Early Stages Of Wound Healing Research Articles

The Use of Light Photobiomodulation on the Treatment of Second-Degree Burns: A Histological Study of a Rodent Model

The aim of this investigation was to compare, by light microscopy, the effects of the use of laser photobiomodulation (LPBM) and polarized light (PL) on second-degree burns on rodents. Burns are severe injuries that result in the loss of tissue fluids, destruction of tissues, infection, and shock. With severe and widespread third-degree burns death may occur. Several light sources have been suggested as being effective for improving wound healing. Forty five rats were used in this study. A second-degree burn was created on the dorsum of each animal, and the animals were divided into four groups: PL (400-2000 nm, 40 mW, 2.4 J/cm(2)/min); LPBM-1 (780 nm, 35/40 mW, theta approximately 2 mm, 4 x 5 J/cm(2)); LPBM-2 (660 nm, 35/40 mW, theta approximately 2 mm, 4 x 5 J/cm(2)); and untreated animals acted as controls. The treatment was started immediately post-burn at four points around the burned area (laser: 5 J/cm(2) per site). The illumination with PL was performed according to the manufacturer's instructions. Treatments were repeated at 24-h intervals for 7 d. The animals were sacrifice at 3, 5, and 7 d post-burn. The specimens were routinely cut and stained and analyzed by light microscopy using hematoxylin and eosin and Sirius red. The analysis of the results demonstrated that the damaged tissue was able to efficiently absorb and process the light at all tested wavelengths. LPBM at 660 nm showed better results at early stages of wound healing. However, the use of 780-nm laser light had beneficial effects throughout the experimental period, with the animals growing newly-formed tissue similar to normal dermis. Despite our findings that the use of both types of light energy improved the healing of second-degree burns at the early stages, long-term assessment is needed to verify if this improvement will influence the final results of treatment.

028 Early ProInflammatory Cytokine Expression During Mucosal Wound Healing

Proinflammatory cytokines (such as IL-1 [alpha], IL-1 [beta]) have been shown to play a significant role in early stages of wound healing. Previous studies also indicate that mucosal wounds heal faster in males. The purpose of this study was to assess gene expression of inflammatory cytokines before and during wound healing. Younger and older volunteers received two small oral wounds on the hard palate. One of the wounds (3.5 mm in diameter) was videographed daily until healed. The other wound (1 × 5 mm) was biopsied either 6 or 24 h postwounding. Real-time PCR (ABI Prism 7000 SDS) was performed on the unwounded and wounded tissues to study gene expression. Preliminary analyses revealed that compared to unwounded tissue, wounding induced rapid gene expression for these cytokines. In males, IL-1 [beta] gene expression peaked at 6 h and was higher than in females. Since males have been shown to heal this type of wound faster than females, this earlier peak of IL-1 [beta] may be beneficial to wound healing. Regardless of gender, IL-1ra and IL-1 [alpha] levels peaked at 6 and 24 h, respectively. Interestingly, cytokine levels in unwounded tissue correlated with wound healing parameters (e.g., wound sizes, time to heal), but in females only. Specifically, women with higher basal levels of IL-1 [beta] and IL-1ra, and lower levels of IL-1 [alpha] tended to heal faster. Thus, the expression of inflammatory cytokine genes in unwounded mucosal tissue may play a determinant role in wound healing outcomes. (Supported by NIH P50 DE-13749)

Nerve-induced ectopic limb blastemas in the axolotl are equivalent to amputation-induced blastemas

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.

Effect of Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) Nanofiber Matrices Cocultured With Hair Follicular Epithelial and Dermal Cells for Biological Wound Dressing

We tested the effects on the early-stage wound healing of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofiber matrices cultured with hair follicular cells. PHBV only, PHBV/collagen, and PHBV/gelatin at a 7/3 weight ratio were produced by electrospinning, and their in vitro cell culture and in vivo wound healing as biological dressings were examined. In cell attachment and growth on matrices, dermal sheath (DS) cells attached to hydrophilic PHBV/collagen and PHBV/gelatin faster than hydrophobic PHBV at the early incubation stage (up to 6 h). From 6- to 24-h incubation, PHBV/collagen showed the best results in cell culture. Furthermore, PHBV/collagen cocultured for 3-5 days with DS and epithelial outer root sheath (ORS) cells expressed more extracellular materials, such as type I collagen, elastin, and alpha-smooth muscle actin, than cocultured PHBV with the same cells. However, there was no significant difference between PHBV and PHBV/collagen in the amounts of cytokeratin 8 expressed. Grafting of PHBV and PHBV/collagen matrices cocultured with ORS/DS cells for 3-5 days showed that PHBV promoted wound closure and re-epithelization more obviously than PHBV/collagen in both cocultured matrices and matrices alone. Cocultured matrices would heal wounds better than the corresponding matrices alone. Thus, PHBV cocultured with ORS/DS cells could be used as a cell-seeded biological dressing, thereby reducing preparation time as well as regenerating the epidermis efficiently during the early stage of wound healing.

Molecular Changes in the Vasculature of Injured Tissues

We have explored molecular specialization of the vasculature of regenerating wound tissue in the skin and tendons to identify a different repertoire of markers from that obtained by studying tumor vasculature. We screened a phage-displayed peptide library for peptides that home to wounds in mice and identified two peptides that selectively target phage to skin and tendon wounds: CARSKNKDC (CAR) and CRKDKC (CRK). CAR is homologous to heparin-binding sites in various proteins and binds to cell surface heparan sulfate and heparin. CRK is similar to a segment in thrombospondin type 1 repeat. Intravenously injected CAR and CRK phage, as well as fluorescein-labeled CAR and CRK peptides, selectively accumulated at wound sites, where they partially co-localized with blood vessels. The CAR peptide showed a preference for early stages of wound healing, whereas the CRK favored wounds at later stages of healing. The CAR peptide was internalized into the target cells and delivered the fluorescent label into the cell nuclei. These results identify new molecular markers in wound tissues and show that the expression of these markers in wound vasculature changes as healing progresses. The peptides recognizing these markers may be useful in delivering treatments into regenerating tissues.

Human monocyte adhesion onto RGD and PHSRN peptides delivered to the surface of a polycarbonate polyurethane using bioactive fluorinated surface modifiers

Fluorinated oligomers, when blended into polyurethane, have been shown to migrate to the surface and generate an interface that minimizes protein denaturation and reduces cell activation. This type of surface modification can be achieved with ppm quantities of a bioactive fluorinated surface modifier (BFSM), enabling the introduction of bioactive agents onto a surface in one manufacturing step. In the current study, two BFSMs were synthesized with covalently conjugated RGD and PHSRN peptides near the fluorine terminal groups, and were shown to be surface active in polyurethane blends. CyQuant cell enumeration, scanning electron microscopy, and cell viability assays all indicated that the bioactive (and fluorinated) substrates supported enhanced monocyte interaction. The simplicity of the surface modification technique and the demonstrated ability of the peptide BFSMs to influence cell attachment and spreading indicate the potential benefits and practical value of the BFSM technology in tailoring surfaces for biomaterial applications. This was specifically highlighted for human blood monocytes, a key cell involved in the early stages of wound healing.

Bandages, dressings, and cranial neurosurgery

Bandages and dressings are commonly applied to incisional scalp wounds to prevent complications, particularly infection, during the early stages of wound healing. Bandaging cranial incisional wounds requires resources, consumes healthcare workers' time, and incurs expense; it is therefore important to examine its efficacy. All cranial operations (excluding shunt placements, procedures on the scalp alone, and bur hole procedures) performed between June 30, 2001 and January 1, 2006, by two neurosurgeons at either of two hospitals, one adult and one pediatric institution, were reviewed. Surgical site infections (SSIs) and other postoperative complications were investigated with respect to the use of bandaging for incisional wounds and other aspects of postoperative wound management. The operations were classified into four categories based on wound type: "clean," "clean-contaminated," "contaminated," and "dirty," according to the criteria of the Centers for Disease Control and Prevention. A total of 702 operations were performed in 577 patients; only five patients received any type of surgical bandaging. There were four SSIs (0.57%; 95% confidence interval [CI] 0.16-1.45). The postoperative infection rate in the 626 clean cases was 0.48% (95% CI 0.10-1.39) and was 2.63% (95% CI 0.07-13.81) in the 38 clean-contaminated cases. The data obtained in this investigation is consistent with the position that bandaging incisional scalp wounds after cranial surgery adds little if any benefit beyond the easier, simpler, and cheaper practice of using antibiotic ointment as a dressing without bandaging.

Histological and immunohistochemical analysis of initial and early osseous integration at chemically modified and conventional SLA® titanium implants: preliminary results of a pilot study in dogs

The aim of the present pilot study was to investigate initial and early tissue reactions to modified (mod) and conventional sand-blasted, large grit and acid-etched (SLA) titanium implants. Implantation of modSLA and SLA implants was performed bilaterally in both the mandible and maxilla of dogs. The animals were sacrificed after a healing period of 1, 4, 7 and 14 days, respectively. Peri-implant tissue reactions were assessed in non-decalcified tissue sections using conventional histology (Toluidine blue-TB and Masson Goldner Trichrome stain-MG) and immunohistochemistry using monoclonal antibodies to transglutaminase II (TG) (angiogenesis) and osteocalcin (OC). Bone density (BD) and bone to implant contact (BIC) were assessed histomorphometrically. Day 1 revealed an early TG antigen reactivity in the provisional fibrin matrix adjacent to both implant surfaces. Day 4 was characterized by the formation of a collagen-rich connective tissue (MG), which revealed the first signs of OC synthesis adjacent to modSLA surfaces. Immunohistochemical staining for TG revealed a direct correlation between angiogenesis and new bone formation, which was clearly identifiable after 7 days by means of increasing BD, BIC and OC values. After 14 days, modSLA surfaces seemed to be surrounded by a firmly attached mature, parallel-fibered woven bone. Within the limits of the present study, it might be concluded that the combination of immunohistochemical and conventional histological stainings in non-decalcified tissue sections is a valuable technique to evaluate the initial and early stages of wound healing around endosseous titanium implants.

Decreased Macrophage Number and Activation Lead to Reduced Lymphatic Vessel Formation and Contribute to Impaired Diabetic Wound Healing

Impaired wound healing is a common complication of diabetes. Although it is well known that both macrophages and blood vessels are critical to wound repair, the role of wound-associated lymphatic vessels has not been well investigated. We report that both the presence of activated macrophages and the formation of lymphatic vessels are rate-limiting to the healing of diabetic wounds. We have previously shown that macrophages contribute to the lymphatic vessels that form during the acute phase of corneal wound healing. We now demonstrate that this is a general phenomenon; cells that co-stain for the macrophage marker F4/80 and the lymphatic markers LYVE-1 (lymphatic vascular endothelium hyaluronate receptor) and podoplanin contribute to lymphatic vessels in full-thickness wounds. LYVE-1-positive lymphatic vessels and CD31-positive blood vessels were significantly reduced in corneal wound healing in diabetic mice (db/db) (P < 0.02) compared with control (db/+) mice. Glucose treatment of control macrophages led to the down-regulation of the lymphatic-specific receptor VEGFR3 and its ligands, vascular endothelial growth factor-C and -D (VEGF-C, -D). Interleukin-1beta stimulation rescued diabetic macrophage function; application of interleukin-1beta-treated db/db-derived macrophages to wounds in db/db mice induced lymphatic vessel formation and accelerated wound healing. These observations suggest a potential therapeutic approach for healing wounds in diabetic patients.

Antiadhesive effect of fibrinogen: a safeguard for thrombus stability

AbstractThe recruitment of phagocytic leukocytes to sites of vessel wall injury plays an important role in thrombus dissolution by proteases elaborated on their adhesion. However, leukocyte adhesion to the fibrin clot can be detrimental at the early stages of wound healing when hemostatic plug integrity is critical for preventing blood loss. Adhesion of circulating leukocytes to the insoluble fibrin(ogen) matrix is mediated by integrins and occurs in the presence of a high concentration of plasma fibrinogen. In this study, the possibility that soluble fibrinogen could protect fibrin from excessive adhesion of leukocytes was examined. Fibrinogen was a potent inhibitor of adhesion of U937 monocytoid cells and neutrophils to fibrin gel and immobilized fibrin(ogen). An investigation of the mechanism by which soluble fibrinogen exerts its influence on leukocyte adhesion indicated that it did not block integrins but rather associated with the fibrin(ogen) substrate. Consequently, leukocytes that engage fibrinogen molecules loosely bound to the surface of fibrin(ogen) matrix are not able to consolidate their grip on the substrate; subsequently, cells detach. This conclusion is based on the evidence obtained in adhesion studies using various cells and performed under static and flow conditions. These findings reveal a new role of fibrinogen in integrin-mediated leukocyte adhesion and suggest that this mechanism may protect the thrombus from premature dissolution.

Chemisch modifizierte, ultra-hydrophile Titanimplantatoberflächen

Recently, a chemically modified ultra-hydrophilic sand-blasted, large grit and acid-etched (modSLA) titanium surface has been introduced in order to enhance bone apposition. Indeed, preliminary preclinical and clinical data have indicated that modSLA implants may enhance bone apposition during early stages of wound healing. These positive effects on bone regeneration might be probably mainly due to the hydrophilic surface properties noted for modSLA which ensured a stabilization of the blood clot. The aim of the present review article is to evaluate, based on the currently available evidence, the potential impact of modSLA surfaces for implant dentistry.

Fibrin/platelet plug counteracts cutaneous wound contraction: The hypothesis of “skipping stone”

Cutaneous wound contraction and epithelialization act collaboratively to minimize the exposed wound surface. However excessive wound contraction is undesirable due to the resultant disfigurement and scarring. Fibrin clot has greater stiffness than surrounding tissue and mechanical strain further enhances its stiffness. On the contrary, skin exhibits diminished stiffness when affected by high strain rates. Therefore during early stages of wound healing, the contractile wound border is confronted by fibrin clot forming a high strain region in the interface of contractile tissue and fibrin clot--which is evidenced by computer simulation. Due to the stress relaxation property of skin, the contractile strain is partly neutralized. Meanwhile, gradually the stiffness of fibrin clot decreases which is followed by another cycle of wound contraction. This cyclic pattern of contraction resembles the movement of a stone over water or "skipping stone". The stone bounces repeatedly when thrown across the surface of water with reduction of jumping altitude with each bounce till the stone stops completely. This hypothesis is further supported by the observed initial delay in wound contraction and the chronological correlation of enhanced wound contraction with loss of superficial eschar and substitution of fibrin clot with granulation tissue. Also there is evidence that fibrin inhibits fibroblast-mediated contraction of collagen. Furthermore, modest increase in wound contraction rate in fibrinogen deficient mice and fibrin-mediated diminished wound contraction are agreement with the proposed hypothesis.

Wound Healing in Tenascin-X Deficient Mice Suggests that Tenascin-X is Involved in Matrix Maturation Rather than Matrix Deposition

Tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence in humans leads to a recessive form of Ehlers-Danlos Syndrome (EDS). TNX deficient patients have hypermobile joints and fragile skin, but unlike the classical type of EDS, no atrophic scars were observed. Anecdotal evidence suggested that wound healing in TNX deficient patients is abnormal, but no detailed study has been performed so far. To address the role of TNX in wound healing, we analyzed skin wound morphology and mechanical properties of scars in TNX knockout (KO) mice. Breaking strength of unwounded skin of KO mice is significantly lower (<50%) than that of wild-type (WT) mice. In the early stage of wound healing when TNX is hardly expressed in WT wounds (day 7), WT and KO skin are of similar strength. After 14 days, when TNX starts to be expressed at moderate levels in wounds of WT mice, the WT scars gain a further increase in breaking strength, whereas KO scars do not progress beyond the mechanical strength of uninjured KO skin. No obvious differences between KO and WT mice were noted in the rate of wound closure, or in expression of fibrillar collagens during wound healing. We conclude that TNX is unlikely to be involved in matrix deposition in the early phase of wound healing, but it is required in the later phase when remodeling and maturation of the matrix establishes and improves its biomechanical properties.

Influence of the application of platelet‐enriched plasma in oral mucosal wound healing

The aim of this study was to describe and quantify the therapeutic value of platelet concentrate on the capillary density in oral mucosal wound healing. The subjects included 10 healthy edentulous patients who underwent bilaterally a sinus floor elevation procedure and a buccal onlay graft with autologous iliac crest bone for maxillary reconstruction. During surgery, platelet-rich plasma (PRP) was prepared from a blood sample taken from the patient. After randomization in a split-mouth design, at one side PRP was administered in the wound and at the contralateral side a placebo. At baseline, microvascular capillary density was scored with the orthogonal polarization spectral (OPS) imaging technique and repeated measurements were performed postoperatively on a daily basis until the tenth day, after which measurements were continued weekly until the fifth postoperative week. Ten patients, five males and five females, were included in the study with a mean age of 54.2+/-9.1 years for females and 57.6+/-6.9 years for males. Donor platelet counts from whole blood had a mean value of 248.5+/-13.5 x 10(9)/l, while the value of platelet counts in the PRP had a mean of 975.9+/-97.9 x 10(9)/l. Wound healing was significantly accelerated in the PRP-treated mucosal wounds during the first 10 postoperative days. After the second week, no obvious differences between the PRP or placebo side could be noted. PRP has a strong stimulant effect on capillary regeneration in wound healing. These effects are mainly noticeable during the early stages of wound healing.

Effect of surgery on monocyte function.

In vitro monocyte lysozyme production (IVMLP) was assessed in 10 male patients preoperatively and at intervals postoperatively. A similar procedure was carried out in 6 sex- and age-matched controls. In contrast to the control group where the IVMLP was constant, the IVLMP levels in the surgical group showed a marked increase at 6-10 h postoperatively, falling to control levels after 10 days. In addition, peripheral monocyte counts were carried out using a Technicon Hemalog D counter which produces accurate reproducible counts for cells such as monocytes which occur at low frequency. The peripheral monocyte counts were stable in the control group, whilst in the surgical patients there were significantly higher readings at 36-48 h, which persisted for up to 10 days compared with preoperative counts. This study suggests that surgery has both a qualitative (IVMLP) and quantitative (monocyte counts) effect on the mononuclear phagocytes. These data may be relevant when considering factors that influence the early stages of wound healing.

Evidence That Fibrinogen Inhibits Leukocyte Adhesion to Fibrin Clot and Immobilized Fibrinogen by Binding to the Substrate but Not to Integrins.

The recruitment of phagocytic leukocytes to sites of injured vessel wall plays an important role in thrombus remodeling during normal vascular repair and in the pathophysiology of thrombosis. Fibrin and fibrinogen, present in the thrombus, are potent adhesive substrates for neutrophils and monocytes. They support cellular attachment by binding cell surface receptors that belong to the β2 subfamily of integrins. Adhesive interactions of neutrophils and monocytes with polymerized fibrin and insoluble fibrinogen matrix in vivo occur in the presence of high concentrations of circulating plasma fibrinogen (~2–4 mg/ml). One important property of fibrinogen that would have a major bearing on leukocyte adhesion is its capacity to form complexes with fibrin. Therefore, by virtue of its binding to the fibrin clot and/or immobilized fibrinogen, soluble plasma fibrinogen can influence leukocyte adhesion to these substrates. In this study, the possibility that soluble fibrinogen could protect fibrin from adhesion of leukocytes was examined. Fibrinogen was an efficient inhibitor of adhesion of U937 monocytoid cells and neutrophils to fibrin gel and immobilized fibrin(ogen). An investigation of the mechanism by which fibrinogen exerts its influence on leukocyte adhesion indicated that it did not block integrins but rather associated non-covalently and weakly with fibrin(ogen) substrates. Consequently, leukocyte integrins that engage fibrinogen molecules loosely bound to the fibrin(ogen) matrix are not able to consolidate their grip on the substrate; subsequently, cells detach. This conclusion is based on the evidence obtained in adhesion studies using various β2-integrin bearing cells and performed under static and flow conditions. Furthermore, surface plasmon resonance studies, undertaken to determine the Kd of fibrinogen-fibrin interactions under flow conditions, indicated that fibrinogen formed complexes with fibrin(ogen) with micromolar affinities. Thus, these findings reveal a new role of fibrinogen in integrin-mediated leukocyte adhesion. They also imply that the anti-adhesive effect of fibrinogen may protect the thrombus from an excessive leukocyte accumulation and premature dissolution at the early stages of wound healing when hemostatic plug integrity is critical for preventing blood loss.

Electrospinning of collagen nanofibers: Effects on the behavior of normal human keratinocytes and early-stage wound healing

Electrospinning of type I collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to fabricate a biomimetic nanofibrous extracellular matrix for tissue engineering was investigated. The average diameter of collagen nanofibers electrospun from 8% collagen solution in HFIP was 460 nm (range of 100–1200 nm). The as-spun collagen nanofibrous matrix was chemically cross-linked by glutaraldehyde vapor with a saturated aqueous solution and then treated with aqueous 0.1 m glycine to block unreacted aldehyde groups. With vapor phase cross-linking for 12 h, porosity of the collagen matrix decreased from 89% to 71%. The collagen nanofibrous matrix showed good tensile strength, even in aqueous solution. Effects on cytocompatibility, cell behavior, cell and collagen nanofiber interactions, and open wound healing in rats were examined. Relatively low cell adhesion was observed on uncoated collagen nanofibers, whereas collagen nanofibrous matrices treated with type I collagen or laminin were functionally active in responses in normal human keratinocytes. Collagen nanofibrous matrices were very effective as wound-healing accelerators in early-stage wound healing. Our results indicate that cross-linked collagen nanofibers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical applications, such as wound dressing and scaffolds for tissue engineering.

Periodontal regeneration in intrabony defects after application of enamel matrix proteins with guided tissue regeneration: An experimental study in dogs

The aim of this study was to evaluate the effects of enamel matrix proteins (EMP) at the early stage of wound healing in the periodontal tissues by a combination treatment with guided tissue regeneration (GTR). Intrabony defects were produced surgically at the distal aspects of both mandibles in six beagle dogs. At 12 weeks following the surgery, the defects were exposed using a full thickness mucoperiosteal flap procedure. Subsequently, the defects were treated by the following treatments: a control group treated with GTR alone, and an experimental group treated with a combination of GTR and EMP. After one, two, four and eight weeks of the treatment, the animals were sacrificed, and sections of the tissue were stained and evaluated microscopically. After one and two weeks, the proliferating cell nuclear antigen (PCNA)-positive cell ratios of the experimental group were significantly greater than that of the control group. After 2 and 4 weeks, new bone and new cementum formation in the experimental group were significantly greater than those in the control group (P < 0.05). However, after 8 weeks, no statistical difference was found between the two groups in new bone or cementum formation. The study results suggest that a maturation of periodontal ligament cells might contribute, during the early stage of periodontal healing, to stimulate a proliferation of periodontal ligament cells.

Cell component changes in an acute and chronic vocal fold (VF) scar rat model

Problem: Knowledge of wound healing is essential to treatment of VF scarring. Fibroblasts and myofibroblasts are thought to be important constituents in wound healing; the former produces extracellular matrix (ECM), and the latter is closely related to wound contraction. Yet little is known about their expression patterns following VF injury. Methods: Using a 1.9 mm diameter telescope for guidance, VF stripping was performed unilaterally in 48 Sprague-Dawley rats. Larynges were harvested at 8 time points (1 day, 3 days, 5 days, 7 days, 2 weeks, 4 weeks, 8 weeks, and 12 weeks) and the scarred and normal VFs were histologically analyzed. Immunohistochemical staining for vimentin, and alfa smooth muscle actin (SMA), a marker of myofibroblast, were performed. Results: In normal VFs, vimentin-positive cells existed in the deep layer and the basement membrane zone of the lamina propria (LP). Most of the alfa-SMA-positive cells highlighted capillaries in the deep and middle layer of the LP and very few single cells were evident. Vimentin-positive cells increased from day 3 to day 14, and then returned to the normal level after 8 weeks. Alfa-SMA-positive cells increased from day 3 to 4 weeks and then returned to normal levels after 8 weeks. At day 5 and day 7, many single cells were evident in the scarred VFs in addition to many collections of cells that depicted the capillaries. These single cells disappeared by day 14. Conclusion: Myofibroblasts were evident at day 5 and day 7 after injury, which suggests that wound contraction in VF occurs in an early phase of wound healing. Significance: Our findings imply that there may be a critical window for VF scar prevention present in the early stage of wound healing. Support: None reported.

030 A Rabbit Ear Excessive Scarring Model Using Growth Factor Stimulators

Research on abnormal scarring, such as excessive scars, has been hampered by the lack of relevant animal models. Recent studies suggest that TGF‐β1, PDGF‐BB and FGF‐2 are stimulators of fibrosis or scarring. We developed a rabbit ear cutaneous excessive scarring model using the growth factor stimulators TGF‐β1 (0.25, 0.5 or 1 μg per wound), PDGF‐BB (1, 2 or 3 μg per wound) and FGF‐2 (0.5, 1, 3 or 5 μg per wound). Three 8‐mm diameter wounds were created on each ear in NZW rabbits (n = 6 wounds). The test growth factors were injected intradermally immediately after wounding. Controls consisted of PBS, BSA and wounding only without injections. Scar thickness of wounds was measured with a micrometer at days 14 and continued twice a week thereafter until sacrifice at day 28. Results showed that the greatest measurement of elevated scars was observed at day 14. Significant differences of scar thickness were observed in TGF‐β1 at a dose of 0.25 μg, PDGF‐BB at a dose of 3 μg, and FGF‐2 at a dose of 3 μg per wound groups when compared to PBS and wounding only groups (p &lt; 0.05). Interestingly, BSA, a protein stabilizer for TGF‐β1 and FGF‐2 proteins, also significantly elevated scar thickness at day 14 over PBS alone (p &lt; 0.05). Trichrome stained tissue sections on day 28 showed an increase in cellularity and thicker epithelium in all doses of TGF‐β1, PDGF‐BB and FGF‐2 protein treated groups when compared to wounding only, PBS or BSA treatments. In general, FGF‐2 treated wounds showed more vascularity than TGF‐β1 and PDGF‐BB treated wounds. By contrast, PDGF‐BB treated wounds showed richer collagen deposition than TGF‐β1 and FGF‐2 treated wounds. Results from these studies demonstrated that TGF‐β1, PDGF‐BB and FGF‐2 contributed to the scar formation in the rabbit ear scar model. BSA may contribute in stimulating scar formation in the early stage of wound healing in this model. Enhancing elevated scar formation in the rabbit ear model will be useful in evaluating anti‐scarring agents for excessive scars such as hypertrophic scars and keloids.