Unilateral Palatal Hypoplasia: A Case Report With a Long-Term Follow-Up.

Isolated unilateral palatal hypoplasia is an extremely rare congenital disorder that usually causes velopharyngeal incompetence. We herein report a case with isolated unilateral palatal hypoplasia that was treated and followed up over nine years. No hypernasality or articulation errors were observed through the postoperative period. Here the intraoperative and postoperative findings are described.

Protection of rat artery grafts from tissue damage by voltage-applied supercooling.

Supercooling preservation below 0 °C allows the storage of the transplantable sources in an unfrozen state. This can improve the safety and efficacy of storage by improving the inhibition of metabolism and organ preservation in comparison with conventional preservation at 4 °C. We have developed a supercooling technique using a voltage-applied apparatus without perfusion. We examined the preservation effects of our supercooling preservation technique in a rat model of artery transplantation. Our technique produces a supercooled state at - 2 °C with application of 1000 V. The viability of tissue cells from rat arteries was found to be higher with storage using the proposed method than that under ordinary conditions. Damage to the vascular endothelium of the femoral artery preserved by voltage-applied supercooling at - 2 °C was reduced compared to storage under ordinary conditions. Artery graft revival was successfully achieved with graft patency after supercooling preservation, and 1 week outcomes for post-transplanted grafts, including thrombosis, were better with supercooling preservation than with conventional 4 °C preservation. Supercooling artery preservation at - 2 °C with 1000 V promises to greatly prolong preservation time and improve post-transplant outcomes.

GALA-Modified Lipid Nanoparticles for the Targeted Delivery of Plasmid DNA to the Lungs.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.

Simple and efficient method for consecutive inactivation-cryopreservation of porcine skin grafts.

We previously reported that inactivation treatment by high hydrostatic pressurization (HHP) has potential utility as a novel skin regeneration therapy for various skin tumors. In this study, we evaluated whether glycerol-cryopreservation could be applied in order to preserve inactivated skin by HHP using a porcine model. Twenty full-thickness skin grafts (1.5 × 1.5 cm) were prepared from a minipig. The skin samples were inactivated by the HHP in normal saline or glycerol/fructose solution, followed by cryopreservation for 5 weeks at - 80 °C in each same solution. Another 10 grafts immediately after inactivation were prepared as non-cryopreserved controls. Nine grafts in each group were randomly implanted on the fascia of a host pig and removed at 1, 4 and 11 weeks after grafting. All grafts showed engraftment macroscopically. Hematoxylin eosin staining showed the cellular components in all areas of the dermis at 4 and 11 weeks after grafting, and immunohistochemical staining for CD31 showed the presence of capillaries in the grafts in all groups. The surface and cross-sectional areas of grafts in the normal saline solution cryopreserved group decreased between 1 and 11 weeks, whereas these areas in the glycerol cryopreserved group did not decrease significantly. Glycerol cryopreservation may therefore be a simple and efficient method for preserving porcine skin inactivated by HHP.

Improved viability of murine skin flaps using a gelatin hydrogel sheet impregnated with bFGF.

Basic fibroblast growth factor (bFGF) promotes epithelial cell proliferation and angiogenesis but its clinical applications are limited by its short half-life and low retention. Recently developed gelatin hydrogel sheets able to release physiologically active substances in a controlled manner have the potential to overcome these issues. In this study, the effects of gelatin hydrogel sheets impregnated with bFGF on flap survival and angiogenesis were examined in a murine skin flap model. A flap of 1 × 3 cm was generated on the backs of 60 C57BL/6 mice. The mice were divided into five groups (n = 12/group): Group I, untreated; Group II, treated with a gelatin hydrogel sheet impregnated with saline; Group III, treated with bFGF (50 µg) without sheets; Groups IV and V, treated with gelatin hydrogel sheets impregnated with 50 and 100 µg of bFGF, respectively. On the seventh day after surgery, the flap survival area and vascular network were examined and hematoxylin and eosin and von Willebrand factor staining were used for histological examinations. The flap survival areas were significantly larger in Groups IV and V than in other groups. The area of new vessels was significantly larger in Group IV than in the other groups. In the murine skin flap model, gelatin hydrogel sheets impregnated with bFGF promoted angiogenesis and improved flap survival. These findings support the use of bFGF-impregnated gelatin hydrogel sheets for improving ischemic flap survival in clinical settings.

Effects of Fibroblast Growth Factor-2 Combined With a Collagen/Gelatin Sponge for Adipogenesis in the Mouse Subcutis.

INTRODUCTION: A collagen/gelatin sponge (CGS) is a new scaffold that promotes wound healing by slowly releasing fibroblast growth factor (FGF)-2. FGF-2 induces mitogenesis, angiogenesis, and adipogenesis. In this study, the adipogenesis-inducing effects of CGS combined with FGF-2 in the subcutis of mice were evaluated. METHODS: Collagens/gelatin sponges (10 × 5 mm) were impregnated with 50 µL of FGF-2 solution (10 or 100 µg/mL). A CGS (Gunze Corp, Osaka, Japan) combined with FGF-2 was implanted subcutaneously into the thoracic region of mice. At 1, 2, 3, and 4 weeks, samples were collected for hematoxylin and eosin staining, von Willebrand factor immunostaining, and perilipin immunostaining to examine adipose tissue localization and angiogenesis. A CGS with only saline solution was prepared as a control. RESULTS: Adipocytes in the collagen fibers appeared at 3 weeks, and a zonal fat layer was noted under the panniculus carnosus at 4 weeks in the FGF-2-combined CGS groups. The fat layer was significantly thicker in the FGF-2 (100 µg/mL) group than in the FGF-2 (10 µg/mL) group. In the control group, no fat pad was newly formed. The number of newly formed vessels in the FGF (10 µg/mL) and (100 µg/mL) groups was significantly greater in the FGF-2 group than in the control group. CONCLUSION: This study presents a promising method to enhance adipogenic effects in the murine subcutis using CGS combined with FGF-2, representing a potential technique for soft tissue reconstruction.

Efficacy of Gelatin Hydrogel Impregnated With Concentrated Platelet Lysate in Murine Wound Healing.

BACKGROUND: The efficacy of a gelatin hydrogel (GH) sheet impregnated with platelet-rich plasma in full-thickness wound healing has been reported. Human platelet lysate is another potential natural product for use in wound healing. The present study examined the effects of a GH sheet impregnated with concentrated freeze-dried platelet lysate on wound healing after storage for 9 mo. MATERIALS AND METHODS: Platelet concentrates were subjected to three freeze-thaw cycles and freeze-dried then preserved at 4°C. Reconstitution with saline was then performed to produce 1-fold (hPL1), 2-fold (hPL2), and 3-fold (hPL3) concentrations of preserved platelet lysate. Full-thickness wounds were made on the back of male C57Bl6J/Jcl mice. Wounds were treated with saline, hPL1, or a GH sheet impregnated with saline, hPL1, hPL2, or hPL3. Histologic examinations using hematoxylin-eosin, Azan, and anti-CD31 staining were performed on days 4, 7, and 14 to assess neoepithelialization and granulation tissue and capillary formation. RESULTS: This study showed that the GH sheet itself or the simple administration of hPL1 did not accelerate the healing process. However, the GH sheet impregnated with hPL1 accelerated the granulation tissue formation to some extent, and the GH sheet impregnated with hPL2 or hPL3 clearly accelerated the capillary formation and the granulation tissue formation. In addition, the GH sheet impregnated with hPL3 had the longest epithelium formation. CONCLUSIONS: A GH sheet impregnated with long-term preserved 2-fold or 3-fold concentrated platelet lysate enhances the wound healing process.

FGF-2 combined with bilayer artificial dermis composed of collagen matrix prompts generation of fat pad in subcutis of mice.

Fibroblast growth factor (FGF)-2 induces mitogenesis, angiogenesis and adipogenesis. In this study, the adipogenesis-inducing effects of FGF-2 combined with bilayer artificial dermis in mice were evaluated. FGF-2-impregnated bilayer artificial dermis composed of collagen matrix, PELNAC (Gunze Corp., Osaka, Japan) was implanted subcutaneously into the thoracic region of mice. At 1, 2, 3, and 4 weeks, samples were collected for H&E staining, von Willebrand factor immunostaining, and perilipin immunostaining to examine adipose tissue localization and angiogenesis. The collagen matrix-implanted group without the addition of FGF-2 was prepared as a control. At 2 weeks after the implantation of FGF-2 combined with dermal substitutes, adipocytes appeared in the collagen fibers. At 3-4 weeks, a fat pad was generated with neovascularization. The thickness of the fat pad had significantly increased at 2, 3, and 4 weeks. The remaining collagen was decreased by absorption over time. In the control group, no fat pad was newly formed. This study has identified a promising method to enhance adipogenic effects in the murine subcutis, representing a potential technique for soft tissue reconstruction.

A case report of the first application of culture epithelial autograft (JACE®) for giant congenital melanocytic nevus after its approval in Japan.

In Japan, the JACE® cultured epidermal autograft (CEA) was approved and covered by public healthcare insurance for use in the treatment of giant congenital melanocytic nevus (GCMN) in 2016. We herein report the results of the application of JACE® after curettage and Q-switched ruby laser therapy. The current patient was the first patient with GCMN to be treated with JACE® since its approval. A 3-month-old girl had a hairy GCMN of 9.5 cm in diameter from her cheek to her temple on the left side of her face. We first performed curettage of the nevus on the temple and applied irradiation using a Q-switched ruby laser; however, erosion relapsed at 2 months after first surgery. After preparing JACE®, we performed curettage a second time at 7 months with irradiation of a Q-switched ruby laser and the application of the CEA. The CEA took successfully and the wound was completely epithelized at 1 week after grafting. Re-pigmentation is an important issue that remains to be solved; however, overcoming this would allow for a deeper abrasion or more intense laser irradiation to be performed in cases in which CEA will be subsequently applied.

Adipose-derived stem cell (ASC)-enriched fat grafting: experiments using White rabbits and an automated cell processing apparatus.

The grafting of fat mixed with adipose-derived stem cells (ASCs) is being increasingly applied to compensate for the disadvantages of previous fat grafting methods. Devices that automatically isolate fat stem cells also have recently been developed. ASCs were isolated from the inguinal region of White rabbits using Icellator®, and the number of cells and their viability were measured. The cell count per fat graft (mL) was adjusted to the following concentrations and subcutaneously transplanted into the back: Control group, Fat + PBS; Fat + ASCs (×0.5) group, 1.6 × 105 cells/mL; and Fat + ASCs (×1) group, 3.2 × 105 cells/mL. Grafted fat weight was measured after 8 weeks, and histological, immunohistological, and specifically stained sections were prepared. Fat absorption was reduced in Fat + ASCs (×0.5) and Fat + ASCs (×1) groups. The number of blood vessels was higher in Fat + ASCs (×1) than in the control group, and blood vessel areas were higher in Fat + ASCs (×0.5) and Fat + ASCs (×1) groups than in the control group. The usefulness of the automated cell processing apparatus, Icellator®, was confirmed, and the results obtained suggest that grafted ASCs promote the vascularization and engraftment of fat grafts.

Latent TGF-ß binding protein-2 is essential for the development of ciliary zonule microfibrils.

Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Verification of the Inactivation of Melanocytic Nevus in vitro Using a Newly Developed Portable High Hydrostatic Pressure Device.

High hydrostatic pressure (HHP) technology is a physical method for inactivating tissue. We reported that nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could be used as autologous dermis for covering skin defects. In this study, we verified the inactivation of nevus specimens using a newly developed portable HHP device which will be used in a clinical trial. Nevus tissue specimens were obtained from 5 patients (mean age 7.2 years, range 1-19). We cultured fibroblasts and nevus cells from the tissue specimens and then evaluated their inactivation after HHP at 200 MPa by confirming the attachment of the suspensions and by the live/dead staining of the suspensions, through the dissociation of the cells on chamber slides and by the live/dead staining of the remaining cells. The cells were also quantitatively evaluated by WST-8 assay. We then confirmed the inactivation of the nevus specimens after HHP using explant culture. Our results indicated that fibroblasts and nevus cells were inactivated after HHP at 200 MPa, with the exception of a small percentage of green-colored cells, which reflected the remaining activity of the cellular esterases after HHP. No cells migrated from the nevus specimens after HHP at 200 MPa. We verified the inactivation of fibroblasts and nevus cells cultured from nevus specimens, and in the nevus samples themselves after pressurization at 200 MPa using this device. This device could be used in clinical trials for giant congenital melanocytic nevi and may thus become useful in various medical fields.

Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

Latent TGF-ß binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5.

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-ß binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

Elastofibroma dorsi: a case report with an immunohistochemical and ultrastructural studies.

Elastofibroma is a rare, benign, fibrous tumor formed by the proliferation of characteristic elastic fibers that commonly occurs between the lower margin of the scapula and the ribcage. We undertook a histochemical, immunohistochemical, and ultrastructural study of an elastofibroma dorsi beneath the right scapula of a 77-year-old woman. Tumor cells comprised collagen fiber bundles, numerous elastic fibers, and spindle cells resembling fibroblasts. The elastic and collagen fibers in the tumor were stained positively with Elastic van Gieson and Masson trichrome staining, respectively. Immunostaining showed that the fibroblasts were strongly positive for CD34, positive for vimentin, and weakly positive for α-smooth muscle actin. Ultrastructural observations revealed elastin and microfibrils between numerous irregularly arranged collagen fiber bundles. Signs suggestive of elastin deposition were also evident in the tangled collagen fibers themselves. The fibroblasts contained a large amount of rough endoplasmic reticulum and were surrounded on the outside of cells by microfibrils and collagen fibers. Although fibroblasts may produce large quantities of elastin, microfibrils, and collagen, our findings suggested that the deposition of elastin on collagen fibers may be involved in the formation of abnormal elastic fibers.

Combined use of fenestrated-type artificial dermis and topical negative pressure wound therapy for the venous leg ulcer of a rheumatoid arthritis patient.

We report a case of circumferential venous leg ulcer in a rheumatoid arthritis patient. Mesh skin grafting was performed in another hospital, but the graft failed and the patient was referred to our hospital. This ulcer was treated by the combination therapy of a fenestrated-type artificial dermis with negative pressure wound therapy (NPWT) and secondary mesh grafting using our 'grip tape technique'. NPWT was started at -100 mmHg and continued until the formation of dermis-like tissue. A section stained using haematoxylin and eosin and an anti-αSMA (α smooth muscle actin) immunohistological section of the biopsy from dermis-like tissue showed an abundant infiltration of fibroblasts and capillary formation beneath the fenestration of the silicone sheet. Threefold mesh skin grafting was subsequently performed and it was taken up completely. The fenestrated-type artificial dermis in combination with NPWT produced good results without infection in the treatment of complex wounds. In addition, our 'grip tape technique' was useful to apply polyurethane foam to the entire surface of the lower leg.

Effect of aging on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in rats.

BACKGROUND: Bone morphogenetic proteins may hold broad potential for use in the reconstruction of bone defects resulting from tumor resection or trauma and in assisting bone healing thanks to methods enabling the synthesis of recombinant human bone morphogenetic protein-2 (rhBMP-2). METHODS: rhBMP-2 was implanted with atelopeptide type I collagen as a carrier into the calf muscles of 3-, 8-, and 48-wk-old Wistar/ST male rats. After 21 d, the formation of ectopic neoplastic bone was examined in soft x-ray imaging, and the bone mineral content, bone area, and bone mineral density (BMD) were measured by dual-energy x-ray absorptiometry. In addition, hematoxylin-eosin and von Kossa staining and proliferation cell nuclear antigen immunostaining were performed. RESULTS: BMD values determined by dual-energy x-ray absorptiometry were 29.40 (standard deviation ±5.47), 24.15 (±2.33), and 19.01 (±2.02) mg/cm(2) in the 3-, 8-, and 48-wk-old rats, respectively, demonstrating that BMD significantly decreased with aging (P < 0.05). The von Kossa stain-positive area decreased significantly with aging (P < 0.01). The number of proliferation cell nuclear antigen-positive cells also decreased significantly with aging (P < 0.01). CONCLUSIONS: The capacity of rhBMP-2 to induce ectopic bone formation decreases with aging. These findings will be of considerable benefit in the development of clinical treatments for the regeneration of cranio-maxillofacial bone in elderly patients.

Sex differences in neurophysiological responses are modulated by attentional aspects of impulse control.

The amplitudes of the N2 and P3 components of event-related potentials (ERPs) may be influenced by personality traits such as impulsivity, and male/female differences may also have an effect. However, few studies have assessed the interaction between personality traits and the sex of the subject in these components. Therefore, in this study we evaluated sex differences in the amplitudes of the N2 and P3 ERP components during a continuous performance task, and their relation to impulse control. Twenty-seven healthy participants were asked to perform an AX-type continuous performance task, also known as a Go/Nogo task, during electroencephalographic recording. Participants then completed the Barratt impulsiveness scale (version 11; BIS-11), and the effortful control (EC) scale to self-report personality measures related to impulse control. We found that in the Nogo condition, males showed significantly larger N2 amplitudes than females in the frontal area. Interestingly, Nogo-N2 amplitudes were positively correlated with BIS-attentional subscale scores, but were negatively correlated with EC-attentional subscale scores, and both correlations were observed only in males. These results suggest that attentional aspects of impulse control modulate Nogo-N2 amplitude only in males. This modulatory effect may be related to a sex-specific inhibitory control mechanism acting during early stimulus evaluation.

Gelatin hydrogel impregnated with platelet-rich plasma releasate promotes angiogenesis and wound healing in murine model.

Platelet-rich plasma (PRP) contains numerous growth factors to promote wound healing and angiogenesis. The objective of this study was to explore the efficacy of biodegradable gelatin hydrogel impregnated with PRP releasate (PRPr) in the wound healing process compared with the single application of PRPr prepared from mouse PRP centrifuged by a double-spin method. Gelatin hydrogel disks with an isoelectric point of 5.0 were used in this study. A total of 180 mice (n = 45/group) were randomly assigned to the following 4 experimental groups: control group, biodegradable gelatin hydrogel group, PRPr group and gelatin hydrogel impregnated with PRPr (PRPrG) group. Wound area and epithelialization were compared on days 1, 5, 7, 14 and 21 post-wounding. After complete epithelialization, wound contraction was also evaluated. Neovascularization using immunohistochemical staining of von Willebrand factor was analyzed on day 14. The wound area of PRPrG on days 5, 7 and 14 was smaller than that in the other groups (p < 0.01). The epithelialization lengths of PRPrG on days 7 and 14 were significantly longer than the others (p < 0.01). The capillary formation of PRPrG was also superior to those in all other groups on day 14. On day 21, all wounds were completely epithelialized and PRPrG prevented wound contraction the most. It is concluded that the sustained-release system of gelatin impregnated with PRPr can stimulate angiogenesis and accelerate wound healing compared with the single application of PRP.

Comparison of neovascularization in dermal substitutes seeded with autologous fibroblasts or impregnated with bFGF applied to diabetic foot ulcers using laser Doppler imaging.

A bilayered artificial dermis (AD) composed of an upper silicone sheet and a lower collagen sponge has been widely applied for skin defects. After application, fibroblasts and capillaries infiltrate the AD and the collagen sponge is replaced by host dermal tissue within a few weeks. However, this delay and the high incidence of infection are concerns regarding the use of AD in the treatment of chronic ulcers. In this study, we compared the neovascularization of conventional AD seeded with autologous fibroblasts (cultured dermis: CD) and collagen/gelatin sponge (CGS), which is a novel artificial dermis capable of sustained release of basic fibroblast growth factor (bFGF) after application using laser Doppler imaging (LDI). CD (n = 5) and CGS impregnated with bFGF (n = 6) were applied to diabetic foot ulcers after debridement. Perfusion units (PUs) were measured just after, and 1, 2 and 3 weeks after application, and complete healing rates within 16 weeks were compared. No significant differences in PUs were seen 1, 2 and 3 weeks after application and in healing rates within 16 weeks between the two groups. This study suggested that CD and CGS treatments were effective, but there were no significant differences between them in the treatment of diabetic ulcers .