Radiation-impaired Wound Healing Research Articles

Role of TGF-β and FGF in the Treatment of Radiation-Impaired Wounds Using a Novel Drug Delivery System

Despite refinements in radiotherapy, radiation-impaired wound healing continues to be a major source of postoperative morbidity with few treatment options. The application of polypeptide growth factors has been investigated in both the clinical and laboratory settings. The authors used a novel sustained-release delivery system to examine the effect of transforming growth factor (TGF)-beta and fibroblast growth factor (FGF) on radiation-impaired wound healing in a rodent model. Eighty Sprague-Dawley rats underwent dorsal skin surface irradiation of 2500 cGy using a medical linear accelerator producing energy of 6 MeV followed by creation of a full-thickness skin incision. Six groups of 16 animals underwent either sham irradiation (irradiation control); irradiation only; irradiation and unimpregnated delivery system only; or irradiation and either TGF-beta, FGF, or TGF-beta plus FGF combined. Four animals from each group were euthanized at 4, 7, 14, and 28 days, and the harvested specimens underwent ultimate tensile strength testing and histologic evaluation. All five irradiated groups had significantly lower ultimate tensile strength than the sham-irradiated control group at all time points (p < 0.05), thus validating the authors' model of radiation-impaired wound healing. Functional analysis demonstrated that all three growth factor-treated groups had significantly higher tensile strengths than either of the untreated irradiated groups at 14 days after wounding (p < 0.05). Histologic evaluation of the irradiated groups revealed increased cellularity and more organized collagen architecture of all treated groups when compared with the untreated groups, with the most pronounced differences seen at 7 days and 14 days after wounding. This study effectively demonstrates that TGF-beta and FGF act individually and synergistically when delivered locally by means of a sustained release system to improve ultimate tensile strength in an acute postirradiation impaired wound-healing model.

Improvement of the breaking strength of wound by combined treatment with recombinant human G‐CSF, recombinant human M‐CSF, and a TGF‐β1 receptor kinase inhibitor in rat skin

Effective doses of ionizing radiation during preoperative radiotherapy occasionally cause wound complications after subsequent surgery. The authors attempted to accelerate radiation-impaired wound healing in animal models. Recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human macrophage colony-stimulating factor (rhM-CSF), and an inhibitor of transforming growth factor (TGF)-beta1 receptor kinase, SB431542, were injected s.c. into a full-thickness incisional wound site in the dorsal skin of rats after local irradiation of X-ray (30 Gy). Wound healing of irradiated skin was assessed using the breaking strength of the wound and histological analyses. The impaired wound healing in irradiated skin was found to be associated with impaired mobilization of bone marrow-derived cells and enhanced expression of TGF-beta1 mRNA. The breaking strength of the wound in the irradiated skin was approximately one-eighth of that in the non-irradiated skin; however, following combined treatment with the above three compounds the breaking strength increased to approximately one-half of that in the non-irradiated skin. Histological analysis of the wounded skin revealed an increase in formation of collagen fibers and the panniculus carnosus following the combined treatment. Moreover, the increased breaking strength was associated with an increase in a subpopulation of fibrocytes (collagen I/ED1 double positive cells). These findings suggested that a combined treatment with rhG-CSF, rhM-CSF, and SB431542 is promising as a means of improving radiation-impaired wound healing.

Role of T-lymphocytes in Radiation-Impaired Wound Healing

Role of T-lymphocytes in Radiation-Impaired Wound Healing

Model of radiation‐impaired healing of a deep excisional wound

Despite many well-recognized benefits, administration of ionizing radiation before surgical resection of malignancies is associated with a high risk of wound-healing complications. Most animal models investigating techniques to improve wound healing use a superficial wound. The goal of this study was to develop a novel model of radiation-impaired healing using a deep excisional wound, which is closer to the clinical situation. In the first part of this study, female Lewis rats were exposed to 0, 12, 15, or 18 Gy single-fraction radiation to the buttocks. Three weeks later, deep wounds were created by excision of the gluteus maximus muscle. Irradiated wounds had a lower rate of healing of the surgically created defect than unirradiated wounds (p<0.001), but there was no significant difference between the different doses of radiation. Impaired healing was still evident at 12 weeks. The second part of this study investigated the ability of porcine small-intestinal submucosa (SIS) to improve healing in this animal model. At 6 weeks, wounds implanted with SIS showed improved healing at all doses of radiation compared with unimplanted irradiated wounds. However, higher doses of radiation were still associated with a lower rate of healing. SIS induced a cellular response that was not evident in defects that did not receive SIS, suggesting that SIS has the potential to stimulate repair. This reproducible model of radiation-impaired wound healing closely resembles the clinical setting. The results indicate that this model can be used to investigate new biomaterials as possible therapeutic agents to enhance wound healing.

Expression and possible mechanism of c‐ski, a novel tissue repair‐related gene during normal and radiation‐impaired wound healing

C-ski is a complicated regulating factor for fibroblast proliferation and an important co-repressor of Smad3. Although inhibiting Smad3 activity can markedly promote wound healing because Smad3 mediates the role of transforming growth factor-beta in inhibiting cell proliferation and inducing cell apoptosis; there has been no report on whether c-ski is expressed during wound healing and the relationship between its expression and wound healing. By establishing animal models of normal and radiation-impaired wound healing and using immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction, we found that c-ski was expressed after wounding and reached its peak on day 9 and then significantly decreased. C-ski was present in all repair cells, and was especially prominent in fibroblasts. Compared with the control side, the irradiated side showed a lower expression of c-ski on postwound days 3-9, but higher on day 15, and not significantly different after the wound was healed. The expression of Smad3 was in contrast to the c-ski and cellular proliferation was similar to that of c-ski expression. The apoptosis index was significantly higher on the irradiated side on days 3-9 compared with the control side. In vitro, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results showed that c-ski could reverse the inhibitory role of Smad3 on fibroblast proliferation. Flow cytometry analysis found that c-ski also diminished fibroblast apoptosis induced by Smad3 transfection. These results suggest that there is not only obvious expression of this regulatory protein but there is also a significant change in the levels of c-ski during wound healing. Its in vivo expression pattern and experiments in vitro suggest that c-ski may be involved in tissue repair by repressing Smad3 activity. Radiation can reduce c-ski and increase Smad3 expression, resulting in elevated Smad3 activity, resulting in diminished cell proliferation, cell apoptosis, and wound-healing delays.

Plasminogen activator inhibitor-I–related regulation of procollagen I (α 1 and α 2) by antitransforming growth factor-β 1 treatment during radiation-impaired wound healing

Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-β₁ (TGF-β₁)–related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of α(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti–TGF-β₁ treatment specifically inhibits α(I)-procollagen synthesis. We used an experimental wound healing study to address anti–TGF-β₁–associated influence on α(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (<i>n</i> = 8) surgery alone; Group 2 (<i>n</i> = 14) irradiation and surgery; Group 3 (<i>n</i> = 8) irradiation and surgery and anti–TGF-β₁ treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, <i>in situ</i> hybridization for TGF-β₁, immunohistochemistry, and immunoblotting for PAI-1, α₁/α₂(I)-procollagen. Results: Anti–TGF-β₁ significantly reduced TGF-β₁ mRNA (<i>p</i> < 0.05) and PAI-1 expression (<i>p</i> < 0.05). Anti–TGF-β₁ treatment <i>in vivo</i> significantly reduced α₁(I)-procollagen protein (<i>p</i> < 0.05) and the number of expressing cells (<i>p</i> < 0.05) in contrast to significantly increased (<i>p</i> < 0.05) α₂(I)-procollagen expression. Conclusion: These results emphasize anti–TGF-β₁ treatment to reduce radiation-induced fibrosis by decreasing α₁(I)-procollagen synthesis <i>in vivo</i>. α₁(I)-procollagen and α₂(I)-procollagen might be differentially regulated by anti–TGF-β₁ treatment. Increased TGF-β signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti–TGF-β₁ treatment.

Transforming growth factor beta 1 dependent regulation of Tenascin-C in radiation impaired wound healing

Background Following preoperative radiotherapy prior to ablative surgery of squamous epithelial cell carcinomas of the head and neck region fibrocontractive wound healing disorders occur. Tenascin-C is significantly increased in fibrotic tissue conditions and can be stimulated by the transcription factor NFκB p65. Previous studies showed a reduction of irradiation induced fibrosis during the wound healing process by anti-TGFβ 1-treatment. The aim of the study was to clarify the question whether Tenascin-C expression is elevated in radiation impaired wounds and whether anti-TGFβ 1-treatment is capable to influence Tenascin-C and NFκB expression. Material and methods Wistar rats (male, weight 300–500 g) underwent preoperative irradiation of the head and neck region with 40 Gy, fractionated four times 10 Gy (16 animals), whereas 8 non-irradated animals served as a control. Four weeks after irradiation a free myocutaneous gracilis flap taken from the groin was transplanted to the neck. Eight animals additionally received 5 μg anti-TGFβ 1 into the graft bed by intradermal injection prior to each fraction of irradiation and on days 1–7 post-operation. On day 14 and 28 following surgery immunohistochemistry (ABC–POX method) was performed assessing the cytoplasmic NFκB and Tenascin-C staining in the transition area between transplant and graft bed. For quantitative considerations the labeling index (ratio: positive cells/total cells) was determined. Results A significantly altered expression of Tenascin-C in the preirradiated tissue was observed following anti-TGFβ 1-treatment. NFκB protein was upregulated in irradiated animals and was significantly reduced in the anti-TGFβ 1 treated group on day 28 after transplantation. Conclusions Tenascin-C expression is prolonged in irradiated animals as compared to non-irradiated tissue. Tenascin-C seems to be regulated by TGFβ 1 as the application of TGFβ 1-neutralizing antibodies reduces Tenascin-C expression. Tenascin-C is a potentially useful marker for tissue remodeling due to its restricted distribution in adult and healthy tissue and a hallmark for developing fibrosis.

Differential Expression of Inflammatory Mediators in Radiation-Impaired Wound Healing

Background. Radiation impairs healing, although the underlying mechanisms are not clearly defined. Normal healing requires a fine balance of promoting and inhibiting factors. We hypothesize that there may be a down-regulation of promoting factors (nitric oxide) and, in turn, an up-regulation of healing inhibiting factors (TNF-α and IFN-γ) in the wound after radiation. Material and methods. Groups of 10 rats were irradiated using single dose 12 or 24 Gy electron radiation at the dorsal skin. Control rats were sham-irradiated. On Day 5 a skin incision in the irradiated area was performed and polyvinyl alcohol sponges were inserted subcutaneously. Rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitrite and nitrate (index of NO synthesis), TNF-α, and IFN-γ were measured within the wound fluid. Expression of the inducible NO-synthase (iNOS) was investigated by immunohistochemistry. Wound-derived fibroblasts were tested in vitro for NO and collagen synthesis. Results. Irradiation significantly reduced wound collagen deposition and wound-breaking strength ( P < 0.05). Impaired healing was reflected in diminished wound NO synthesis and iNOS expression ( P < 0.01). TNF-α and IFN-γ were increased in irradiated wounds ( P < 0.05). Ex vivo, NO synthesis and collagen deposition by fibroblasts from irradiated rats were decreased ( P < 0.01). In vitro irradiation of fibroblasts from nonirradiated rats decreased both NO and collagen production ( P < 0.01). Conclusion. Radiation-impaired healing is reflected in an imbalance of promoting and inhibiting factors, leading to increased levels of TNF-α and IFN-γ and decreased NO expression in the wound.

Differential Expression of Inflammatory Mediators in Radiation-Impaired Wound Healing

Background. Radiation impairs healing, although the underlying mechanisms are not clearly defined. Normal healing requires a fine balance of promoting and inhibiting factors. We hypothesize that there may be a down-regulation of promoting factors (nitric oxide) and, in turn, an up-regulation of healing inhibiting factors (TNF-α and IFN-γ) in the wound after radiation. Material and methods. Groups of 10 rats were irradiated using single dose 12 or 24 Gy electron radiation at the dorsal skin. Control rats were sham-irradiated. On Day 5 a skin incision in the irradiated area was performed and polyvinyl alcohol sponges were inserted subcutaneously. Rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitrite and nitrate (index of NO synthesis), TNF-α, and IFN-γ were measured within the wound fluid. Expression of the inducible NO-synthase (iNOS) was investigated by immunohistochemistry. Wound-derived fibroblasts were tested in vitro for NO and collagen synthesis. Results. Irradiation significantly reduced wound collagen deposition and wound-breaking strength (P < 0.05). Impaired healing was reflected in diminished wound NO synthesis and iNOS expression (P < 0.01). TNF-α and IFN-γ were increased in irradiated wounds (P < 0.05). Ex vivo, NO synthesis and collagen deposition by fibroblasts from irradiated rats were decreased (P < 0.01). In vitro irradiation of fibroblasts from nonirradiated rats decreased both NO and collagen production (P < 0.01). Conclusion. Radiation-impaired healing is reflected in an imbalance of promoting and inhibiting factors, leading to increased levels of TNF-α and IFN-γ and decreased NO expression in the wound.

Expression of MMP1 in Surgical and Radiation-Impaired Wound Healing and Its Effects on the Healing Process

Radiation-impaired wound is characterized by delayed healing, nonhealing, and carcinogenesis. The mechanism remains unclear. Matrix metalloproteinases (MMPs) are one family of key regulators of the process of wound healing. Their abnormal expression plays important roles in the formation of some chronic skin ulcers. The objective of this project was to study the expression of MMP1 in surgical and radiation-impaired wound healing and its effects on the healing process and tissue remodeling. A rat model of radiation-impaired wound healing was used. Routine light microscopy, electron microscopy, immunohistochemistry, and in situ hybridization, all of which enabled the detection of MMP1 expression during the healing process, were performed. The wound healing process was impaired and delayed. In rats receiving 25Gy gamma-ray locally, the irradiated wounds healed 6 days later than the nonirradiated controls. The following changes in MMP1 expression were found: (1) In the early inflammatory phase and in the period of granulation tissue formation, MMP1 expression was only slightly if at all affected in the newly formed epidermis of irradiated wounds compared with controls. Later, the epidermal expression of MMP1 in radiation wounds was comparatively increased following the delay of the healing process. (2) MMP1 expression in irradiated wounds was markedly decreased in fibroblasts, endothelial cells, and macrophages compared with controls. The expression phase was prolonged because of the delay of the healing process. The reduced expression of MMP1 in granulation tissue retards such important processes as cell migration, angiogenesis, and tissue remodeling, thus slowing the healing process. The expression ofMMP1 in the proliferating keratinocytes may help re-epithelialization. However, in the late healing period, overexpression of MMP1 in the epidermis may hinder the establishment of basal membrane and the formation of granulation tissue, and affect the tissue remodeling process.

Healing impairment of open wounds by skin irradiation.

Ionizing radiation is frequently used for the treatment of malignancy. Sequelae of therapeutic radiation frequently present clinical problems in the form of poor wound healing and easy injury of treated tissue in response to mild trauma. We describe a radiation-impaired wound healing model in guinea pigs, developed to determine the effect of cutaneous irradiation on wound contraction. Guinea pigs were anesthetized and a flap of skin was isolated and treated with x-rays to 18 Gy. Circular wounds of identical size were made in irradiated and control skin, and average wound size was recorded. Our results demonstrate that a statistically significant wound healing deficit is produced in open wounds by surface irradiation of skin. Radiation of skin results in slower healing of open wounds, and provides an in vivo system for evaluation of topical dressings and growth factors in radiation-impaired wounds.