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-1ß (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.

Real-time quantification of proteins secreted by artificial connective tissue made from uni- or multidirectional collagen I scaffolds and oral mucosa fibroblasts.

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

Controlled release of an extract of Calendula officinalis flowers from a system based on the incorporation of gelatin-collagen microparticles into collagen I scaffolds: design and in vitro performance.

Aiming to develop biological skin dresses with improved performance in the treatment of skin wounds, acellular collagen I scaffolds were modified with polymeric microparticles and the subsequent loading of a hydroglycolic extract of Calendula officinalis flowers. Microparticles made of gelatin-collagen were produced by a water-in-oil emulsion/cross-linking method. Thereafter, these microparticles were mixed with collagen suspensions at three increasing concentrations and the resulting mixtures lyophilized to make microparticle-loaded porous collagen scaffolds. Resistance to enzymatic degradation, ability to associate with the C. officinalis extract, and the extract release profile of the three gelatin-collagen microparticle-scaffold prototypes were assessed in vitro and compared to collagen scaffolds without microparticles used as control. Data indicated that the incorporation of gelatin-collagen microparticles increased the resistance of the scaffolds to in vitro enzymatic degradation, as well as their association with the C. officinalis flower extract. In addition, a sharp decrease in cytotoxicity, as well as more prolonged release of the extract, was attained. Overall results support the potential of these systems to develop innovative dermal substitutes with improved features. Furthermore, the gelatin-collagen mixture represents a low-cost and scalable alternative with high clinical transferability, especially appealing in developing countries.