Remodeling Phase Of Wound Healing Research Articles

CXCL11 Expression by Keratinocytes Occurs Transiently Between Reaching Confluence and Cellular Compaction.

Objective: To investigate whether differentiation or cellular confluence is responsible for CXCL11 expression patterns in re-epithelialization. Approach:In vitro model systems of re-epithelialization using the HaCaT keratinocyte cell line were utilized in monitoring expression of differentiation markers, including desmoplakin and various cytokeratins while evaluating for an association with chemokine CXCL11 expression. Results: CXCL11 expression was elevated in sparse culture with peak expression near the time of confluence. This somewhat followed the accumulation of desmoplakin in detergent-insoluble pool of proteins. However, in postconfluent, despite continued accumulation of desmoplakin within cells, CXCL11 expression decreased to baseline levels. This biphasic pattern was also seen in low calcium culture, an environment that inhibits keratinocyte differentiation and accumulation of desmosomal proteins. Highest CXCL11-expressing areas best correlated with newly confluent areas within culture expressing basal keratin 14, but also activated keratin 6. Innovation: Achievement of a threshold cellular density induces cell signaling cascade through CXCR3 that, in addition to other undiscovered pathways, can progress cutaneous wounds from the proliferative into the remodeling phases of cutaneous wound healing. Conclusion: These results suggest that the achievement of confluence with increased cellular density by migrating keratinocytes at the wound edge triggers expression of CXCL11. Since CXCR3 stimulation in endothelial cells results in apoptosis and causes neovascular pruning, whereas stimulation of CXCR3 in fibroblasts results decreased motility and cellular contraction, we speculate that CXCL11 expression by epidermal cells upon achieving cellular confluence could be the source of CXCR3 stimulation in the dermis ushering a transition from proliferative to remodeling phases of wound healing.

Local release of pioglitazone (a peroxisome proliferator‐activated receptor γ agonist) accelerates proliferation and remodeling phases of wound healing

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily known for its anti-inflammatory and macrophage differentiation effects, as well as its ability to promote fat cell differentiation and reduce insulin resistance. Pioglitazone (Pio) is a PPARγ agonist used clinically as an anti-diabetic agent for improving insulin sensitivity in patients with diabetes. The objective of this study was to develop a drug delivery system (DDS) for the local release of Pio to promote wound healing. Pio of low aqueous solubility was water-solubilized by micelles formed from gelatin grafted with L-lactic acid oligomers, and incorporated into a biodegradable gelatin hydrogel. An 8-mm punch biopsy tool was used to prepare two skin wounds on either side of the midline of 8-week-old mice. Wounds were treated by the hydrogels with (Pio-hydrogel group) or without (control group) Pio, and the wound area were observed 1, 4, 7, and 14 days after treatment. In addition, a protein assay and immunohistological stain were performed to determine the effects of the Pio-hydrogel on inflammation and macrophage differentiation. The Pio-hydrogels promote wound healing. Moreover, Western blotting analysis demonstrated that treatment with Pio-hydrogels resulted in decreased levels of the cytokines MIP-2 and TGF-β, and increased levels of glucose-regulating adiponectin. It is concluded that Pio-incorporated hydrogels promote the proliferation and remodeling phases of wound healing, and may prove to be effective as wound dressings.

Spiral ganglion cells and macrophages initiate neuro-inflammation and scarring following cochlear implantation.

Conservation of a patient's residual hearing and prevention of fibrous tissue/new bone formation around an electrode array are some of the major challenges in cochlear implant (CI) surgery. Although it is well-known that fibrotic tissue formation around the electrode array can interfere with hearing performance in implanted patients, and that associated intracochlear inflammation can initiate loss of residual hearing, little is known about the molecular and cellular mechanisms that promote this response in the cochlea. In vitro studies in neonatal rats and in vivo studies in adult mice were performed to gain insight into the pro-inflammatory, proliferative, and remodeling phases of pathological wound healing that occur in the cochlea following an electrode analog insertion. Resident Schwann cells (SC), macrophages, and fibroblasts had a prominent role in the inflammatory process in the cochlea. Leukocytes were recruited to the cochlea following insertion of a nylon filament in adult mice, where contributed to the inflammatory response. The reparative stages in wound healing are characterized by persistent neuro-inflammation of spiral ganglion neurons (SGN) and expression of regenerative monocytes/macrophages in the cochlea. Accordingly, genes involved in extracellular matrix (ECM) deposition and remodeling were up-regulated in implanted cochleae. Maturation of scar tissue occurs in the remodeling phase of wound healing in the cochlea. Similar to other damaged peripheral nerves, M2 macrophages and de-differentiated SC were observed in damaged cochleae and may play a role in cell survival and axonal regeneration. In conclusion, the insertion of an electrode analog into the cochlea is associated with robust early and chronic inflammatory responses characterized by recruitment of leukocytes and expression of pro-inflammatory cytokines that promote intracochlear fibrosis and loss of the auditory hair cells (HC) and SGN important for hearing after CI surgery.

The Antifibrosis Effects of Peroxisome Proliferator-Activated Receptor δ on Rat Corneal Wound Healing after Excimer Laser Keratectomy.

Corneal stromal fibrosis characterized by myofibroblasts and abnormal extracellular matrix (ECM) is usually the result of inappropriate wound healing. The present study tested the hypothesis that the ligand activation of peroxisome proliferator-activated receptor (PPAR) δ had antifibrosis effects in a rat model of corneal damage. Adult Sprague-Dawley rats underwent bilateral phototherapeutic keratectomy (PTK). The eyes were randomized into four groups: PBS, GW501516 (a selective agonist of PPARδ), GSK3787 (a selective antagonist of PPARδ), or GW501516 combined with GSK3787. The agents were subconjunctivally administered twice a week until sacrifice. The cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and postmortem histology. A myofibroblast marker (α-smooth muscle actin) and ECM production (fibronectin, collagen type III and collagen type I) were examined by immunohistochemistry and RT-PCR. At the early stages of wound healing, GW501516 inhibited reepithelialization and promoted angiogenesis. During the remodeling phase of wound healing, GW501516 attenuated the activation and proliferation of keratocytes, which could be reversed by GSK3787. GW501516 decreased transdifferentiation from keratocytes into myofibroblasts, ECM synthesis, and corneal haze. These results demonstrate that GW501516 controls corneal fibrosis and suggest that PPARδ may potentially serve as a therapeutic target for treating corneal scars.

Limiting prolonged inflammation during proliferation and remodeling phases of wound healing in streptozotocin-induced diabetic rats supplemented with camel undenatured whey protein

BackgroundImpaired diabetic wound healing occurs as a consequence of excessive reactive oxygen species (ROS) and inflammatory cytokine production. We previously found that whey protein (WP) was able to normally regulate the ROS and inflammatory cytokines during the inflammatory phase (first day) in streptozotocin (STZ)-diabetic wound healing. This study was designed to assess the effect of WP on metabolic status, the inflammation and anti-inflammation response, oxidative stress and the antioxidant defense system during different phases of the wound healing process in diabetic rats. WP at a dosage of 100 mg/kg of body weight, dissolved in 1% CMC, was orally administered daily to wounded normal (non-diabetic) and STZ-induced diabetic rats for 8 days starting from the 1st day after wounding.ResultsThe data revealed that WP enhanced wound closure and was associated with an increase in serum insulin levels in diabetic rats and an alleviation of hyperglycemic and hyperlipidemic states in diabetic animals. The increase in insulin levels as a result of WP administration is associated with a marked multiplication of β-cells in the core of islets of Langerhans. WP induced a reduction in serum TNF-α, IL-1β and IL-6 levels and an increase in IL-10 levels, especially on the 4th day after wounding and treatment. WP also suppressed hepatic lipid peroxidation and stimulated the antioxidant defense system by increasing the level of glutathione and the activity of glutathione-S-transferase, glutathione peroxidase and superoxide dismutase (SOD) in wounded diabetic rats.ConclusionsWP was observed to enhance wound closure by improving the diabetic condition, limiting prolonged inflammation, suppressing oxidative stress and elevating the antioxidant defense system in diabetic rats.

Cellular confluence and cohesion regulates CXCL11/IP9 expression during keratinocyte re‐epithelialization

A role for CXCL11/IP9/I‐TAC in coordinating cellular events to transition the regenerative phase to the remodeling phase of wound healing has been demonstrated. However, the physiologic signals regulating CXCL11 expression remain unknown. Of specific interest are the factors that stimulate and later downregulate expression of this transiently‐expressed chemokine. To investigate keratinocyte‐mediated CXCL11 expression, in vitro cell culture and skin organ culture model systems were used. CXCL11 expression was found to be upregulated soon after cellular confluence was reached. In addition, CXCL11 was not dependent upon calcium‐dependent differentiation as the increased expression was also noted in confluent cultures grown in low calcium conditions. These results suggest that achievement of cellular confluence results in the upregulation of chemokine CXCL11 likely as a paracrine to the underlying dermis that covering re‐epithelialization has occurred.

The effect of intravitreal anti-VEGF agents on peripheral wound healing in a rabbit model

PurposeTo investigate the effect of intravitreal pegaptanib, bevacizumab, and ranibizumab on blood-vessel formation during cutaneous wound healing in a rabbit model and to compare this effect to placebo controls.MethodsForty New Zealand albino rabbits underwent full thickness cutaneous wounds using 6-mm dermatologic punch biopsies. The rabbits were assigned to four groups of ten, each receiving intravitreal injections of pegaptanib, bevacizumab, ranibizumab, or no injection (untreated controls). Five rabbits from each group underwent wound harvesting on day 7 and five from each group on day 14. The skin samples were stained with hematoxylin and eosin (HE), Masson’s trichrome (MT), and CD34 for vascular endothelial cells. Semiquantitative evaluation of HE- and MT-stained slides was performed by one pathologist. Quantitative assessment of mean neovascularization (MNV) scores was obtained from five contiguous biopsy margin 400× fields of CD34-stained sections by four independent observers.ResultsWeek 1 MNV scores in CD-34 stained sections were: untreated controls: 11.51 ± 4.36; bevacizumab: 7.41 ± 2.82 (P = 0.013); ranibizumab: 8.71 ± 4.08 (P = 0.071); and pegaptanib: 10.15 ± 5.59 (P = 0.378). Week 2 MNV data were: untreated controls: 6.14 ± 2.25; bevacizumab: 7.25 ± 2.75 (P = 0.471); ranibizumab: 4.53 ± 3.12 (P = 0.297); and, pegaptanib: 6.35 ± 3.09 (P = 0.892). Interobserver variability using intraclass correlation coefficient was 0.961.ConclusionsAt week 1, all three anti-VEGF agents had suppressed MNV scores compared to controls. Although not statistically significant, there was an inhibitory trend, particularly with bevacizumab and ranibizumab. These effects were diminished at 2 weeks, reflecting a transition between the proliferative and remodeling phases of wound healing.

Mast Cells Are Required in the Proliferation and Remodeling Phases of Microdeformational Wound Therapy

Mast cells are important in numerous inflammatory processes. They are also mechanosensitive and likely play an important role in wound healing. The authors hypothesized that mechanical alteration of the wound environment with a distributed suction device could link mast cells to the healing cascade. Controlled uniform full-thickness wound surface microdeformations were induced by suction combined with an open-pore polyurethane foam (microdeformational wound therapy) in mast cell-deficient WWv mice and their mast cell-sufficient littermates. Wound healing parameters were assessed in the inflammatory, proliferative, and remodeling phases of healing. Wound tissue granulation, cell proliferation, blood vessel sprouting, and collagen maturation were found to be mast cell-dependent throughout the proliferating and remodeling stages of healing. Mast cells are critical in the robust granulation tissue response seen in microdeformational wound therapy and in the modulation of the remodeling phase of wound healing.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α–smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

Non-invasive assessment of healing of bacteria infected and uninfected wounds using optical coherence tomography

Bacterial infection is one of the main predisposing factors for the delay in wound healing. To facilitate a timely decision for correct therapy, it is important to accurately monitor the morphological changes in the infected wounds using noninvasive tools. In the present study, we have explored the use of optical coherence tomography (OCT) for monitoring the healing of superficial wounds infected with Staphylococcus aureus in mice under in vitro and in vivo conditions and studied the changes in collagen birefringence in the infected wounds. The tape stripping method was used for generating superficial skin wounds in mice and wounds were infected with S. aureus. For in vitro studies, infected and uninfected wound tissues were resected, back scattered intensity and birefringence changes in collagen during wound healing were studied on the 2, 4 and 10th day of postinfection using polarization-sensitive (PS) OCT and images were compared with histology. Real-time OCT was used for studying the kinetics of healing of infected wounds under in vivo conditions. From the PS-OCT images, different phases of wound healing such as inflammation, reepithelialization and collagen remodeling could be identified. The edematic regions appeared prominent in infected wounds. Compared with uninfected wounds, reepithelization and collagen remodeling phases of wound healing were delayed significantly in the infected wounds. These changes were comparable with the different stages of wound healing observed under in vivo conditions. OCT imaging can provide a rapid assessment of the morphological changes associated with bacteria-infected and uninfected wounds and thereby aid in timely treatment planning.

Metalloproteinase Expression is Associated with Traumatic Wound Failure

Matrix metalloproteinases (MMPs) are crucial in the inflammatory and remodeling phases of wound healing. We previously reported the correlation between pro-inflammatory cytokines and timing of successful combat-wound closure. We now extend our studies to investigate the correlation between wound-remodeling MMP expression and wound healing. Thirty-eight wounds in 25 patients with traumatic extremity combat wounds were prospectively studied. Surgical debridement with vacuum-assisted closure (VAC) device application was repeated every 48 to 72h until surgical wound closure. Wound effluent and patient serum were collected at each wound debridement and analyzed for five matrix metalloproteinases using the Luminex multiplex system; Millipore Corp, Billerica, MA. The primary outcome was wound healing within 30 d of definitive wound closure. Impairment was defined as delayed wound closure (>21 d from injury) or wound dehiscence. MMP expression was compared between impaired and normal healing wounds. Elevated levels of serum MMP-2 and MMP-7 and reduced levels of effluent MMP3 were seen in impaired wounds (n = 9) compared with wounds that healed (n = 29; P<0.001). Receiver operating characteristic (ROC) curve analysis yielded area-under-the-curve (AUC) of 0.744, 0.783, and 0.805, respectively. Impaired wound healing is characterized by pro-inflammatory MMP-2 and MMP-7. Serum and effluent concentrations of MMP-2, MMP-3, and MMP-7 can effectively predict the outcome of traumatic war wounds and can potentially provide decision-supportive, objective evidence for the timing of wound closure.

Gene expression of transforming growth factor‐β1 and hepatocyte growth factor during wound healing of injured rat vocal fold

To investigate the expression of genes coding transforming growth factor (TGF)-beta1, hepatocyte growth factor (HGF), and c-Met, its membrane-spanning tyrosine kinase receptor, during the inflammatory, proliferative, and remodeling phases of wound healing in the injured rat vocal fold. Prospective animal study. Thirty five rats were involved in this study. Bilateral vocal fold wounds were created in 30 rats. Injured vocal fold specimens were harvested on postinjury day 1, 3, 7, 14, 28, and 56. Real-time polymerase chain reaction (PCR) was used to quantify mRNA expression of TGF-beta1, HGF, and c-Met. Five uninjured rats were used to establish PCR control. Results of analysis of variance revealed a significant main effect for TGF-beta1 (P = .000), HGF (P = .000), and c-Met (P = .000) expression across time points. Post-hoc testing revealed that TGF-beta1 expression increased significantly on postinjury day 7 (P = .001) compared to control. HGF expression decreased significantly on postinjury day 1 (P = .001), and increased significantly on postinjury day 14 (P = .000). c-Met expression decreased significantly on postinjury day 1 (P = .000), day 3 (P = .000), and day 56 (P = .000), and increased significantly on postinjury day 28 (P = .000). Results revealed time-dependent changes in the regulation of genes coding TGF-beta1, HGF, and c-Met during wound healing in the injured rat vocal fold. These patterns of gene expression correspond well with previously reported histologic changes of the rat vocal fold after injury.

Effects of low-level laser therapy on mast cell number and degranulation

Mast cells have been shown to participate in the wound healing process. We investigated the effects of low-level laser therapy (LLLT) on mast cell number in the inflammation, proliferation, and remodeling phases of the wound healing process of experimental burns. Sixty rats subjected to third-degree burns were divided into four groups: two laser-treated, one control, and one nitrofurazone-treated group. In the two laser-treated groups, burned areas received LLLT with a helium-neon laser at energy densities of 38.2 J/cm(2) and 76.4 J/cm(2), respectively. The effects on mast cell number and degranulation were assessed 7, 16, and 30 days postburn (inflammation, proliferation, and remodeling phases of wound healing, respectively). Intact and degranulated mast cells were counted. Five rats with no burns were used for baseline studies. On day 7 in the first laser group, the total number of mast cells was significantly higher than in the other groups. On day 16 in the nitrofurazone-treated group, the total number of mast cells was significantly higher than in the control, first laser, and normal groups. LLLT on the experimental third-degree burns significantly increased the total number of mast cells during the inflammation phase of wound healing; also, topical application of 0.2% nitrofurazone ointment on the same burns significantly increased the total number of mast cells during the proliferation phase of burn healing.

Knee loading accelerates bone healing in mice.

Knee loading is an anabolic loading modality that applies lateral loads to the knee. This study shows that loads applied to the proximal tibial epiphysis stimulate healing of surgically generated wounds in the tibial diaphysis. Wound healing is sensitive to mechanical stimulation such as various forms of stress and different magnitudes of strain. Knee loading has been shown to induce anabolic responses to murine tibias and femora when a strain of 10-20 mustrain is applied at the site of new bone formation. The object of this study was to address a question: does knee loading accelerate closure of open wounds in the tibia? Fifty-three C57/BL/6 female mice were used. A surgical wound (0.5 mm in diameter) was generated in the left tibia (loaded) and the right tibia (sham-loaded control). From the fourth postoperative day, knee loading was performed to the left knee with a custom-made piezoelectric loader for 3 min/d for 3 consecutive days. The peak-to-peak force was 0.5 N. Animals were killed 1, 2, or 3 wk after surgery, and the healing process was evaluated with muCT, pQCT, and bone histomorphometry with calcein labeling. The measured strain was <20 mustrain with 0.5-N force regardless of the presence or absence of surgical wounds. Compared with sham-loaded controls, the results showed load-driven acceleration of wound healing. First, muCT data revealed that knee loading reduced the size of surgical wounds by 13% (p < 0.01; 1 wk), 25% (p < 0.001; 2 wk), and 15% (p < 0.01; 3 wk). Second, pQCT data indicated that total BMD and BMC and cortical BMD and BMC were significantly increased in the third postoperative week. Last, bone histomorphometry revealed that bone formation was stimulated from the site proximal (close to the knee) to the wound. The reparative and remodeling phases of wound healing were enhanced by loads applied to the knee without inducing significant in situ strain at the site of wounds. Noninvasive knee loading might therefore be useful clinically to stimulate bone healing in the entire tibia along its length (including cast immobilized wounds).

205: The effects of engineered EGF mutants on fibroblast biological functions

Introduction: Epidermal growth factor (EGF) is one of the major growth factors involved in the re-epithelialization and remodeling phase of wound healing. In addition, local and sustained presence of EGF has been shown to accelerate wound healing. However, previous attempts to develop EGF for clinical applications have been limited by its short circulating half-life. We have previously developed a technique utilizing directed evolution to engineer EGF mutants (mtEGFs) with enhanced and persistent binding affinity to human EGFR (hEGFR).

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high ( p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.

A TGF-β1-Dependent Autocrine Loop Regulates the Structure of Focal Adhesions in Hypertrophic Scar Fibroblasts

Following injury, fibroblasts migrate into wounds and differentiate into alpha smooth muscle cell actin (SMCA)-positive cells, termed myofibroblasts, that assemble and remodel the scar. Cultured myofibroblasts assemble larger focal adhesions than do normal dermal fibroblasts and these focal adhesions attach to alpha SMCA-rich stress fibers. Following severe traumatic or thermal injury to the dermis, hypertrophic scars (HTSs) often develop and these scar fibroblasts (HTSFs) express alpha SMCA persistently. We now report that HTSFs stably display large focal adhesions as a consequence of both the autocrine production and activation of transforming growth factor beta1 (TGF-beta1). We also observe that myofibroblasts elaborating larger focal adhesions adhere more tightly to fibronectin. Conditioned medium from HTSFs induces focal adhesion growth in normal fibroblasts and this is blocked by pre-incubation with a soluble TGF-beta1 receptor mimetic. Human foreskin fibroblasts transduced with a retrovirus encoding active TGF-beta1 elaborate large focal adhesions, whereas fibroblasts overexpressing normal, latent TGF-beta1 do not. We conclude that the large focal adhesions found in pathogenic myofibroblasts arise through an autocrine loop involving the production and activation of TGF-beta1; these adhesions likely mediate both tighter adhesion to wound matrix and the exuberant wound contraction observed in pathogenic scars.

Mechanical strain causes hypertrophic scarring in vivo by blocking fibroblast apoptosis in the proliferative phase of wound healing

Bhatt, Kirit A. MD; Aarabi, Shahram BA; Bastidas, Nicholas BA; Lin, Shin-e BA; Tabbal, Georges BA; Gali, Shadi MD; Bonillas, Robert MD; Gurtner, Geoffrey MD Author Information

Effect of in vitro mechanical compression on Epilysin (matrix metalloproteinase‐28) expression in hypertrophic scars

Epilysin, designated matrix metalloproteinase (MMP)-28, is the newest member of this family of proteases expressed by keratinocytes in response to an injury. MMP-28's physiological role and specific substrates are unknown, but its expression pattern suggests that it may serve a role in both tissue homeostasis and wound healing. The aim of this preliminary study was to observe the presence of MMP-28 protein in normotrophic and hypertrophic scars and to evaluate the effect of in vitro mechanical compression on its expression. Biopsies from normotrophic and hypertrophic scars resulting from burns were divided into two samples, one to be used as control (uncompressed) and the other to be compressed in an oxygenated organ chamber for 24 hours in the presence of a serum-free medium, using an electromechanical load transducer (stable pressure = 35 mmHg). Analysis of MMP-28 protein secretion, assessed by Western blot and beta-casein zymography in scar conditioned media, revealed that normotrophic scar did not release MMP-28 in any condition while hypertrophic scar released active MMP-28 both in control conditions and after compression. MMP-28 immunohistochemistry revealed a light protein presence in normotrophic scar keratinocytes and a strong MMP-28 positivity in hypertrophic scar keratinocytes in control conditions, while compression increased MMP-28 staining in normotrophic scar and induced a significant reduction of the protein presence in hypertrophic scar keratinocytes. As it has been suggested that MMP-28 may restructure the skin basal membrane (Saarialho-Kere et al., 2002), our data indicate that mechanical compression directly acts to modulate the remodeling phase of wound healing, altering release and activity of MMP-28 in hypertrophic scars.

Both Constitutive and Inducible Prostaglandin H Synthase Affect Dermal Wound Healing in Mice

In an attempt to define the roles of prostaglandin H synthase 1 (PGHS-1, cyclooxygenase-1, COX-1) and prostaglandin H synthase 2 (PGHS-2, cyclooxygenase-2, COX-2) in wound healing, we investigated the healing of incisional dermal wounds in wild-type, PGHS-1 null, and PGHS-2 null mice. We measured tensile strength of the wounds, levels of PGHS-1 and PGHS-2 mRNA in the wound site, and histologic markers for the inflammatory, proliferative, and remodeling phases of wound healing. Although no gross visible differences were noted among healed wounds of the different mouse types, measurement of tensile strength showed that both PGHS-1 and PGHS-2 null wounds were weaker (75% and 70%, respectively) than wild-type wounds at 12 days after incision. At Day 8 the endothelial staining was 70% greater in the wounds of PGHS-2 null mice compared with their wild-type counterparts. In contrast at Day 12, staining for macrophages and myofibroblasts was less in PGHS-1 null wounds compared with wild-type and PGHS-2 null tissue. Compensatory expression of the alternate PGHS mRNA could be demonstrated by RT-PCR in the wounds of PGHS null mice on Days 1 and 4. We conclude that both PGHS-1 and PGHS-2 genes play distinct roles in the process of dermal wound healing.