Liane Moura

Recent advances on the development of wound dressings for diabetic foot ulcer treatment—A review

Diabetic foot ulcers (DFUs) are a chronic, non-healing complication of diabetes that lead to high hospital costs and, in extreme cases, to amputation. Diabetic neuropathy, peripheral vascular disease, abnormal cellular and cytokine/chemokine activity are among the main factors that hinder diabetic wound repair. DFUs represent a current and important challenge in the development of novel and efficient wound dressings. In general, an ideal wound dressing should provide a moist wound environment, offer protection from secondary infections, remove wound exudate and promote tissue regeneration. However, no existing dressing fulfills all the requirements associated with DFU treatment and the choice of the correct dressing depends on the wound type and stage, injury extension, patient condition and the tissues involved. Currently, there are different types of commercially available wound dressings that can be used for DFU treatment which differ on their application modes, materials, shape and on the methods employed for production. Dressing materials can include natural, modified and synthetic polymers, as well as their mixtures or combinations, processed in the form of films, foams, hydrocolloids and hydrogels. Moreover, wound dressings may be employed as medicated systems, through the delivery of healing enhancers and therapeutic substances (drugs, growth factors, peptides, stem cells and/or other bioactive substances). This work reviews the state of the art and the most recent advances in the development of wound dressings for DFU treatment. Special emphasis is given to systems employing new polymeric biomaterials, and to the latest and innovative therapeutic strategies and delivery approaches.

Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured with Endothelial Cells

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34+ cells), by co-culturing the stem cells in a 3D fibrin gel with CD34+-derived endothelial cells (ECs). ECs differentiated from CD34+ cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34+-derived ECs on CD34+ cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34+ cells. We also show that the endothelial differentiation of CD34+ cells in co-culture with CD34+-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34+ cells with CD34+-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.

Regulatory aspects on nanomedicines

Nanomedicines have been in the forefront of pharmaceutical research in the last decades, creating new challenges for research community, industry, and regulators. There is a strong demand for the fast development of scientific and technological tools to address unmet medical needs, thus improving human health care and life quality. Tremendous advances in the biomaterials and nanotechnology fields have prompted their use as promising tools to overcome important drawbacks, mostly associated to the non-specific effects of conventional therapeutic approaches. However, the wide range of application of nanomedicines demands a profound knowledge and characterization of these complex products. Their properties need to be extensively understood to avoid unpredicted effects on patients, such as potential immune reactivity. Research policy and alliances have been bringing together scientists, regulators, industry, and, more frequently in recent years, patient representatives and patient advocacy institutions. In order to successfully enhance the development of new technologies, improved strategies for research-based corporate organizations, more integrated research tools dealing with appropriate translational requirements aiming at clinical development, and proactive regulatory policies are essential in the near future. This review focuses on the most important aspects currently recognized as key factors for the regulation of nanomedicines, discussing the efforts under development by industry and regulatory agencies to promote their translation into the market. Regulatory Science aspects driving a faster and safer development of nanomedicines will be a central issue for the next years.

Chitosan-based dressings loaded with neurotensin—an efficient strategy to improve early diabetic wound healing

One important complication of diabetes mellitus is chronic, non-healing diabetic foot ulcers (DFUs). This study aims to develop and use dressings based on chitosan derivatives for the sustained delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. Three different derivatives, namely N-carboxymethyl chitosan, 5-methyl pyrrolidinone chitosan (MPC) and N-succinyl chitosan, are presented as potential biomaterials for wound healing applications. Our results show that MPC has the best fluid handling capacity and delivery profile, also being non-toxic to Raw 264.7 and HaCaT cells. NT-loaded and non-loaded MPC dressings were applied to control/diabetic wounds to evaluate their in vitro/in vivo performance. The results show that the former induced more rapid healing (50% wound area reduction) in the early phases of wound healing in diabetic mice. A NT-loaded MPC foam also reduced expression of the inflammatory cytokine TNF-α (P<0.001) and decreased the amount of inflammatory infiltrate on day 3. On day 10 MMP-9 was reduced in diabetic skin (P<0.001), significantly increasing fibroblast migration and collagen (COL1A1, COL1A2 and COL3A1) expression and deposition. These results suggest that MPC-based dressings may work as an effective support for sustained NT release to reduce DFUs.

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1β (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.

Angiogenesis and Inflammation Signaling Are Targets of Beer Polyphenols on Vascular Cells

Emerging evidence indicates that chronic inflammation and oxidative stress cluster together with angiogenic imbalance in a wide range of pathologies. In general, natural polyphenols present health‐protective properties, which are likely attributed to their effect on oxidative stress and inflammation. Hops used in beer production are a source of polyphenols such as xanthohumol (XN), and its metabolites isoxanthohumol (IXN) and phytoestrogen 8‐prenylnaringenin (8PN). Our study aimed to evaluate XN, IXN, and 8PN effects on angiogenesis and inflammation processes. Opposite in vitro effects were observed between 8PN, stimulating endothelial and smooth muscle cell (SMC) growth, motility, invasion and capillary‐like structures formation, and XN and IXN, which inhibited them. Mouse matrigel plug and rat skin wound‐healing assays confirmed that XN and IXN treatments reduced vessel number as well as serum macrophage enzymatic activity, whereas 8PN increased blood vessels formation in both assays and enzyme activity in the wound‐healing assay. A similar profile was found for serum inflammatory interleukin‐1β quantification, in the wound‐healing assay. Our data indicate that whereas 8PN stimulates angiogenesis, XN and IXN manifested anti‐angiogenic and anti‐inflammatory effects in identical conditions. These findings suggest that the effects observed for individual compounds on vascular wall cells must be carefully taken into account, as these polyphenols are metabolized after in vivo administration. The modulation of SMC proliferation and migration is also of special relevance, given the role of these cells in many pathological conditions. Furthermore, these results may provide clues for developing useful therapeutic agents against inflammation‐ and angiogenesis‐associated pathologies. J. Cell. Biochem. 111: 1270–1279, 2010.

Neurotensin downregulates the pro-inflammatory properties of skin dendritic cells and increases epidermal growth factor expression.

In the last decades some reports reveal the neuropeptide neurotensin (NT) as an immune mediator in the Central Nervous System and in the gastrointestinal tract, however its effects on skin immunity were not identified. The present study investigates the effect of NT on signal transduction and on pro/anti-inflammatory function of skin dendritic cells. Furthermore, we investigated how neurotensin can modulate the inflammatory responses triggered by LPS in skin dendritic cells. We observed that fetal-skin dendritic cells (FSDCs) constitutively express NTR1 and NTR3 (neurotensin receptors) and that LPS treatment induces neurotensin expression. In addition, NT downregulated the activation of the inflammatory signaling pathways NF-κB and JNK, as well as, the expression of the cytokines IL-6, TNF-α, IL-10 and the vascular endothelial growth factor (VEGF), while the survival pathway ERK and epidermal growth factor (EGF) were upregulated. Simultaneous dendritic cells exposure to LPS and NT induced a similar cytokine profile to that one induced by NT alone. However, cells pre-treated with NT and then incubated with LPS, completely changed their cytokine profile, upregulating the cytokines tested, without changes on growth factor expression. Overall, our results could open new perspectives in the design of new therapies for skin diseases, like diabetic wound healing, where neuropeptide exposure seems to be beneficial.

Neurotensin Modulates the Migratory and Inflammatory Response of Macrophages under Hyperglycemic Conditions

Diabetic foot ulcers (DFUs) are characterized by an unsatisfactory inflammatory and migratory response. Skin inflammation involves the participation of many cells and particularly macrophages. Macrophage function can be modulated by neuropeptides; however, little is known regarding the role of neurotensin (NT) as a modulator of macrophages under inflammatory and hyperglycemic conditions. RAW 264.7 cells were maintained at 10/30 mM glucose, stimulated with/without LPS (1 μg/mL), and treated with/without NT(10 nM). The results show that NT did not affect macrophage viability. However, NT reverted the hyperglycemia-induced impair in the migration of macrophages. The expression of IL-6 and IL-1β was significantly increased under 10 mM glucose in the presence of NT, while IL-1β and IL-12 expression significantly decreased under inflammatory and hyperglycemic conditions. More importantly, high glucose modulates NT and NT receptor expression under normal and inflammatory conditions. These results highlight the effect of NT on cell migration, which is strongly impaired under hyperglycemic conditions, as well as its effect in decreasing the proinflammatory status of macrophages under hyperglycemic and inflammatory conditions. These findings provide new insights into the potential therapeutic role of NT in chronic wounds, such as in DFU, characterized by a deficit in the migratory properties of cells and a chronic proinflammatory status.

Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH.

The effect of neurotensin in human keratinocytes--implication on impaired wound healing in diabetes.

Diabetic foot ulcers are an important complication of diabetes mellitus characterized by chronic, non-healing ulcers resulting from poor proliferation and migration of fibroblasts and keratinocytes, thus impairing a correct re-epithelialization of wounded tissues. This healing process can be modulated by neuropeptides released from peripheral nerves; however, little is known regarding the role of neurotensin (NT) as a modulator of human keratinocyte function under hyperglycemic conditions. Therefore, this work is focused on the effect of NT in human keratinocytes, under normal and hyperglycemic conditions at different functional levels, namely NT receptors, cytokine, and growth factor expression, as well as proliferation and migration. Human keratinocyte cells were maintained at either 10/30 mM glucose and treated with or without NT (10 nM). The results show that NT did not affect keratinocyte viability. In addition, NT and all NT receptor expression levels were significantly reduced by hyperglycemia; however, NT treatment stimulated expression of NT and neurotensin receptor 2 (NTR2) while neurotensin receptor 1 (NTR1) and neurotensin receptor 3 (NTR3) expression levels were unchanged. Keratinocyte proliferation was not affected by NT and hyperglycemia, while cell migration was reduced by NT treatment. These results demonstrated that hyperglycemic conditions strongly impaired endogenous NT and NTR2 expression in keratinocytes. Despite the addition of exogenous NT to stimulate the endogenous NT and NTR2 expression, these changes do not translate into functional modifications on keratinocytes, particularly in terms of migration, proliferation, and production of cytokines or growth factors. These results suggest that NT production by keratinocytes may exert a paracrine effect on other skin cells, namely fibroblasts, macrophages, and dendritic cells for correct wound healing.