amnion/chorion and umbilical tissue, in two in vivo injury models. Laser Doppler data from rodent ischemia hindlimbs treated with FPF revealed significant tissue perfusion improvements compared to control ischemic hindlimbs. To further corroborate FPF's effects, we used a rodent ischemic bipedicle skin flap wound model. FPF treatment significantly increased the rate of wound closure and the quality of wound healing. FPF-treated wounds displayed reduced inflammation and an increase in angiogenesis. Furthermore, quantitative PCR and next-generation sequencing analysis confirmed these changes in the FPF-treated group at both the gene and transcriptional level. The observed modulation in miRNAs was associated with angiogenesis, regulation of inflammatory microenvironment, cell migration and apoptosis, reactive oxygen species generation, and restoring epithelial barrier function, all processes involved in impaired tissue healing. Taken together, these data validate the tissue regenerative properties of the flowable placental formulation configuration tested.

Comprehensive Comparison of Amnion Stromal Cells and Chorion Stromal Cells by RNA-Seq.

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.

Exosomes Secreted from Amniotic Membrane Contribute to Its Anti-Fibrotic Activity.

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFß1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFß1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes.

Structural and Functional Equivalency Between Lyopreserved and Cryopreserved Chorions with Viable Cells.

Objective: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. Approach: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. Results: Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. Innovation: Characterization of structural and functional properties of VLCM is reported for the first time. Conclusion: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.

A Flowable Placental Formulation Prevents Bleomycin-Induced Dermal Fibrosis in Aged Mice.

Fibrosis, the thickening and scarring of injured connective tissue, leads to a loss of organ function. Multiple cell types, including T-cells, macrophages, fibrocytes, and fibroblasts/myofibroblasts contribute to scar formation via secretion of inflammatory factors. This event results in an increase in oxidative stress and deposition of excessive extracellular matrix (ECM), characteristic of fibrosis. Further, aging is known to predispose connective tissue to fibrosis due to reduced tissue regeneration. In this study, we investigated the anti-fibrotic activity of a flowable placental formulation (FPF) using a bleomycin-induced dermal fibrosis model in aged mice. FPF consisted of placental amnion/chorion- and umbilical tissue-derived ECM and cells. The mice were injected with either FPF or PBS, followed by multiple doses of bleomycin. Histological assessment of FPF-treated skin samples revealed reduced dermal fibrosis, inflammation, and TGF-ß signaling compared to the control group. Quantitative RT-PCR and Next Generation Sequencing analysis of miRNAs further confirmed anti-fibrotic changes in the FPF-treated group at both the gene and transcriptional levels. The observed modulation in miRNAs was associated with inflammation, TGF-ß signaling, fibroblast proliferation, epithelial-mesenchymal transition and ECM deposition. These results demonstrate the potential of FPF in preventing fibrosis and may be of therapeutic benefit for those at higher risk of fibrosis due to wounds, aging, exposure to radiation and genetic predisposition.

A Viable Lyopreserved Amniotic Membrane Modulates Diabetic Wound Microenvironment and Accelerates Wound Closure.

Objective: Wound healing is a complex process involving the dynamic interplay of various types of cells and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of the process, resulting in non-healing chronic wounds. Human amniotic membrane (AM) has been shown to be effective in the management of chronic wounds. Recently, a viable lyopreserved AM (VLAM) has been developed. The VLAM retains the structural, molecular, and functional properties of fresh AM with the advantage of a long shelf life for living tissue at ambient temperatures. The objective of this study was to evaluate the effects of VLAM on the impaired wound microenvironment and wound closure in db/db mice. Approach: VLAM or saline gel (control) was applied weekly to 7-mm excisional wounds in diabetic (db/db) mice. Wound appearance and size were assessed weekly. Inflammation and redox state in wounds were tested by cytokine gene and protein expression, and by catalase and glutathione peroxidase activities, respectively. Wound tissue granulation and neovascularization were assessed histologically. Results: Diabetic wounds treated with VLAM closed faster than control wounds. On an average, VLAM-treated wounds closed 4 days faster than the control wounds, with a significantly faster rate of closure at days 7 and 14 as compared with control wounds. The faster closure correlated with a decrease in the expression of proinflammatory factors and oxidative stress, and an increase in angiogenesis and dermal thickness. Innovation: Effects of VLAM on a chronic wound microenvironment and underlying molecular mechanisms were investigated for the first time. Conclusion: VLAM accelerates wound closure in db/db mice by decreasing inflammation and oxidative stress and supporting wound tissue granulation, neovascularization, and re-epithelialization.

Endogenous viable cells in lyopreserved amnion retain differentiation potential and anti-fibrotic activity in vitro.

Human amniotic membrane (AM) has intrinsic anti-inflammatory, anti-fibrotic and antimicrobial properties. Tissue preservation methods have helped to overcome the short shelf life of fresh AM allowing "on demand" use of AM grafts. Cryopreserved AM that retains all native tissue components, including viable cells, has clinical benefits in treating chronic wounds. However, cryopreservation requires ultra-low temperature storage, limiting the use of cryopreserved products. To overcome this limitation, a new lyopreservation method has been developed for ambient storage of living tissues. The goal of this study was to investigate the viability and functionality of AM cells following lyopreservation. Fresh AM and devitalized lyopreserved AM (DLAM) served as positive and negative controls, respectively. Using live/dead staining, we confirmed the presence of living cells in viable lyopreserved AM (VLAM) and showed that these cells persisted up to 21 days in culture medium. The functionality of cells in VLAM was assessed by their differentiation potential and anti-fibrotic activity in vitro. With osteogenic induction, cells in VLAM deposited calcium within the membrane, a marker of osteogenic cells, in a time-dependent manner. The migration of human lung fibrotic fibroblasts in a scratch wound assay was reduced significantly in the presence of VLAM-derived conditioned medium. Quantitative PCR analyses indicated that VLAM reduced the expression of pro-fibrotic factors such as type I collagen and increased the expression of anti-fibrotic factors such as hepatocyte growth factor and anti-fibrotic microRNA in fibrotic fibroblasts. Taken together, these results demonstrate that endogenous cells in VLAM remain viable and functional post-lyophilization. STATEMENT OF SIGNIFICANCE: This study, for the first time, provides direct evidence showing that tissue viability and functional cells can be preserved by lyophilization. Similar to fresh amniotic membrane (AM), viable lyopreserved AM (VLAM) retains viable cells for extended periods of time. More importantly, these cells are functional and maintain their osteogenic differentiation potential and anti-fibrotic activity. Our results confirmed that the novel lyophilization method preserves tissue viability.

Viable cryopreserved umbilical tissue (vCUT) reduces post-operative adhesions in a rabbit abdominal adhesion model.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) "point of care" allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at -80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.

Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage.

Human amniotic membrane (AM) has a long history of clinical use for wound treatment. AM serves as a wound protective barrier maintaining proper moisture. AM is anti-inflammatory, anti-microbial and antifibrotic, and supports angiogenesis, granulation tissue formation and wound re-epithelialization. These properties of AM are attributed to its native extracellular matrix, growth factors, and endogenous cells including mesenchymal stem cells. Advances in tissue preservation have helped to overcome the short shelf life of fresh AM and led to the development of AM products for clinical use. Viable cryopreserved amnion (VCAM), which retains all native components of fresh AM, has shown positive outcomes in clinical trials for wound management. However, cryopreservation requires ultra-low temperature storage and shipment that limits widespread use of VCAM. We have developed a lyopreservation technique to allow for ambient storage of living tissues. Here, we compared the structural, molecular, and functional properties of a viable lyopreserved human amniotic membrane (VLAM) with properties of VCAM using in vitro and in vivo wound models. We found that the structure, growth factors, and cell viability of VLAM is similar to that of VCAM and fresh AM. Both, VCAM and VLAM inhibited TNF-α secretion and upregulated VEGF expression in vitro under conditions designed to mimic inflammation and hypoxia in a wound microenvironment, and resulted in wound closure in a diabetic mouse chronic wound model. Taken together, these data demonstrate that VLAM structural and functional properties are equivalent to VCAM but without the constraints of ultra-low temperature storage.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens.

Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

Insulin regulates multiple signaling pathways leading to monocyte/macrophage chemotaxis into the wound tissue.

Wound healing is a complex process that involves sequential phases that overlap in time and space and affect each other dynamically at the gene and protein levels. We previously showed that insulin accelerates wound healing by stimulating faster and regenerative healing. One of the processes that insulin stimulates is an increase in monocyte/macrophage chemotaxis. In this study, we performed experiments in vivo and in vitro to elucidate the signaling transduction pathways that are involved in insulin-induced monocyte/macrophage chemotaxis. We found that insulin stimulates THP-1 cell chemotaxis in a dose-dependent and insulin receptor-dependent manner. We also show that the kinases PI3K-Akt, SPAK/JNK, and p38 MAPK are key molecules in the insulin-induced signaling pathways that lead to chemoattraction of the THP-1 cell. Furthermore, both PI3K-Akt and SPAK/JNK signaling involve Rac1 activation, an important molecule in regulating cell motility. Indeed, topical application of Rac1 inhibitor at an early stage during the healing process caused delayed and impaired healing even in the presence of insulin. These results delineate cell and molecular mechanisms involved in insulin-induced chemotaxis of monocyte/macrophage, cells that are critical for proper healing.

Understanding the Impact of Preservation Methods on the Integrity and Functionality of Placental Allografts.

INTRODUCTION: Human placental membranes (hPMs) have a long history in treating burns and wounds. The composition of hPMs includes structural matrix, growth factors, and neonatal cells, all of which contribute to their regenerative potential. However, most hPM products are devitalized after dehydration and irradiation. We compared the functionality of single-layer viable cryopreserved human amniotic membrane (vCHAM) with multilayer devitalized dehydrated human amnion/chorion membrane (dHACM) in wound-relevant models to determine the effect of different processing methods on hPMs. METHODS: Viable cryopreserved human amniotic membrane and dHACM were compared with fresh hPM for structural integrity and viability. Viable cell persistence in vCHAM over time was evaluated in vitro and in vivo in a diabetic chronic wound mouse model. Proliferation of cells within fresh hPM and vCHAM was evaluated with bromodeoxyuridine and Ki-67 staining, and proliferation of isolated cells in culture was evaluated. Growth factor release over time and in vitro response to chronic wound stimuli (tumor necrosis factor α, lipopolysaccharide, and hypoxia) were used to compare the functionality of vCHAM and dHACM. RESULTS: The structure and thickness of fresh hPM were retained in vCHAM but were compromised in dHACM. Similar to fresh hPM, vCHAM contained viable cells, whereas dHACM did not. Cells in vCHAM remained viable after 4 and 7 days in culture and in an in vitro chronic wound environment and after 4 and 8 days in vivo after application to a mouse chronic wound. Staining for bromodeoxyuridine and Ki-67 did not reveal proliferative cells within fresh hPM and vCHAM. However, isolated cells proliferated in culture. Viable cryopreserved human amniotic membrane increased platelet-derived growth factor BB, hepatocyte growth factor, and epidermal growth factor levels over time and responded to chronic wound stimuli in vitro by significantly increasing levels of vascular endothelial growth factor and prostaglandin E2. Dehydrated human amnion/chorion membrane showed no significant accumulation of growth factors and did not respond to chronic wound stimuli. CONCLUSIONS: These results indicate that vCHAM retains intact, native matrix, and viable, active cells and responds to chronic wound stimuli in vitro. The inclusion of multiple layers of hPM does not compensate for structural degradation and loss of viability caused by dehydration as evidenced by a lack of functional response by dHACM. The clinical significance of these results remains to be answered.

Platelet Hyperactivity in TNFSF14/LIGHT Knockout Mouse Model of Impaired Healing.

Objective: Impaired and chronic wounds occur due to defects in one or more of the overlapping stages of healing. However, problems related to the vascular system are critical for nonhealing, and chronic wounds in humans often show the presence of fibrin cuffs/clots. We hypothesized that these clots are due to alterations in platelet function; hence, we have investigated whether alterations in platelet function are present during impaired healing. Approach: Platelets were subjected to different agonists to determine the rate of aggregation and evaluate the molecules involved in adhesion and aggregation that could lead to faster thrombosis and potentially contribute to impaired wound healing. Results: We show that wounding of TNFSF14/LIGHT-/- mice, which have impaired healing, leads to an enhanced response in platelet aggregation and a faster time to blood vessel occlusion (thrombosis). In addition, after wounding, platelets from these mice have increased levels of P-selectin, integrin αIIbß3, and phosphatidylserine, molecules that contribute to platelet adhesion. They also have more extensive open canalicular system than platelets of control mice, suggesting increased surface area for interactions upon activation. Innovation: These results show a novel function for TNFSF14/LIGHT during wound healing. Conclusion: The absence of TNFSF14/LIGHT from the cell surface of platelets causes rapid platelet aggregation and thrombus formation that may contribute to impaired healing by reducing the ability of the blood vessels to transport nutrients and oxygen and other molecules needed for proper healing.

Tobacco toxins deposited on surfaces (third hand smoke) impair wound healing.

Third hand smoke (THS) is the accumulation of second hand smoke (SHS) toxins on surfaces in homes, cars, clothing and hair of smokers. It is known that 88M US nonsmokers ≥3 years old living in homes of smokers are exposed to THS toxicants and show blood cotinine levels of ≥0.05 ng/ml, indicating that the toxins are circulating in their circulatory systems. The goal of the present study is to investigate the mechanisms by which THS causes impaired wound healing. We show that mice living under conditions that mimic THS exposure in humans display delayed wound closure, impaired collagen deposition, altered inflammatory response, decreased angiogenesis, microvessels with fibrin cuffs and a highly proteolytic wound environment. Moreover, THS-exposed mouse wounds have high levels of oxidative stress and significantly lower levels of antioxidant activity leading to molecular damage, including protein nitration, lipid peroxidation and DNA damage that contribute to tissue dysfunction. Furthermore, we show that elastase is elevated, suggesting that elastin is degraded and the plasticity of the wound tissue is decreased. Taken together, our results lead us to conclude that THS toxicants delay and impair wound healing by disrupting the sequential processes that lead to normal healing. In addition, the lack of elastin results in loss of wound plasticity, which may be responsible for reopening of wounds.

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats.

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischaemia, inflammation and infection costing $7.5 billion/year in the U.S.A. alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA [poly(D,L-lactic-co-glycolic acid)] microparticles that provides a sustained release of bioactive insulin for >20 days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring healing. Using heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04 mg insulin/cm(2) every 3 days for 9 days have faster closure, a higher rate of disintegration of dead tissue and decreased oxidative stress. In addition, in insulin-treated wounds, the pattern of neutrophil inflammatory response suggests faster clearing of the burned dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibres organized more like a basket weave (normal skin) than aligned and cross-linked (scar tissue). In summary, application of ASD-containing insulin-loaded PLGA particles on burns every 3 days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Improved burn wound healing by the antimicrobial peptide LLKKK18 released from conjugates with dextrin embedded in a carbopol gel.

Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol®, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerated wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduced oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulated angiogenesis (increased VEGF and microvessel development in vivo). Moreover, collagen staining evaluated by Masson's Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. Overall, we generated an effective, safe and inexpensive formulation that maintains a moist environment in the wound, easy to apply and remove, and with potential to prevent infection due to the presence of an antimicrobial peptide. These findings propel us to further study this LLKKK18-containing formulation, setting the foundations towards a potential therapeutic approach for burn wound treatment. STATEMENT OF SIGNIFICANCE: This work presents a newly developed formulation that holds great potential as a therapeutic approach for burn treatment. It is based on the sustained delivery of an antimicrobial peptide - LLKKK18 - from conjugates with dextrin, after degradation of dextrin backbone upon exposure to wound α-amylases. Conjugates were further embedded in Carbopol®, a commercially available hydrogel, suitable for topical administration and that provides a moist environment to the wound. Overall, we obtained an efficient, safe and non-expensive formulation that improves burn wound healing, maintains a moist environment within the wound, is easy to apply-and-remove, and has potential to prevent infection due to the presence of an antimicrobial peptide. Importantly, this is the first time the wound healing ability of LLKKK18 is demonstrated and that its main mechanisms of action are identified.

Arachidonic acid-derived signaling lipids and functions in impaired healing.

Very little is known about lipid function during wound healing, and much less during impaired healing. Such understanding will help identify what roles lipid signaling plays in the development of impaired/chronic wounds. We took a lipidomics approach to study the alterations in lipid profile in the LIGHT(-/-) mouse model of impaired healing which has characteristics that resemble those of impaired/chronic wounds in humans, including high levels of oxidative stress, excess inflammation, increased extracellular matrix degradation and blood vessels with fibrin cuffs. The latter suggests excess coagulation and potentially increased platelet aggregation. We show here that in these impaired wounds there is an imbalance in the arachidonic acid (AA) derived eicosonoids that mediate or modulate inflammatory reactions and platelet aggregation. In the LIGHT(-/-) impaired wounds there is a significant increase in enzymatically derived breakdown products of AA. We found that early after injury there was a significant increase in the eicosanoids 11-, 12-, and 15-hydroxyeicosa-tetranoic acid, and the proinflammatory leukotrienes (LTD4 and LTE) and prostaglandins (PGE2 and PGF2α ). Some of these eicosanoids also promote platelet aggregation. This led us to examine the levels of other eicosanoids known to be involved in the latter process. We found that thromboxane (TXA2 /B2 ), and prostacyclins 6kPGF1α are elevated shortly after wounding and in some cases during healing. To determine whether they have an impact in platelet aggregation and hemostasis, we tested LIGHT(-/-) mouse wounds for these two parameters and found that, indeed, platelet aggregation and hemostasis are enhanced in these mice when compared with the control C57BL/6 mice. Understanding lipid signaling in impaired wounds can potentially lead to development of new therapeutics or in using existing nonsteroidal anti-inflammatory agents to help correct the course of healing.

Third-hand Smoke: Impact on Hemostasis and Thrombogenesis.

Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first-hand smoke and passive/second-hand smoke exposure are known to increase the risk of coronary thrombosis. Although recently a new risk has been identified and termed third-hand smoke (THS), which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished, it remains to be determined whether it can also enhance the risk of thrombogenesis, much like first-hand smoke and second-hand smoke. Therefore, the present studies investigated the impact of THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation and secretion responses as well as enhanced integrin GPIIb-IIIa activation. Furthermore, it was found that THS exposure shortens the tail bleeding time and the occlusion time in a model of thrombosis. Thus, our data demonstrate for the first time (at least in mice) that THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets.