Reparative Collagen Deposition Research Articles

CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice.

Macrophages are essential for wound repair after myocardial infarction (MI). CD226, a member of immunoglobulin superfamily, is expressed on inflammatory monocytes, however, the role of CD226 in infarct healing and the effect of CD226 on macrophage remain unknown.Methods: Wild type and CD226 knockout (CD226 KO) mice were subjected to permanent coronary ligation. CD226 expression, cardiac function and ventricular remodeling were evaluated. Profile of macrophages, myofibroblasts, angiogenesis and monocytes mobilization were determined.Results: CD226 expression increased in the infarcted heart, with a peak on day 7 after MI. CD226 KO attenuated infarct expansion and improved infarct healing after MI. CD226 deletion resulted in increased F4/80+ CD206+ M2 macrophages and diminished Mac-3+ iNOS+ M1 macrophages accumulation in the infarcted heart, as well as enrichment of α-smooth muscle actin positive myofibroblasts and Ki67+ CD31+ endothelial cells, leading to increased reparative collagen deposition and angiogenesis. Furthermore, CD226 deletion restrained inflammatory monocytes mobilization, as revealed by enhanced retention of Ly6Chi monocytes in the spleen associated with a decrease of Ly6Chi monocytes in the peripheral blood, whereas local proliferation of macrophage in the ischemic heart was not affected by CD226 deficiency. In vitro studies using bone marrow-derived macrophages showed that CD226 deletion potentiated M2 polarization and suppressed M1 polarization.Conclusion: CD226 expression is dramatically increased in the infarcted heart, and CD226 deletion improves post-infarction healing and cardiac function by favoring macrophage polarization towards reparative phenotype. Thus, inhibition of CD226 may represent a novel therapeutic approach to improve wound healing and cardiac function after MI.

Nitric oxide restores impaired healing in normoglycaemic diabetic rats

Hyperglycaemia impairs wound healing. However, little is known about the underlying cellular mechanisms that lead to diminished wound repair in insulin-controlled and non-insulin-controlled diabetes. This study investigated the role of endogenous and exogenous nitric oxide on incisional wound healing in diabetic rats. Groups of 10 wild-typeWistar control rats - 10 genetically diabetic BioBreeding rats and 10 genetically diabetic BioBreeding rats treated with subcutaneous insulin implants to render them normoglycaemic - underwent dorsal skin incision followed by subcutaneous insertion of polyvinyl alcohol sponges. The rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitric oxide, an important mediator in diabetic wound healing and collagen synthesis, was measured in wound fluid. Wound-derived fibroblasts were tested for ex vivo synthesis of nitric oxide and collagen. Exogenous nitric oxide was used for the therapeutic interventions. Wound-breaking strength and wound collagen deposition were significantly impaired in the hyperglycaemic diabetic animals (p<0.01). Wound nitric-oxide synthesis and ex vivo wound fibroblast nitric-oxide production were reduced in the hyperglycaemic rats (p<0.01). Insulin treatment partially reversed some of the effects of hyperglycaemia on wound repair (p<0.05). Exogenous nitric oxide further restored wound mechanical strength, collagen deposition and fibroblast collagen synthesis (p<0.01) in insulin-treated (normoglycaemic) diabetic animals. Wound healing is impaired in hyperglycaemic and normoglycaemic diabetic rats. This is reflected in impaired wound fibroblast nitric-oxide synthesis. Used in combination with insulin, exogenous nitric oxide further improves healing outcomes, making it a potential target for therapeutic intervention in insulin-treated normoglycaemic diabetes.

Sirolimus impairs wound healing

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. 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. 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. Experimentally, our data show that sirolimus impairs wound healing, and this is reflected by diminished expression of VEGF and nitric oxide in the wound.

MHC-Class-II-Deficiency Impairs Wound Healing

MHC-class-II-deficient mice lack T helper cell dependent immune reactions. T cell related immune functions are critical for normal wound healing. We hypothesized that MHC-II-deficiency compromises wound repair by affecting the normal wound immune response. Groups of 10 male MHC-class II-knockout mice and wild-type controls underwent dorsal skin incision. Polyvinyl alcohol sponges were then inserted subcutaneously. The mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Activity of T cells and macrophages isolated from the spleens and from the healing wounds was investigated. Fibroblasts derived from the wounds were tested ex vivo for proliferative activity and collagen synthesis. Wound collagen deposition and wound breaking strength were impaired in MHC-class-II-knockout mice (P < 0.05). Impaired healing was reflected in diminished mitogen-reactivity of splenic T-cells (P < 0.01), and decreased CD4 expression in wounds. In addition, basal and LPS + IFN-gamma-induced synthesis of TNF-alpha and nitric oxide by wound-derived macrophages was impaired. Exvivo, fibroblast proliferation and fibroblast collagen production from MHC-II-deficient mice was decreased. MHC-II-deficiency compromises wound healing. This may be a reflection of impaired wound immune cell function and decreased activity of wound fibroblasts.

Regulation of Nitric Oxide Synthesis in Wounds by IFN-γ Depends on TNF-α

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-γ plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-γ -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IFN-γ was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-γ, TNF-α, and anti-TNF-α antibody. In a separate experiment, IFN-γ -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-γ-knockout mice (p <. 05). Impaired healing was reflected in diminished synthesis of TNF-α and NO in wounds (p <. 05). In vivo treatment with molsidomine reversed impaired healing in IFN-γ-deficient mice. Ex vivo, addition of IFN-γ stimulated the synthesis of TNF-α and NO in wound-derived macrophages. IFN-γ -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-α-antibody-treatment, which could be fully reversed by exogenous TNF-α. Thus we conclude that IFN-γ-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-γ on macrophage NO production depends on endogenous TNF-α synthesis.

Erythropoietin Stimulates Wound Healing and Angiogenesis in Mice

Erythropoietin exerts hematopoietic effects by stimulating proliferation of early erythroid precursors. Nonhematopoietic effects of erythropoietin have also been shown. It may act as a new angiogenic factor in wound healing. This study aimed to investigate the effect of systemic administration of recombinant human erythropoietin on wound healing in mice. Dorsal incisional wounds were performed in mice, which were then divided into two groups; a group treated for 7 days with recombinant human erythropoietin, and a control group. Sacrificing animals on day 7, the wound tissues were collected for analysis of wound breaking strength, malondialdehyde, a marker of lipid peroxidation, hydroxyproline, an index of reparative collagen deposition, reduced glutathione levels, and for histological evaluation. The immunohistochemical determination of vascular endothelial growth factor (VEGF) which is believed to be the most prevalent angiogenic factor throughout the skin repair process, was also studied. The treatment significantly increased wound breaking strength by decreasing malondialdehyde and increasing hydroxyproline levels on day 7 after wounding. No statistically meaningful change was observed in reduced glutathione content. VEGF was immunostained significantly more on wound tissue of treated animals compared to the control group. Recombinant human erythropoietin treatment may be effective in wound healing due to inhibition of lipid peroxidation, deposition of collagen, and VEGF expression in wound area.

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)-β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ 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-β, TNF-α, IFN-γ, and CD4 and CD8 in the anastomosis was investigated. Tacrolimus impaired dermal healing (p <. 05). This was paralleled by decreased expression of TGF-β (stimulates healing) and increased expression of IFN-γ and TNF-α (both inhibit healing) (p <. 05). In contrast, tacrolimus did not inhibit healing of colonic anastomoses. No effect was seen on the expression of TGF-β, TNF-α, IFN-γ, 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.

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.

Effect of a selective aldosterone receptor antagonist in myocardial infarction.

Myocardial infarction (MI) initiates adaptive tissue remodeling, which is essential for heart function (such as infarct healing) but is also important for maladaptive remodeling (for example, reactive fibrosis and left ventricular dilation). The effect of aldosterone receptor antagonism on these processes was evaluated in Sprague-Dawley rats using eplerenone, a selective aldosterone receptor antagonist. Infarct healing and left ventricular remodeling were evaluated at 3, 7, and 28 days after MI by determination of the diastolic pressure-volume relationship of the left ventricle, the infarct-thinning ratio, and the collagen-volume fraction. Eplerenone did not affect reparative collagen deposition as was evidenced by a similar collagen volume fraction in the infarcted myocardium between eplerenone and vehicle-treated groups at 7 and 28 days post-MI. In addition, the thinning ratio, which is an index of infarct expansion, was comparable between the eplerenone and vehicle-treated animals at 7 and 28 days post-MI. A protective effect of eplerenone was demonstrated at 28 days post-MI, where reactive fibrosis in the viable myocardium was reduced in eplerenone-treated animals compared with vehicle-treated animals. Thus aldosterone receptor antagonism does not retard infarct healing but rather protects against maladaptive responses after MI.

Inhibition of nitric oxide synthesis in wounds: pharmacology and effect on accumulation of collagen in wounds in mice.

To investigate the effect of systemic inhibition of nitric oxide (NO) synthesis in wounds on collagen accumulation. Randomised experimental study. Teaching hospital, USA. 240 Balb/C mice divided into groups of 10 animals each. Polyvinyl alcohol sponges were inserted subcutaneously through a dorsal skin incision. Beginning on the day of wounding, N omega-nitro-L-arginine-methylester (L-NAME), NG-L-monomethyl-arginine (L-NMMA), aminoguanidine hemisulphate (AGU), and S-methyl isothiouronium (MITU) were given orally or intraperitoneally. The mice were killed 10 days later. Nitrite and nitrate concentrations, both stable end products of NO, were measured in wound fluid. Sponge hydroxyproline content was assayed as an index of reparative collagen deposition. NOS inhibitors given orally in the drinking water or by daily intraperitoneal injection had no effect on wound nitrite/nitrate concentrations or deposition of collagen in wounds. When given continuously through intraperitoneally-placed osmotic pumps, AGU (500 mg/kg/day) (p < 0.001) and MITU (p < 0.01) significantly reduced wound fluid nitrite/nitrate concentrations in a dose dependent manner. Inhibition of wound nitric oxide synthase by 500 mg AGU/kg/day and 100 mg MITU/kg/day was paralleled by lowered accumulation of collagen in wounds (p < 0.01). NO is beneficial in wound healing.

The effect of tacrolimus on colon anastomotic healing

We have shown that the immunosuppressant tacrolimus (TA), used in solid organ transplantation, decreases wound mechanical strength and collagen deposition in dermal wound healing~ Failure of colonic healing has a major impact on surgical morbidity and mortality. Whether tacrolimus affects the outcome of colonic healing, however, is not known. We, therefore, studied the effect of systemic treatment with tacrolimus on standard parameters of colonic wound healing in rats. Groups of eight Sprague Dawley rats underwent laparotomy and single layer 6-0 prolene inverted left colon anastomosis. Beginning at the day of wounding, rats were treated once daily by s.c. injections with 2.0, or 5.0 mg TA/kg. Rats were sacrificed at postoperative day five for determination of colonic bursting pressure (BP) and hydroxyproline content of anastomoses (OHP), an index of reparative collagen deposition. Splenic lymphocytes were tested for proliferative activity (3H-thymidine uptake ). Tacrolimus levels in blood, obtained by cardiac puncture at the day of sacrific e, were measured by ELISA.

Diabetes-impaired healing and reduced wound nitric oxide synthesis: A possible pathophysiologic correlation

Background. Nitric oxide (NO) is synthesized in wounds, but its role in the healing process is not fully understood. The inhibition of NO production during wound healing is accompanied by decreased wound reparative collagen deposition. To further define the role of NO in reparative collagen accumulation, we studied its production during diabetes-induced wound healing impairment. Methods. Male Sprague-Dawley rats (290 to 310 gm) were rendered diabetic by intraperitoneal streptozotocin administration. Seven days after induction of diabetes (blood glucose greater than 300 mg/dl), the rats underwent dorsal skin incision and subcutaneous implantation of polyvinyl alcohol sponges. Beginning on the day of wounding, 21 diabetic animals were treated with 3 units/day insulin via intraperitoneally implanted miniosmotic pumps. Ten days after injury, wound breaking strength was determined, and wound collagen accumulation and types I and III collagen gene expression were measured in subcutaneously implanted polyvinyl alcohol sponges. NO synthesis, as measured by nitrite/nitrate accumulation, was determined in wound fluid and in supernatants of wound cell cultures. Results. Streptozotocin-induced diabetes markedly impaired wound breaking strength and collagen deposition. A parallel decrease occurred in wound NO synthesis as reflected by decreased nitrite/nitrate concentration in wound fluid and in diminished ex vivo NO production by wound cells. Insulin treatment partially but significantly improved wound mechanical strength (p < 0.01) and collagen accumulation (p < 0.001). Decreased wound NO accumulation and ex vivo NO production by wound cells were also partially restored by insulin treatment. Conclusions. Impaired diabetic wound healing is paralleled by decreased wound NO synthesis, supporting the hypothesis that NO plays a significant role in wound reparative collagen accumulation.

Effect of growth hormone replacement on wound healing in healthy older men

The secretion of growth hormone, an important anabolic agent, declines with aging. We hypothesize that growth hormone levels (measured as insulin-like growth factor-1) correlate with postoperative tissue repair in otherwise healthy, elderly persons. The goal was to determine whether growth hormone supplementation can improve wound healing in this circumstance. We conducted a randomized controlled double-blind trial of 6 months of growth hormone replacement or placebo in 28 healthy older men (>69 years of age) with low baseline plasma insulin-like growth factor-1. Growth hormone doses were adjusted to elevate insulin-like growth factor-1 to levels expected in younger adults. Wound healing was tested by implanting 10 cm expanded polytetrafluoroethylene porous tubes for 10 days, then measuring the content of collagen (as hydroxyproline), DNA, and total protein. Hydroxyproline content was 15% greater in the wounds of the growth hormone group (n = 13) compared with the placebo group (n = 15), (4.52 +/- 0.94 versus 3.92 +/- 0.78 microg/cm; p = 0.04). Therefore, healthy older men who took growth hormone had enhanced reparative collagen deposition during the wound healing process. This action may be clinically useful after selected surgery or trauma in the elderly.

Nitric Oxide Regulates Wound Healing

Nitric oxide (NO) synthesis occurs during wound healing, but its role has not been defined. To study the effect of NO on wound repair,S-methyl isothiouronium (MITU, a competitive inhibitor of NO synthase) was administered at a dose of 10, 50, and 100 mg/kg body weight/day, using intraperitoneally implanted miniosmotic pumps. Groups of 10 male Balb/C mice underwent a dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. The animals were sacrificed 10 days postwounding and wound breaking strength and hydroxyproline content of sponges, an index of reparative collagen deposition, were determined. Some sponges were used to harvest wound fluid and infiltrating cells, which were then incubated overnight with or without 1 mMMITU. Nitrite and nitrate, stable end products of NO, were measured in wound fluid and in wound cell culture supernatants. Continuous intraperitoneal infusion of MITU significantly decreased wound fluid nitrite/nitrate concentrations in a dose dependent manner (P< 0.01). Inhibition of wound NO synthesis by 100 mg MITU/kg/day was paralleled by lowered wound collagen accumulation (P< 0.01) and wound breaking strength (P< 0.01).In vitroNO synthesis by wound cells obtained from animals treated with 100 mg MITU/kg/day was not significantly different from controls (12.6 ± 1.2 vs 10.7 ± 0.6 nmole NO2+ NO3/μg DNA), reflecting the reversible inhibition of NO synthase by MITU. However, NO production was equally inhibited in wound infiltrating cells by thein vitroaddition of MITU (83% vs 85%, respectively). These data suggest that nitric oxide synthesis is critical to wound collagen accumulation and acquisition of mechanical strength.

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.

Cyclosporine a impairs wound healing in rats

Cellular immune responses may play an important role in the early inflammatory and cellular phases of wound healing. Cyclosporine A (CSA), a new immunosuppressive agent, impairs cellular immunity and T-cell-dependent humoral immunity. Therefore, the effect of CSA-induced immunosuppression in a rat wound-healing model was studied. Sprague-Dawley rats underwent a standardized skin incision and subcutaneous implantation of sterile polyvinyl alcohol sponges. CSA was dissolved in olive oil and given by gavage to one group of animals at a total dose of 125 mg/kg/10 days. The control group received an equivalent volume of olive oil. Ten-day-old wounds were weaker in CSA-treated animals, both in the fresh state (282 ± 19 g vs 380 ± 27 g, P < 0.01), and after formalin fixation (1111 ± 74 g vs 1419 ± 57 g, P < 0.01). In addition, CSA-treated rats accumulated significantly less hydroxyproline in the wound sponge granuloma, an index of reparative collagen deposition. The impairment in wound healing occurred without differences in body weight gain or organ weights. There was a profound immunosuppression in the animals receiving CSA as determined by thymic lymphocyte blastogenesis in response to Con A and PHA. These findings suggest that immunosuppression in otherwise healthy animals impairs wound healing.

Pathogenesis of the collagen defect in experimental scurvy.

In the quarter century since Wolbach's studies 1 on the disturbance in collagen formation in experimental scorbutus, extensive research has been performed in this field. Much of it was stimulated by the hope that manipulation of the collagen defect might provide a lead to better understanding of collagen metabolism, wound healing, and the collagen diseases. In 1956, Dunphy and his associates 2 reported on a preliminary trial of polyvinyl sponge implants into the abdominal wall of experimental animals in this field. Others 3,4 had used this material previously but not in experimental scurvy. The technique has certain advantages over experimental linear or excised wounds in studies of reparative collagen deposition. For example, with multiple implants in the same animal one sponge can be submitted for chemical analysis and the other for pathologic evaluation. Our experience with the simultaneous implantation of polyvinyl sponges into the abdominal wall and anterior chamber of