Supplemental vitamin A prevents the tumor-induced defect in wound healing.

To test our hypothesis that supplemental vitamin A would mitigate the impaired healing that occurs in tumor-bearing animals, six groups of C3H mice, eight per group, eating a standard commercial mouse chow ad libitum that supports normal growth, reproduction, and longevity were innoculated with 200,000 C3HBA cells. When tumors measured approximately 6 mm in diameter, the mice were anesthesized and wounded (dorsal skin incisions and subcutaneous polyvinyl alcohol sponges). Twenty-four hours later, two groups (one continued on the chow and the other started on the chow supplemented with 150,000 IU vitamin A/kg chow) underwent local tumor irradiation; two groups, one ingesting the chow, the other the vitamin A supplemented chow, were started on cyclophosphamide therapy; two groups, one ingesting the chow, the other the vitamin A supplemented chow, received neither local tumor irradiation nor cyclophosphamide therapy. An additional two groups ingesting the chow, one group neither innoculated with tumor nor wounded, the other wounded by not innoculated, served as controls. Wound breaking strength and sponge reparative collagen accumulation (assessed by hydroxyproline proline measurement) were used as indicators of wound healing. The mice were killed 12 days after wounding. Tumor presence decreased wound breaking strength and sponge hydroxyproline content; these effects were largely negated by supplemental vitamin A. Local tumor irradiation diminished the adverse effect of tumor on sponge reparative collagen content but to a lesser extent than the supplemental vitamin A. Supplemental vitamin A added to the irradiation effect on healing but irradiation did not add to the vitamin A effect. Cyclophosphamide, a systemic radiomimetic anti-tumor agent, did not alter the impaired wound healing of the tumor-bearing mice. Supplemental vitamin A mitigated the impaired wound healing in the cyclophosphamide-treated tumor-bearing mice. Supplemental vitamin A also moderated the effects of wounding, tumor, and tumor therapies (local irradiation and cyclophosphamide) on the increase in adrenal size, leukopenia, thrombocytopenia, and thymic involution (except the last was not moderated in the cyclophosphamide-treated tumor-bearing rats). The splenic enlargement in the untreated tumor-bearing wounded rats and in those treated with cyclophosphamide was lessened by supplemental vitamin A. We hypothesize that these anti-stress effects of vitamin A underlie, in part, its action in mitigating the impaired wound healing of tumor-bearing mice, including those treated by local irradiation or cyclophosphamide. These findings have implications for the care of patients with malignant tumors.     

Influence of vitamin A on wound healing in rats with femoral fracture.

Groups of healthy wounded rats with and without comminuted femoral fractures, and maintained on nutritionally complete commercial rat chow with and without supplemental vitamin A, were studied. The test wounds were standard dorsal skin incisions and s.c. polyvinyl alcohol sponge implants. In some experiments the rats were pair-fed; the rats with femoral fracture not receiving supplemental vitamin A were the lead group for determining food allowanced. In other experiments, the rats were allowed food ad libitum. We found that wound healing of rats with femoral fracture was increased when supplemental vitamin A was given, but the supplemental vitamin A did not completely obviate the adverse effects of fracture. The ratio of the breaking strengths of the skin incisions after formalin fixation to the breaking strengths of the incisions in the fresh state was higher in the unsupplemented rats, supporting the results of our earlier experiments that vitamin A increases the rate of collagen cross-linking.     

Local instillation of Staphylococcus aureus peptidoglycan at operation prevents wound healing impairment after trauma

BACKGROUND: Considerable experimental evidence and limited clinical evidence indicate that wound healing is impaired after trauma. Because Staphylococcus aureus peptidoglycan (SaPG) accelerates healing in normal rats and prevents wound healing impairment induced by glucocorticoids, cyclophosphamide, and streptozotocin-diabetes, we hypothesized that SaPG would prevent the impaired wound healing after trauma. METHODS: In each of two experiments, 18 Sprague-Dawley male rats were divided into two groups, nine rats each, paired by weight; one group received unilateral comminuted femoral fracture and wounding (two dorsal skin incisions and six subcutaneous polyvinyl alcohol [PVA] sponges), and the other group was only wounded. The incision and PVA sponges on one side were inoculated at operation with saline (200 microL/incision, 50 microL/sponge) and on the other side with SaPG in saline (860 microg of SaPG per centimeter of incision, 0.5 mg of SaPG per sponge). Rats ate chow and drank tap water ad libitum and were killed 7 days postoperatively. RESULTS: In both experiments, the wound breaking strength (WBS) of saline-inoculated incisions was significantly lower in rats with femoral fracture; histologically, reparative granulation tissue was looser and less prominent. WBS of SaPG-inoculated incisions in rats with and without femoral fracture was significantly higher than that of saline-inoculated incisions and, histologically, reparative tissue was more prevalent, more closely packed, and more mature. WBS of SaPG-inoculated incisions in rats with femoral fracture was similar to that of saline-inoculated incisions in rats without femoral fracture. Reparative tissue hydroxyproline and histologic findings of saline-inoculated PVA sponge reparative tissue were similar in all rats, as were the increases induced by SaPG inoculation. CONCLUSION: Wound breaking strength and histologic findings of skin incisions (impaired in rats with unilateral femoral fracture) are more sensitive to the adverse effects of trauma than accumulation of PVA sponge reparative tissue. A single inoculation of SaPG at operation increased wound incision healing in rats both without and with femoral fracture and notably prevented the impaired healing in rats with femoral fracture.     

Staphylococcus aureus peptidoglycan ameliorates cyclophosphamide-induced impairment of wound healing.

Cyclophosphamide given systemically to rats leads to impaired wound healing, characterized by decreases in the inflammatory reaction, fibroplasia, neovascularization, reparative collagen accumulation, and wound breaking strength. In contrast, the local application of Staphylococcus aureus peptidoglycan at the time of wounding increases all of these processes in normal rats. Accordingly, we hypothesized that inoculation of S. aureus peptidoglycan into wounds of cyclophosphamide-treated rats would ameliorate the otherwise impaired healing. Dorsal bilateral skin incisions and subcutaneous implantation of polyvinyl alcohol sponges (two on each side) were performed on male Sprague-Dawley rats receiving either saline or cyclophosphamide (24 mg/kg) intraperitoneally at the time of operation, on postoperative days 1, 2, 3, 4 (for rats killed on postoperative day 7), and also on day 8 (for rats killed on postoperative day 14). The incisions on one side were inoculated at the time of closure with 0.2 ml of saline solution, and the incisions on the other side with 6 mg S. aureus peptidoglycan in 0.2 ml saline solution (860 microg/cm incision). The sponges were instilled with 0.1 ml saline solution on the saline solution-instilled incision side or with S. aureus peptidoglycan 0.5 mg/sponge) in 0.1 ml saline solution on the other side. In control rats receiving saline solution intraperitoneally, incisions treated with S. aureus peptidoglycan were significantly stronger than saline solution-treated incisions by a factor of 1.8 at 1 week (p < 0.001); at 2 weeks the increase was small and not significant. Cardiac blood leukocytes and platelets fell markedly (90%) in cyclophosphamide- treated rats, and there was a decrease in wound breaking strength of their saline-treated incisions at both 7 and 14 days compared with saline solution-treated incisions of control rats. S. aureus peptidoglycan treatment of the wounds completely prevented this effect at 7 days, and partially at 14 days. Polyvinyl alcohol sponge reparative tissue hydroxyproline, 7 days after surgery, was decreased in cyclophosphamide-treated rats; this was completely prevented by S. aureus peptidoglycan treatment of the sponges. Histologically, the inflammatory response to the wounding, influx of macrophages and fibroblasts, angiogenesis, and collagen accumulation were all reduced at day 7 and 14 after surgery in the sponge reparative tissue of cyclophosphamide- treated rats; this was prevented by S. aureus peptidoglycan treatment of the sponges. In conclusion, a single local application of S. aureus peptidoglycan ameliorates cyclophosphamide-impaired wound healing.     

Supplemental vitamin A prevents the acute radiation-induced defect in wound healing.

Acute radiation injury leads to thymic involution, adrenal enlargement, leukopenia, thrombocytopenia, gastrointestinal ulceration, and impaired wound healing. The authors hypothesized that supplemental vitamin A would mitigate these adverse effects in rats exposed to acute whole-body radiation. This hypothesis was based on previous experiments in their laboratory that showed that supplemental vitamin A is thymotropic for normal rodents and lessens the thymic involution, lymphopenia, and adrenal enlargement that follows stress, trauma, and neoplasia, largely obviates the impaired wound healing induced by the radiomimetic drugs streptozotocin and cyclophosphamide, lessens the systemic response (thymic involution, adrenal enlargement, leukopenia, lymphocytopenia) to local radiation, and shifts the median lethal dose (LD50/30) following whole-body radiation to the right. To test their hypothesis, dorsal skin incisions and subcutaneous implantation of polyvinyl alcohol sponges were performed in anesthetized Sprague-Dawley rats at varying times following sham radiation or varying doses of whole-body radiation (175-850 rad). In each experiment, the control diet [which contains about 18,000 IU vit. A/kg chow (3 X the NRC RDA for normal rats)] was supplemented with 150,000 IU vit. A/kg diet beginning at, before, or after sham radiation and wounding or radiation and wounding. The supplemental vitamin A prevented the impaired wound healing and lessened the weight loss, leukopenia, thrombocytopenia, thymic involution, adrenal enlargement, decrease in splenic weight, and gastric ulceration of the radiated (750-850 rad) wounded rats. This was true whether the supplemental vitamin A was begun before (2 or 4 days) or after (1-2 hours to 4 days) radiation and wounding; the supplemental vitamin A was more effective when started before or up to 2 days after radiation and wounding. The authors believe that prevention of the impaired wound healing following radiation by supplemental vitamin A is due to its enhancing the early inflammatory reaction to wounding, including increasing the number of monocytes and macrophages at the wound site; possible effect on modulating collagenase activity; effect on epithelial cell (and possible mesenchymal cell) differentiation; stimulation of immune responsiveness; and lessening of the adverse effects of radiation.     

Wound healing: captopril, an angiogenesis inhibitor, and Staphylococcus aureus peptidoglycan

BACKGROUND: Captopril, an angiotensin-converting enzyme inhibitor, used for treating hypertension and heart failure, inhibits angiogenesis in the corneas of rats in response to basic fibroblast growth factor, slows the growth of experimental tumors in rats, and leads to the regression of Kaposi's sarcoma. Because angiogenesis is key to wound healing, we hypothesized that captopril would impair wound healing. We hypothesized also that because local application at operation of Staphylococcus aureus peptidoglycan (SaPG) increases angiogenesis and accelerates wound healing in rats, SaPG would prevent or ameliorate the postulated captopril-impaired wound healing. MATERIALS AND METHODS: In each experiment, rats were divided randomly into two groups: one drinking tap water, and the other, tap water containing 0.5 mg captopril/ml. All ate chow and drank ad libitum, pre-operatively (4-12 days) and postoperatively (7 days). In experiments 1 and 2, bilateral paravertebral 5.5-cm skin incisions were made aseptically (intraperitoneal sodium pentobarbital), and closed with interrupted No. 35 stainless-steel sutures. On one side, the wound was immediately inoculated with 157 microliter pyrogen-free isotonic saline and on the other side the wound was inoculated with 157 microliter saline containing 4.7 mg SaPG (860 microgram SaPG/cm incision). In the third experiment, polyvinyl alcohol (PVA) sponges (16-17 mg dry wt each) containing either 50 microliter saline or 0.5 mg SaPG in 50 microliter saline were implanted subcutaneously, two on each side, via 1-cm incisions closed with a single suture. In the fourth experiment, 5.5-cm bilateral skin incisions and subcutaneous implantation of PVA sponges were done as described but all sites were instilled with saline only. All rats were euthanized (CO(2) asphyxia) 7 days postoperatively. RESULTS: Wound breaking strength (WBS) of the saline-treated incisions was significantly higher (P < 0.001) in captopril-treated rats than in controls (172 +/- 13 g vs 105 +/- 6 g) in experiment 1 and higher, but not significantly in captopril-treated rats in experiment 2 (153 +/- 8 g vs 114 +/- 6 g) (PNS). SaPG inoculation of the incisions increased WBS significantly in both control and captopril-treated rats: 187 +/- 11 g vs 105 +/- 6 g (P < 0.001) and 283 +/- 16 g vs 172 +/- 13 g (P < 0.001), respectively, in experiment 1, and 217 +/- 13 g vs 114 +/- 6 g (P < 0.0001) (controls) and 266 +/- 17 g vs 153 +/- 8 g (captopril-treated rats) (P < 0.0001) in experiment 2. In experiment 3, subcutaneous PVA saline-inoculated sponge reparative tissue hydroxyproline (OHP) content was similar in control and captopril-treated rats, and SaPG inoculation increased reparative tissue OHP significantly in both groups: 2458 +/- 218 microgram/100 mg dry sponge vs 3869 +/- 230 microgram/100 mg (P < 0.001) (controls) and 2489 +/- 166 microgram/100 mg vs 4176 +/- 418 microgram/100 mg (P < 0.001) (captopril-treated rats). Histologically, angiogenesis and reparative tissue collagen were similar in control and captopril-treated rats, in both saline-inoculated and SaPG-inoculated sponges. In experiment 4 (all incisions and subcutaneous PVA sponges were saline-inoculated), there was no significant difference in WBS between control and captopril-treated rats (107 +/- 6 g vs 96 +/- 5 g, NS). PVA sponge reparative tissue OHP was significantly higher in captopril-treated rats: 3698 +/- 170 microgram/100 mg dry sponge vs 2534 +/- 100 microgram/100 mg (P < 0.0001). CONCLUSION: Unexpectedly, in four experiments, captopril did not inhibit WBS or PVA sponge reparative tissue angiogenesis or collagen accumulation; in fact, WBS was increased significantly in one of three experiments, and PVA sponge reparative tissue OHP was increased significantly in one of two experiments. Also, captopril did not interfere with the wound healing-accelerating effect of SaPG.     

Single local instillation of nonviable Staphylococcus aureus or its peptidoglycan ameliorates glucocorticoid-induced impaired wound healing.

An excess in glucocorticoid steroids, either from endogenous or exogenous sources, has been shown to inhibit wound repair. Key to this impairment is a diminution of the inflammatory response to wounding, fibroplasia, capillary formation, reparative tissue collagen accumulation, and wound breaking strength. Because a single local application at operation of nonviable Staphylococcus aureus or its peptidoglycan increases all of these processes in normal rats, we hypothesized that nonviable S. aureus and S. aureus peptidoglycan would each ameliorate glucocorticoid-induced impaired healing. Sprague-Dawley male rats aseptically received two 7 cm paravertebral skin incisions and underwent subcutaneous implantation of polyvinyl alcohol sponges. Two glucocorticoids were used: hydrocortisone, 8 mg intramuscularly, daily beginning 1 day before operation and continuing during the postoperative period; or a single dose of a long-acting preparation of methylprednisolone, 6 or 8 mg intramuscularly, on the day before operation. Controls received intramuscular injections of saline solution at the same respective times. At the time of the operation, one incision and the polyvinyl alcohol sponges on one side of the animal were instilled with saline solution while the incision and sponges on the opposite side were instilled with nonviable S. aureus (hydrocortisone study) or S. aureus peptidoglycan (two methylprednisolone studies). The data showed that, at postoperative day 7, the single local application at wounding of nonviable S. aureus or S. aureus peptidoglycan increased wound breaking strength in the control rats by factors of 1.6 in the hydrocortisone experiment and 1.4 and 1.6 in the methylprednisolone studies. These treatments prevented (in hydrocortisone-treated rats) or mitigated (in methylprednisolone-treated rats) the glucocorticoid-induced decrease in wound breaking strength. In addition, these treatments prevented the glucocorticoid-induced decreases in the inflammatory (largely mononuclear cells) response to wounding and in the accumulation within the polyvinyl alcohol sponge of reparative tissue fibroblasts, capillaries, and collagen.     

Single local instillation of Staphylococcus aureus peptidoglycan prevents diabetes-induced impaired wound healing

Diabetes-induced impaired wound healing is characterized by inhibition of the inflammatory response to wounding, macrophage infiltration, angiogenesis, fibroplasia, reparative collagen accumulation, and wound breaking strength. Because all of these processes are accelerated in normal rats by a single local application at operation of Staphylococcus aureus peptidoglycan, we hypothesized that S. aureus peptidoglycan would prevent diabetes-induced impaired wound healing, despite persistent, untreated hyperglycemia, polydipsia, glycosuria, and polyuria. Sprague-Dawley male rats were divided into two groups. One group received an intraperitoneal injection of streptozotocin (65 mg/kg) in citrate solution; the other group received an intraperitoneal injection of an equivalent volume of citrate solution. Seventeen days after the injections, the diabetic and control rats received aseptically two 5.5-cm paravertebral incisions and subcutaneous implantation of six polyvinyl alcohol sponges, three on each side. On one side, each sponge contained 0.5 mg S. aureus peptidoglycan in 50 µl saline solution, and the incision was inoculated along its length with 4.7 mg S. aureus peptidoglycan in 157 µl saline solution (860 µg/S. aureus peptidoglycan/cm incision); on the other side, the same respective volumes of saline were used. During the preoperative and postoperative periods, diabetic rats lost a small amount of weight (2%), were hyperglycemic (363 ± 10 mg/100 ml blood), polydipsic, glycosuric, and polyuric, whereas the controls gained weight (25%) and were normoglycemic (104 ± 5 mg/100 ml blood); these differences were significantly different (p < .001 in each case). In controls, S. aureus peptidoglycan inoculation increased wound breaking strength (by a factor of 2.0) and hydroxyproline content (by a factor of 1.4; p < .001 in each case); in diabetics, there were significant decreases in wound breaking strength (by a factor of 1.7) and hydroxyproline content (by a factor of 1.3) of saline solution-inoculated incisions and sponges compared with the wound breaking strength and hydroxyproline content of saline solution-inoculated incisions and sponges in controls (p < .02 and p < .001, respectively). These decreases were completely prevented when the incisions and polyvinyl alcohol sponges had been inoculated at operation with S. aureus peptidoglycan; S. aureus peptidoglycan inoculation in the diabetic rats increased wound breaking strength by a factor of 2.2 and sponge reparative tissue hydroxyproline by a factor of 1.6 (p < .001 in each case). Thus, diabetes-induced impaired wound healing was prevented completely by a single local instillation at operation of S. aureus peptidoglycan, despite persistent, untreated hyperglycemia, polydipsia, polyuria, and glycosuria.     

Accelerating effects of nonviable Staphylococcus aureus, its cell wall, and cell wall peptidoglycan.

We have previously reported that local application of viable Staphylococcus aureus dramatically accelerates wound healing, but viable Staphylococcus epidermidis does not. Because the S. aureus effect occurred in the absence of infection and because the cell walls of the two bacterial species differ, we hypothesized that nonviable S. aureus, its cell wall, and its cell wall component(s) would accelerate healing. Nonviable S. aureus was prepared by chemical and physical means, and its cell wall and peptidoglycan was prepared from heat-killed cultures. In a large number of experiments, nonviable S. aureus (independent of the strain's protein A content), its cell wall, and peptidoglycan when instilled locally at the time of wounding each significantly increased the breaking strength of rat skin incisions (tested both in the fresh state and after formalin fixation). These agents also enhanced subcutaneous polyvinyl alcohol sponge reparative tissue collagen accumulation, generally by a factor of two. Histologic features of treated and control incisions were similar. In contrast, the reparative tissue of treated sponges contained more neutrophils, macrophages, capillaries, and collagen. These experimental data thus confirm our previous studies, as well as our hypothesis, and extend these observations of enhanced wound healing to specific fractions of the bacterial cell wall.     

Interleukin 2 enhances wound healing in rats

Antigen-stimulated lymphocytes secrete lymphokines which have been shown to enhance in vitro fibroblast migration, proliferation, and protein synthesis. In the present experiments, the effect of human recombinant interleukin 2 (RIL-2) on wound healing was assessed in vivo. Groups of male Lewis rats, 225-250 g, underwent intraperitoneal insertion of osmotic pumps and a 7-cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges under anesthesia. The dorsal wounds were closed with stainless-steel sutures. The dose of RIL-2 administered was 60,000 u/rat/day for 7 days in experiment I, and 140,000 u/rat/day for 7 days in experiment II. Controls received equal volumes of excipient. Animals were sacrificed 10 days post wounding and wound healing was assessed by fresh breaking strength, fixed breaking strength (following 72 hr of Formalin fixation which maximally crosslinks the collagen present), and sponge hydroxyproline content (an index of reparative collagen accumulation). In vivo RIL-2 administration significantly augmented wound fresh and fixed breaking strength and wound collagen synthesis. Higher doses of RIL-2 (experiment II) did not result in further increases in the parameters studied. The data suggest that lymphocytes participate directly in the process of wound healing.     

Inhibition of wound repair by thymic hormones

To further define the role of the thymus in wound healing, we studied the effects of two thymic hormones on fibroplasia in normal euthymic and in nude athymic mice. Groups of 10 mice underwent a 2.5 cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges. Starting on the day of wounding, the following daily injections were given: (1) thymopentin (TP5), an active synthetic pentapeptide of thymopoietin, a naturally occurring thymic hormone (1 microgram/day/IM); (2) thymulin or facteur thymique serique (FTS), a naturally occurring circulating thymic hormone (0.2 microgram/day/IM); (3) control saline solution (0.1 ml/day/IM). All mice were killed 4 weeks after wounding, and wound breaking strength and hydroxyproline content of the sponge granulomas were measured. The results show that both thymic hormones impaired wound breaking strength and reparative collagen synthesis in normal and athymic mice. The magnitude of the wound healing impairment induced by the two hormones was equal in the thymus-bearing and in the nude mice. The data support previous findings, which suggested that the thymus has an inhibitory effect on wound healing.     

A comparison between cyclosporin A and methylprednisolone plus azathioprine on bronchial healing following canine lung autotransplantation

The effects of two low-dose immunosuppressive therapies upon the healing of the bronchial anastomosis and skin wounds following lung autotransplantation were evaluated. Autotransplantation was performed in three groups of dogs: Group 1 (nine dogs) received no immunosuppression, Group 2 (seven dogs) received postoperative immunosuppression with methylprednisolone (2 mg/kg) and azathioprine (1.5 mg/kg), and Group 3 (four dogs) received postoperative immunosuppression with cyclosporin A (20 mg/kg/day). Skin incisions 7 cm in length were made in the dorsal region of each dog. Dogs were put to death 23 days postoperatively, and the breaking strength of the bronchial anastomoses and skin wounds was evaluated with the Instron Universal Testing Machine, with a cross-head speed of 0.5 cm/min. Bronchial breaking strengths were similar in Groups 1 and 3 and significantly higher than in Group 2 (p less than 0.001). Skin breaking strengths were similar in Groups 1 and 3 and significantly higher than in Group 2 (p less than 0.001). Scanning electron microscopic (SEM) studies of both skin and bronchial wounds showed normal formation of collagen bundles in Groups 1 and 3 but a disorganized pattern in Group 2. Our results suggest that low-dose immunosuppression with methylprednisolone and azathioprine significantly affects wound healing and breaking strength of both bronchial anastomoses and healed skin incisions following canine lung autotransplantation. Immunosuppression with cyclosporin A had no adverse effect on either bronchial or skin healing.     

Differential effect of tacrolimus on dermal and intestinal wound healing.

Tacrolimus, used in organ transplantation, inhibits cellular immune function. Little is known about the effect on dermal and colonic healing. Groups of 10 rats underwent dorsal skin incision, and polyvinyl alcohol sponges were implanted subcutaneously. Beginning at the day of wounding, rats were treated intraperitoneal with 1.0 or 2.0 mg tacrolimus/kg/day. Animals were sacrificed 10 d later to determine wound breaking strength and reparative collagen deposition. Expression of transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma was studied in wounds. Groups of 8 rats underwent laparotomy and left colonic anastomosis. These rats were treated by subcutaneous injections with 2.0 or 5.0 mg tacrolimus/kg. Animals were sacrificed 5 d later to test colonic bursting pressure and reparative collagen deposition. Expression of TGF-beta, TNF-alpha, IFN-gamma, and CD4 and CD8 in the anastomosis was investigated. Tacrolimus impaired dermal healing (p < .05). This was paralleled by decreased expression of TGF-beta (stimulates healing) and increased expression of IFN-gamma and TNF-alpha (both inhibit healing) (p < .05). In contrast, tacrolimus did not inhibit healing of colonic anastomoses. No effect was seen on the expression of TGF-beta, TNF-alpha, IFN-gamma, and CD4 and CD8 in colonic anastomoses. We concluded that tacrolimus differentially effects tissue healing and expression of cellular mediators in dermal and intestinal wounds.     

Supplemental L-arginine enhances wound healing in diabetic rats

L-arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L-arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L-arginine can restore the impaired wound healing of diabetic rats. Fifty-six male Lewis rats were used in this study, of which twenty-nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty-seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L-arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L-arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L-arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline-treated counterparts. mRNA for procollagen I and III were elevated by L-arginine treatment in both diabetic rats and controls. Treatment with L-arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L-arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis.     

Significance of T-lymphocytes in wound healing

To determine the importance of T-lymphocytes in wound healing, we examined the effect of T-lymphocyte depletion on the healing of surgical wounds. Thirty Balb/c mice were injected intraperitoneally with 1 mg of rat anti-mouse (IgG2b) cytotoxic monoclonal antibody (30H12) against the Thy1.2 (all T) determinant. Twenty-four hours later animals showed a greater than 95% depletion of Thy1.2 cells in peripheral blood and spleen. Thirty control mice received nonspecific rat immunoglobulin (1 mg). Twenty-four hours after treatment mice underwent a 2.5 cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges. Injections were repeated at weekly intervals. Wound healing was assessed at 2, 3, and 4 weeks by the breaking strength of wound strips and by the hydroxyproline content of sponge granulomas (an index of wound reparative collagen deposition). Thy1.2 depletion at death was 95% to 57% in peripheral blood and 86% to 68% in the spleen. Both groups gained weight equally. We found that T cell depletion significantly impairs wound breaking strength and wound collagen deposition at all times studied. The data strongly suggest that T-lymphocytes modulate fibroblast activity during normal wound healing.     

Nonviable Staphylococcus aureus and its peptidoglycan stimulate macrophage recruitment,angiogenesis, fibroplasia,and collagen accumulation in wounded rats

We have previously shown that local application at the time of operation of Staphylococcus aureus, nonviable S. aureus, its cell wall, or S. aureus peptidoglycan accelerates wound healing. We hypothesized that this effect is due to both direct and indirect mechanisms, among which is an increase in the inflammatory response to wounding, resulting in an increase in macrophages, angiogenesis, and fibroblasts. Twenty-seven Sprague-Dawley male rats were anesthetized, and two 7-cm paravertebral skin incisions were made. Four polyvinyl alcohol sponges, two on each side, containing either 100 µl of isotonic saline or 0.5 mg of nonviable S. aureus or S. aureus peptidoglycan in 100-µl saline were implanted subcutaneously. Nonviable S. aureus or S. aureus peptidoglycan (860 µg/cm incision) in 200-µl saline were inoculated into the incisions at closure. The rats ate a commercial rat chow and drank tap water ad libitum throughout. After days 3 and 7 postwounding, rats were euthanized, and tissues were examined for immunohistochemical features of reparative tissue using ED-1, Factor VIII, and vimentin antibodies, markers for monocyte/macrophages, endothelial cells, and mesenchymal cells (including fibroblasts), respectively. Incisions treated with nonviable S. aureus or S. aureus peptidoglycan showed more macrophages along and deep in the wound tract 7 days postoperatively. Nonviable S. aureus or S. aureus peptidoglycan-treated sponges were surrounded and penetrated by much larger capsules of reparative tissue than saline-treated sponges at both 3 and 7 days. Neutrophil influx was much greater in nonviable S. aureus or S. aureus peptidoglycan-treated sponges, especially in central regions, and there were many more ED-1-stained macrophages in distinct geographic locations, specifically, the more peripheral-cortical areas. Some clustering of macrophages occurred around areas of invasion by reparative tissue into the surrounding subcutaneous fat and within the interstices of the sponges at the interface between reparative tissue and acute inflammatory cells. In contrast, saline-treated sponge reparative tissue had significantly fewer macrophages, much thinner and flimsy reparative tissue, with proportionately fewer macrophages clustering centrally. There were many more mesenchymal cells (notably fibroblasts) and new blood vessels and much more reparative collagen in the nonviable S. aureus or S. aureus peptidoglycan- treated sponges. We conclude that local application of nonviable S. aureus or S. aureus peptidoglycan at wounding induces an increased number and alteration in location of macrophages, increased influx (or proliferation) of mesenchymal cells (notably fibroblasts), and increased angiogenesis and reparative collagen accumulation, as well as increasing the overall acute inflammatory response to wounding.     

Transforming growth factor beta3 promotes fascial wound healing in a new animal model

HYPOTHESIS: Transforming growth factor beta(3) (TGF-beta(3)) promotes fascial wound healing in a new animal model, as measured by wound breaking strength, collagen deposition, and cellular proliferation. DESIGN/INTERVENTION: Bilateral, longitudinal incisions were made in the anterior rectus sheaths of 24 male New Zealand white rabbits. One incision was treated with 1 microg of TGF-beta(3); the contralateral incision served as a control. The wounds were harvested at 1, 2, 3, 4, 6, and 8 weeks after creation ("wounding"). MAIN OUTCOME MEASURES: Wound tissue was tested for breaking strength using a tensiometer and processed for histological examination of collagen deposition and cellular proliferation at all time points after wounding. Collagen deposition and cellular proliferation were measured in histological cross sections of wounds with Masson trichrome staining and proliferating cell nuclear antigen immunohistochemistry, respectively. RESULTS: At all time points after wounding, treatment with TGF-beta(3) significantly increased the wound breaking strength (up to 138%) and collagen deposition (up to 150%) over the control group. Cellular proliferation was increased during the first 3 weeks after wounding (up to 147%), but returned to baseline levels by the fourth week. CONCLUSIONS: Transforming growth factor beta(3) promotes fascial wound healing. In this new animal model of fascial wound healing, TGF-beta(3) increased fascia breaking strength, collagen deposition, and cellular proliferation. These results are similar to findings in cutaneous wound models and demonstrate, for the first time, a pharmacologic agent to accelerate fascial healing.     

Intravenous hyperalimentation with high arginine levels improves wound healing and immune function

The purpose of this study was to evaluate the effect of increased arginine levels in intravenous hyperalimentation (IVH) therapy on wound healing and thymic immune function. Groups of SD rats, 275-325 g, underwent placement of internal jugular catheter, 7-cm dorsal skin wounding, insertion of polyvinyl alcohol sponges subcutaneously, and closure of wounds with stainless-steel sutures. Twenty-four hours later, rats were started on IVH at a rate of 0.8-1 ml/100 g body wt/hr. All IVH solutions contained 20% dextrose, adequate amounts of minerals and vitamins, and two different amino acid mixtures: (A) Fre III (4.05 g ARG/liter) (n = 13); (B) experimental (7.50 g ARG/liter) (n = 11). Solutions were isonitrogenous, and contained similar amounts of essential amino acids. After 7 days of IVH, weight gain did not differ between the two groups; however, cumulative N balance was superior in group A. Wound healing was improved in group B as assessed by fresh wound strip breaking strength, fixed breaking strength, and the amount of reparative collagen deposition as assessed by the hydroxyproline content of the implanted sponges. Group B animals also had improved thymic function as assessed by thymic weight, the total number of thymic lymphocytes/gland and mitogenic reactivity of thymic lymphocytes to PHA and Con A. The experiments indicate that high arginine levels in IVH solutions improve wound healing and thymic immune function following injury.     

Inhibition of tumor necrosis factor-alpha attenuates wound breaking strength in rats.

Exogenous administration of tumor necrosis factor-alpha has been shown to both enhance and attenuate cutaneous healing in a dose-dependent manner. We examined the effects of tumor necrosis factor inhibition in the healing wound by both systemic and local administration of tumor necrosis factor-binding protein. Male Balb/C mice underwent dorsal skin incision with subcutaneous implantation of 20 mg polyvinyl alcohol sponges (4 per animal). In Experiment I, one group (n = 20) received intraperitoneal injections of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding, while another group (n = 20) received saline. Four animals from each group were euthanized on days 1, 3, 5, 7, and 14 postwounding. In Experiment II, one group (n = 10) received an intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding and every third day thereafter. Another group (n = 10) received an intraperitoneal injection of saline at the time of wounding and every third day thereafter. In Experiment III, one group received a single intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding (n = 7), or on postwounding day 4 (n = 7), or day 7 (n = 7). Another group received saline injections at the time of wounding (n = 7), or on postwounding days 4 or 7 (n = 7, respectively). All animals in Experiments II and III were killed at postwounding day 14. Wound breaking strengths were assessed using a tensiometer. Wound fluid collected from the implanted sponges was assayed for tumor necrosis factor-alpha and tumor necrosis factor-binding protein levels using a biological assay and enzyme-linked immunosorbent assay, respectively. Collagen gene expression in sponge granulomata was assessed by Northern analysis. Collagen deposition in sponges was quantified by measuring hydroxyproline content. Wounds were significantly weaker in the animals that received repeated injections of tumor necrosis factor-binding protein with a mean wound breaking strength of 93.1 g vs. 186.6 g in controls (p < 0.05). Wound breaking strength in groups that received a single injection of tumor necrosis factor-binding protein on either day 0, 4, or 7 postwounding were no different than their respective controls. There was no difference in the mean hydroxyproline content of sponges between any of the tumor necrosis factor-binding protein groups and their respective controls. Northern analysis for collagen I and III expression also revealed no differences. These data indicate that continued systemic administration of tumor necrosis factor-binding protein resulted in significantly weaker wounds with no corresponding differences in wound collagen content, and collagen gene expression. This suggests that tumor necrosis factor-alpha inhibition throughout healing leads to a qualitatively impaired wound without a quantitative alteration in collagen deposition.     

Sirolimus impairs wound healing

Background and aims Clinically, the immunosuppressive drug sirolimus, used in organ transplantation, appears to impair wound healing. Little is known about the mechanisms of action. We investigated the effect of sirolimus on wound healing, and we analyzed the expression of stimulating mediators of angiogenesis (VEGF, vascular endothelial growth factor) and collagen synthesis (nitric oxide) in wounds. Materials and methods Groups of ten rats underwent dorsal skin incision, and polyvinyl alcohol sponges were implanted subcutaneously. Beginning at the day of wounding, rats were treated with 0.5, 2.0, or 5.0 mg sirolimus/kg/day. Animals were killed 10 days later to determine wound breaking strength and reparative collagen deposition. Expression of VEGF and nitric oxide was studied in wounds. Results Splenic lymphocyte proliferative activity was significantly decreased by sirolimus (p < 0.05). Sirolimus levels in wound fluid were found to be approximately two- to fivefold higher than blood levels (p < 0.01). Sirolimus (2.0 and 5.0 mg kg⁻¹ day⁻¹) reduced wound breaking strength (p < 0.01) and wound collagen deposition (p < 0.05). This was paralleled by decreased expression of VEGF and nitric oxide in wounds. Conclusion Experimentally, our data show that sirolimus impairs wound healing, and this is reflected by diminished expression of VEGF and nitric oxide in the wound. Best abstracts — Surgical Forum 2007.