Hyaluronan in human acute and chronic dermal wounds

There is growing interest in the relationship of hyaluronan and inflammation in a number of physiologic processes including wound healing. The objective of this study was to make a quantitative comparison of inflammation and hyaluronan expression in human normal healing open wounds and in pressure ulcers. Using an open dermal wound model, myeloperoxidase activity was found to peak at day 3. Hyaluronan levels showed a bimodal distribution with transient peaks occurring on days 1 and 7. Mean levels of myeloperoxidase activity in pressure ulcers were significantly higher than at any time in the acute wounds, whereas hyaluronan levels were significantly lower than at any time in the acute wounds. Levels of hyaluronidase activity increased slightly in the postwound period. Hyaluronidase activity in pressure ulcers was significantly elevated compared with the acute wounds. These results suggest a role for increased enzymatic degradation of hyaluronan as a function of inflammation during wound repair. This is the first reported quantitative examination of hyaluronan expression in human acute dermal wounds and in chronic pressure ulcers.     

Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography

Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2 ± 0.3 mm⁻¹ to 1.3 ± 0.6 mm⁻¹, whereas, in the apoptotic cells, an increase to 6.4 ± 1.7 mm⁻¹ was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment.     

Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds

Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase-8 originating from neutrophils appearing earlier than the fibroblast-derived matrix metalloproteinase-1. Open (6 mm) full-thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase-1 and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase-1 and matrix metalloproteinase-8 in wound exudates with peak levels of matrix metalloproteinase-8 occurring on day 4 and matrix metalloproteinase-1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase-8 were statistically higher than matrix metalloproteinase-1 (100-fold to 200-fold). Tissue inhibitor of metalloproteinases-1 levels declined over time, whereas levels of matrix metalloproteinase-1/tissue inhibitor of metalloproteinase-1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase-8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.     

Comparison studies of the in vivo treatment of full‐thickness excisional wounds and burns by an artificial bilayer dermal equivalent and J‐1 acellular dermal matrix

The effects upon skin repair were compared between a homemade bilayer dermal equivalent (BDE), composed of a collagen/chitosan porous scaffold and a silicone membrane, and J‐1 acellular dermal matrix (ADM), a commercial ADM that is used widely in China to treat various skin defects. Full‐thickness excisional and burn wounds were prepared on the backs of pigs and then treated with the BDE and J‐1 ADM. Biopsy specimens were harvested on days 7, 14, and 21 after surgery for gross, biochemical, and molecular examinations. In comparison with the burn wounds, the excisional wounds showed accelerated granular tissue formation and superior integration with the equivalents, regardless of their type. Immunohistochemical, immunofluorescence, real time quantitative polymerase chain reaction and Western blotting analyses showed that the vascularization rates in the excisional wounds group were also significantly faster than those of the burn group for both dermal equivalents. There was no significant difference between J‐1 ADM and BDE treatment on the formation of newly formed blood vessels for the excisional wounds at days 7, 14, and 21. However, there was a significant difference in the number of nascent blood vessels formed in the burn wounds after treatment with J‐1 ADM compared with BDE. The highest numbers of newly formed and mature blood vessels were present in the J‐1 ADM‐treated excisional wounds after 21 days. Ultrathin skin grafts were further transplanted on to the regenerated dermis for 28 days, resulting in the repair of the full‐thickness wounds and production of a structure similar to normal skin.     

Investigation of the skin repair and healing mechanism of N‐carboxymethyl chitosan in second‐degree burn wounds

N‐carboxymethyl chitosan (NCMC) was synthesized with the modification of chitosan; the substitution degree was measured by titration. The biocompatibility and degradability of the NCMC were studied in vivo and the results showed that the NCMC was nontoxic and biocompatible. The in vivo degradation rate of NCMC in musculature was faster than that in subcutaneous tissue due to the relatively high lysozyme concentration. The NCMC was used as biomaterial to heal deep second‐degree burn wounds. The wound size reduction, histological examination, and the quantification of transforming growth factor‐β₁, tumor necrosis factor‐α and interleukin‐8 protein levels, and Smad3 gene expression were measured to evaluate the healing effects. The results demonstrated that the NCMC was efficient in accelerating wound healing via activating transforming growth factor‐β₁/Smad3 signaling pathway.     

Metalloproteinases in chronic and acute wounds: A systematic review and meta‐analysis

A systematic review and meta‐analysis were undertaken in order to explore the influence of matrix metalloproteinases and their diagnostic methods in chronic and acute wounds. Searches were conducted in the PubMed (Medline) and Embase (Elsevier) databases from inception to late November 2017. We included clinical trials enrolling patients with cutaneous chronic and acute wounds where a validated diagnostic method was employed for metalloproteinases. We excluded in vitro, animal or preclinical studies, nonoriginal articles, and studies without available data for analysis. In addition, references of narrative and systematic reviews were scrutinized for additional articles. Eight studies met the inclusion criteria. Results revealed that the most frequently determined matrix metalloproteinases were MMP‐2 and MMP‐9, and were found in 54.5% of wounds. MMP‐9 was present in more than 50% of the chronic wounds with a range from 37 to 78%. However, metalloproteinases were found in only 20% of acute wounds, and other types of metalloproteinases were also observed (MMP‐2 and MMP‐3). On the basis of the available evidence, high levels of metalloproteinases have been correlated with significantly delayed wound healing in wounds of a variety of etiologies.     

Toll‐like receptor 4 signaling regulates the acute local inflammatory response to injury and the fibrosis/neovascularization of sterile wounds

The role of Toll‐like receptor 4 (TLR4) in the regulation of inflammation and fibrosis in sterile wounds was investigated in TLR4 signal‐deficient (C3H/HeJ or TLR4^?/?) and control mice using the subcutaneously implanted polyvinyl alcohol sponge wound model. Total and differential wound cell counts 1, 3, and 7 days after injury did not differ between C3H/HeJ and C3H/HeOuJ animals. Blood monocytes from both strains expressed CCR2 equally. Day one wounds in C3H/HeJ mice contained fewer Gr‐1^high wound macrophages, CCL3, and CCL5, and more CCL17 than those in controls. The accumulation of CCL2, CX3CL1, tumor necrosis factor‐α, interleukin (IL)‐6, IL‐10, IL‐12, and interferon‐γ in wound fluids was not TLR4 dependent. Wound macrophages from C3H/HeJ and C3H/HeOuJ mice expressed CCR4 and CCR5, but not CCR1 or CCR3. Wound macrophage recruitment was not altered in CCR5^?/? mice or in C3H/HeOuJ animals injected with neutralizing anti‐CCL3 and anti‐CCL5 antibodies. Neutralization of the CCR4 ligand CCL17 in C3H/HeJ mice did not alter wound macrophage populations. There was a twofold increase in collagen content and number of neovessels in 21‐day‐old wounds in C3H/HeJ vs. C3H/HeOuJ mice. There were no differences between strains in the number of myofibroblasts in the wounds 7 or 21 days postwounding. The increased fibrosis and angiogenesis in wounds from /HeJ mice correlated with higher concentrations of transforming growth factor‐β and fibroblast growth factor 2 in wound fluids from these animals. Wound fluids did not contain detectable lipopolysaccharide and did not induce IκBα degradation in J774.A1 macrophages. Results support a role for endogenous ligands of TLR4 in the regulation of inflammation and repair in sterile wounds.     

Combination of stromal cell‐derived factor‐1 and collagen–glycosaminoglycan scaffold delays contraction and accelerates reepithelialization of dermal wounds in wild‐type mice

While dermal substitutes can mitigate scarring and wound contraction, a significant drawback of current dermal replacement technologies is the apparent delay in vascular ingrowth compared with conventional skin grafts. Herein, we examined the effect of the chemokine stromal cell‐derived factor‐1 (SDF‐1) on the performance of a porous collagen–glycosaminoglycan dermal analog in excisional wounds in mice. C57BL/6 mice with 1 cm × 1 cm dorsal full‐thickness wounds were covered with a collagen–glycosaminoglycan scaffold, followed by four daily topical applications of 1 μg SDF‐1 or phosphate‐buffered saline vehicle. Some animals were also pretreated with five daily doses of 300 mg/kg granulocyte colony‐stimulating factor. Animals treated with SDF‐1 and no granulocyte colony‐stimulating factor reepithelialized 36% faster than vehicle controls (16 vs. 25 days), and exhibited less wound contraction on postwounding day 18 (～35% greater wound area) plus three‐fold longer neoepidermis formed than controls. Conversely, granulocyte colony‐stimulating factor promoted contraction and no epidermal regeneration. Early (postwounding Day 3) inflammatory cell infiltration in the SDF‐1‐treated group was 86% less, while the fraction of proliferating cells (positive Ki67 staining) was 32% more, when compared with controls. These results suggest that SDF‐1 simultaneously delays contraction and promotes reepithelialization and may improve the wound‐healing performance of skin substitutes.     

Changes in the extracellular matrix surrounding human chronic wounds revealed by 2‐photon imaging

Chronic wounds are a growing problem worldwide with no effective therapeutic treatments available. Our objective was to understand the composition of the dermal tissue surrounding venous leg ulcers and diabetic foot ulcers (DFU). We used novel 2‐photon imaging techniques alongside classical histology to examine biopsies from the edges of two common types of chronic wound, venous leg ulcers and DFU. Compared to normal intact skin, we found that collagen levels are significantly reduced throughout the dermis of venous leg ulcer biopsies and DFU, with a reduction in both fibril thickness and abundance. Both wound types showed a significant reduction in elastin in the upper dermis, but in DFU, the loss was throughout the dermis. Loss of extracellular matrix correlated with high levels of CD68‐ and CD18‐positive leukocytes. 2‐photon imaging of the extracellular matrix in the intact tissue surrounding a chronic wound with a hand‐held device may provide a useful clinical indicator on the healing progression or deterioration of these wounds.     

Fibronectin‐derived Epiviosamines enhance PDGF‐BB‐stimulated human dermal fibroblast migration in vitro and granulation tissue formation in vivo

Fibronectin (FN) is a multimodular glycoprotein that is a critical component of the extracellular matrix (ECM) anlage during embryogenesis, morphogenesis, and wound repair. Our laboratory has previously described a family of FN‐derived peptides collectively called “epiviosamines” that enhance platelet‐derived growth factor‐BB (PDGF‐BB)‐driven tissue cell survival, speed burn healing, and reduce scarring. In this study, we used an agarose drop outmigration assay to report that epiviosamines can enhance PDGF‐BB‐stimulated adult human dermal fibroblast (AHDF) outmigration in a dose‐dependent manner. Furthermore, these peptides can, when delivered topically, stimulate granulation tissue formation in vivo. A thiol‐derivatized hyaluronan hydrogel cross‐linked with polyethyleneglycol diacrylate (PEGDA) was used to topically deliver a cyclized epiviosamine: cP12 and a cyclized engineered variant of cP12 termed cNP8 to porcine, full‐thickness, excisional wounds. Both cP12 and cNP8 exhibited dose‐dependent increases in granulation tissue formation at day 4, with 600 μM cNP8 significantly enhancing new granulation tissue compared to vehicle alone. In contrast to previous studies, this study suggests that epiviosamines can be used to increase granulation tissue formation without an exogenous supply of PDGF‐BB or any cell‐binding peptides. Thus, epiviosamine may be useful topically to increase granulation tissue formation in acute wounds.     

Role of different negative pressure values in the process of infected wounds treated by vacuum‐assisted closure: an experimental study

Vacuum‐assisted closure (VAC) device is widely used to treat infected wounds in clinical work. Although the effect of VAC with different negative pressure values is well established, whether different negative pressures could result in varying modulation of wound relative cytokines was not clear. We hypothesise that instead of the highest negative pressure value the suitable value for VAC is the one which is the most effective on regulating wound relative cytokines. Infected wounds created on pigs' back were used to investigate the effects of varying negative pressure values of VAC devices. Wounds were treated with VAC of different negative pressure values or moist gauze, which was set as control. The VAC foam, semiocclusive dresses and moist gauze were changed on days 3, 5, 7 and 9 after wounds were created. When changing dressings, tissues from wounds were harvested for bacteria count and histology examination including Masson's trichrome stain and immunohistochemistry for microvessels. Western blot was carried out to test the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Results showed that on days 3 and 5 the number of bacteria in wounds treated by VAC with 75, 150, 225 and 300 mmHg was significantly decreased compared with that in wounds treated by gauze and 0 mmHg pressure value. However, there was no difference in wounds treated with negative pressure values of 75 , 150, 225 and 300 mmHg at any time spot. Immunohistochemistry showed that more microvessels were generated in wounds treated by VAC using 75 and 150 mmHg negative pressure comparing with that using 225 and 300 mmHg on days 3 and 5. However this difference vanished on days 7 and 9. Morphological evaluation by Masson's trichrome staining showed increased collagen deposition in VAC of 75 and 150 mmHg compared with that in VAC of 225 and 300 mmHg. Western blot showed that the expression of VEGF and bFGF significantly increased when the wounds treated with 75 and 150 mmHg negative pressure values compared with the wounds treated with 225 and 300 mmHg on day 5. Treatment using VAC with different negative pressure values more than 75 mmHg has similar efficiency on reducing bacteria in the infected wound. VAC with negative pressure values of 75 and 150 mmHg promote wound healing more quickly than other pressure values. Moreover, comparing with vigorous negative pressure, relatively moderate pressures contribute to wound healing via accelerated granulation growth, increased angiogenic factor production and improved collagen fibre deposition. Further study of this model may show other molecular mechanisms.