Experimental Studies Of Decorin On Wound Healing And Scar Formation Of Rabbit Ear Scar Model

Cutaneous wound healing consists of three phases:inflammatory, tissue formation and tissue remodeling. This complicated process involves increased permeability of blood vessels. inflammatory cell migration to wound site and production of growth factors in a wound, re-epithelialization, and matrix deposition and remodeling mediated by fibroblasts. When successful, wound healing restores normal function with a well organized minimal scar. But when the controlling mechanisms are abnormal, hypertrophic scar formation, characterized by excessive connective tissue formation, can occur.The post-traumatic hypertrophic scar is one of the clinical problems faced by the current lack of effective means of prevention and treatment. Hypertrophic Scar forming mechanism is not clear, generally thought, it is because of various fators causes abnormal fibroblast proliferation and collagen synthesis of substantial, resulting in excessive collagen-induced extracellular matrix deposition.To prevention and treatment for the scar, researchers mainly focuse on blocking the participating process of cells and the role of cytokines, inhibition of excessive extracellular matrix production and accumulation. Of many kinds of growth factors that influence wound repair and induce scar formation, TGF-β1 appears to play the most important role. TGF-β1 can promote fibroblast proliferation, promote extracellular matrix production, and inhibit collagen degradation. In addition, monocytes, macrophages and fibroblasts perpetuate the high concentration of TGF-β1 at the wound site by expressing TGF-β1 in an autocrine manner. TGF-β1 also is over-expressed in cultured fibroblasts derived from hypertrophic scars and keloids. In contrast, fetal skin of animals, which has very low levels of TGF-βq expression, can heal without scar formation after injury. Whereas exogenously added TGF-β1 can induce scar formation in fetus. All these facts indicate that TGF-β1 plays essential role in scar development. In addition, scar formation can decrease by inhibiting the expression and signal transduction of TGF-β1. Thus, a strategy for inhibiting hypertrophic scarring is to block the bioactivity of TGF-β1.Decorin belongs to a family of small leucine-rich proteoglycans and is found in the extracellular matrix, consisting of a 40KD core protein and a single glycosaminoglycan chain. The core protein consists of 10 to 12 repeats of a leucine-rich sequence of 24 amino acids. Decorin is one of the most abundant proteoglycans in normal dermis. But hypertrophic scar in the early stage contains reduced amounts of decorin, Interestingly, the content of decorin in mature scar is similar to that in normal skin. Fibroblasts isolated from reticular dermis reportedly synthesize less decorin than cells from papillary dermis, while fibroblasts responsible for healing may come from deep dermal survival fibroblasts in scar-healing wound. Decorin appears to be expressed early and abundantly in normally healing wounds. These data suggest that reduction of decorin is possibly related to hypertrophic scar formation. Decorin is present in a variety of connective tissues, and is involved in important biological functions. As named for its "decorating" association with collagen fibrils, decorin controls the morphology of collagen fbrils, as demonstrated in mouse knockouts by non-uniform fibril thickness and skin fragility. It also modulates cell adhesion and neutralize TGF-β1 activity.Find and establish an effective animal model is the basis of scar research. Many researchers had established a reproducible in vivo rabbit dermal ear ulcer model that produces hypertrophic scars mimicking the human condition. This model has been successfully used for investigation and modulation of wound healing and hypertrophic scar formation. For example, topical application of wounds with TGF-β1 improves wound healing, without altering scar prominence. In contrast, the targeted inhibition of collagen deposition with a prolyl hydroxylase inhibitor decreases scar hypertrophy, without delaying wound healing.At present, there is no report about the effect of decorin on the hypertrophic scars of the rabbit ear scar model. The purpose of this study was to examine the effect of decorin on wound healing and hypertrophic scar formation in the rabbit dermal wound model. This study will be divided into two parts. In the first part decorin was injected into the hypetrophic scar of rabbit ear right after scar formation, to examine that if decorin could reduce scar hypertrophy and promote scar softening. In the second part, decorin would be ocured on the rabbit dermal wound model in the early wound healing stage and late wound healing stage respectively, to observe that if decorin could promote rabbit ear wound healing and inhibit scar formation. We hope that the findings of this study have clinical implications for patients undergoing procedures in plastic surgery in the future.Part I The inhibition of decorin on the rabbit ear scar model in the early scar formation stageObjective To observe the effect of decorin on inhibiting hypertrophic scar by establishing hypertrophic scar model on the ventral side of rabbit ears.Methods Full-thickness dermal wounds, 8mm in diameter, were made over the ventral side of ears in 12 adult New Zealand white rabbits. Then the rabbits were divided into 3 groups:DCN group is experimental group (decorin, 10μg/ml) and PBS group(PBS) and control group. Observe wound healing and scar formation in each case and compare average wound healing time and scar formation rate of three groups. On the days 20 and 25 after operation, the hypertrophic scar were injected with decorin and PBS intralesionally.30 Samples were harvested 5 days after the last injection (30s day). Half of the specimens were stored for sirrus red staining and type I collagen and TGF-β1 monocolonal antibody immunohistochemistry staing, to measure the Scar Elevation Index (SEI) and quantify the expression level of type I collagen and TGF-β1 protein. The other half of were extracted total RNA and quantified the mRNA expression level of procollagen I and TGF-β1 by real-time polymerase chain reaction.Results:In addition to several infected wounds, the majority of the rabbit ear dermal wounds healed on days 15-17 after wounding. The healing wounds were red or light red, hard and "hill" like scar in the central of the wounds. The average healing wounds time of DCN group was 15.794±1.393 d, that of PBS group and the control group were15.602±1.389d and 15.722±1.389d respectively. Scar formation rate on post-wounding 20 days of DCN group was 80.492±3.731%, PBS group and control group were 79.659±3.222% and 80.038±3.624% respectively. There were no significantly difference among three groups.On the post-wounding days 30 the DCN group had their scars appeared to be flatter, softer and lighter in color, compared with PBS group and control group. Under the microscope, the fibroblasts and more slender collagen of DCN group scar, arranged in rules, without obviously swirling structure. PBS injection group and control group had hypertrophic scars, hook-shaped and hard to be bent. Under the microscope the scars show more thicken epidermis and a large number of fibroblasts and collagen arranged in disorder, or in nodules. The proliferation of capillaries and inflammatory cell infiltration were noticed in the dermis. SEI of DCN injection group was1.716±0.290, significantly lower than PBS group (2.785±0.435)and control group (2.981±0.475). The PBS group and the control group had no significant difference (P>0.05).Immunohistochemical staining showed that collagenⅠprotein expression signals were brown staining and mainly located in the extracellular matrix and the fibroblast cytoplasm. It was noticed that the extracellular matrix of PBS group and control group were filled with dark brown, thick collagen fibrous bundle, or irregular fiber network. The collagen I staining of DCN group were slightly deeper brown. TGF-β1 expression signals were also brown staining, mainly located in the cytoplasm of fibroblasts, extracellular matrix lightly stained. In the fibroblasts cytoplasm of PBS group and the control group, dense brown particles, or clumps of deep brown staining could be seen. In DCN group the number of positive cells were reduced, most of which were lightly and uneven brown staining. The relative protein express level of typeⅠcollagen and TGF-β1 were quantifed with mean absorbance value caculated in computer image analysis system. The results showed that the mean absorbance of typeⅠcollagen and TGF-β1of DCN group were significantly lower than PBS group (P=0.000) and the control group (P=0.000). There was no significant statistical difference between the PBS group and the control group (P>0.05).The pro-collagen I and TGF-β1 mRNA relative expression level were indicated with 2-△Ct value, assessed by real-time PCR. The results showed that the pro-collagen I and TGF-β1 mRNA relative expression level of DCN group were statistically lower than PBS group and control group (P<0.01). There were no significant difference between PBS group and control group (P>0.05).Conclusion This part of the experiment confirmed that exogenous decorin, injected into the rabbit ear hypertrophic scar at the early stage of scar formation, inhibit the scar proliferation and remarkably decrease the degree of fibrosis in the scar.Part II The effect of decorin on the rabbit ear scar model in the different wound healing stageObjective:To investigate the effect of decorin on the wound healing and scar formation of the rabbit dermal ear wound model, the wounds were treated with decorin in the different early and late wound healing time respectively. Methods: 180 full-thickness dermal wounds,8mm in diameter, were made over the ventral side of ears in 15 adult New Zealand white rabbits, each side of rabbit ear had 6 wounds on, a total of 180 wounds in the study. Then the rabbits were divided into 3 groups: early treatment group (E-DCN group) were treated with lOμg/ml decorin on postwounding days 1,3,5; late treatment group (L-DCN group) were treated with 10μg/ml decorin on postwounding days 9,11,13; the wounds of control group were exposed to natural healing. On days 7 and 30 after wounding samples were harvested. Half of the samples stained with immunohistochemistry monocolonal antibodies and sirrus red to quantify Scar Elevation Index (SEI) and the protein expression level of type I collagen and TGF-β1 The other half of samples were stored for subsequent RNA extraction and real-time polymerase chain reaction and examined the mRNA expression level of pro-collagen I and TGF-β1.Results:The average wound healing time of early treatment group was 16.188±1.856 days. Compared with late treatment group (15.338±1.615 d) and the untreated control group (15.388±1.559 d), the difference was statistically significant (P<0.05). There was no significant difference between late treatment group and control group (P> 0.05). On ays 20 after wounding the scar formation rate of three groups were not significantly different.The SEI of early treatment group was 80±6.847%, that of late treatment group was 85±5.590%, and the control group was 82.5±6.847%. On postwounding days 30, SEI of late treatment group was 1.951±0.312, significantly lower than those of early treatment group (2.770±0.519) and control group (2.858±0.358). There was no significant difference between early treatment group and control group (P> 0.05). Immunohistochemical staining of the granulation tissue of 7 days after wounding showed that in the early treatment group, the mean absorbance value of type I collagen and TGF-β1 were lower than late treatment group (P<0.01, P<0.05) and the control group (P<0.01, P<0.05), the difference was significant statistically. No significant difference between late treatment group and control group (P> 0.05). Immunohistochemical staining of the scar of 30 days after wounding indicated that the mean absorbance value of type I collagen and TGF-β1 of late treatment group lower than early treatment group and control group, the difference was statistically significant (P<0.05). No significant difference between early treatment group and control group (P> 0.05).2-△Ct value indicates the relative mRNA expression level of procollagenⅠand TGF-β1. The results displayed that in the early treatment group procollagenⅠ(1.196±0.310) and TGF-β1 mRNA level were significantly lower than in late treatment group (procollagenⅠ:1.619±0.380) and the control group (procollagen I:1.747±0.486) (P<0.01). No significant difference between late treatment group and control group (P>0.05). On days 30 after wounding, the results showed that procollagen I (1.619±0.380) and TGF-β1 mRNA level of the late treatment group were significantly lower than early treatment group (procollagenⅠ3.926±0.666) and the control group(procollagen I 4.012±0.797) (P<0.05). No significant difference between early treatment group and control group (P> 0.05).Compared with days 7 after wounding, the procollagen I mRNA level of each group increased significantly (P<0.01) on postwounding days 30. For early treatment group TGF-β1 mRNA level on postwounding days 7 increased from 0.025±0.087 up to 0.080±0.029 on postwounding days 30, late treatment group decreased from 0.067±0.022 to 0.038±0.012, the difference were significant statistically. Although TGF-β1 mRNA expression level of control group increased from 0.061±0.020 to 0.074±0.032, no significant difference between days 7 and 30 after woundingConclusion:This part of the experiment confirmed that exogenous decorin, injected into the rabbit ear hypertrophic scar at the early stage of wounding healing period, prolonged the wounding healing time, without the effect of inhibiting the scar proliferation. When decorin was choosed at the late stage of wound healing time, the wounds healed without delay and the formation of hypertrophic scar was inhibited successfully.TGF-β1 has been identified as a critical factor in hypertrophic scar formation. Our findings suggest that to decrease hypertrophic scar formation, treatment with TGF-β1 inhibitor, decorin, needs to occur when TGF-β1 mRNA expression level are near their peak numbers, and collagen synthesis is very active. Otherwise, inhibition the activity of TGF-β1 earlier may affect the wound healing process and fail to suppress the role of scar formation. This may be clinically significant, because strategies to decrease scarring can potentially impair healing. If wound healing has progressed sufficiently, various strategies to reduce collagen synthesis may be more successful.