Improvement of the maxillary bone growth suppression in the cleft palate operation with cultured dermal substitute: animal experiment and patient reports in preliminary clinical application.

Cleft palate patients often show impaired maxillary bone growth after cleft-palate-correction surgery. We attempted to investigate and elucidate the effects of using allogeneic, cultured dermal substitute (CDS) to cover an exposed, palatal bone surface in animal experiments. Fibroblasts from the abdominal skin of Wistar rats were cultured. Subsequently, the fibroblasts were seeded onto a matrix that composed of hyaluronic acid and atelo-collagen. Forty Wistar rats (3-week-old males) were assigned to one of four groups: control, open-treatment, matrix and CDS groups. The control group (n=5) received no surgical operations. In the open-treatment group (n=11), the mucosa and periosteum of the left-half of the palate were removed surgically and the bone was exposed. In the matrix group (n=11), the area of exposed bone was covered with only the matrix, excluding any cells. In the CDS group (n=10), the area of exposed bone was covered with CDS. At 9 weeks postoperatively, biopsies of the wounds were obtained. Skull preparations were made and the palatal widths were determined. The palatal widths in the CDS group were significantly wider compared to the matrix and open-treatment groups (P<0.05). However, there were no significant differences when the CDS group was compared to the control group. Haematoxylin, eosin and CD31 immunostaining confirmed a larger number of capillaries in the CDS group. This animal experiment suggested that this procedure might provide an optimum wound-healing condition, thus, reducing the maxillary bone-growth suppression. Therefore, a preliminary clinical application in three patients was performed using the autologous CDS after the pushback method.

The lateral wedged insole with subtalar strapping significantly reduces dynamic knee load in the medial compartment gait analysis on patients with medial knee osteoarthritis.

OBJECTIVE: Two lateral wedged insoles were compared: one with, and the other without, subtalar strapping. METHODS: Twenty-one patients (age 58-83, mean 72) with medial knee osteoarthritis (OA) were enrolled. Thirty-seven knees in the patients were divided into three groups based on the Kellgren and Lawrence OA grading system; grades 2 (cases=20), 3 (cases=11), and 4 (cases=6). The subjects were tested during walking barefoot and during walking with a silicon rubber lateral wedged insole with elevation of 10 mm attached to a barefoot. Gait analysis was performed on a 10 m walkway for each subject under three different walking conditions; barefoot, wearing a conventional insole, and a subtalar strapping insole. Peak knee varus moment during gait was measured under each condition, and compared between the three conditions and between the OA grades. RESULTS: On the whole (cases=37), the peak varus moment was significantly reduced by wearing either of the insoles, compared to walking barefoot. The reduction was more obvious with the strapping insole (-13%, P<0.01), compared with the conventional insole (-8%, P<0.05). In moderate OA patients (grades 2 and 3), the moments were significantly lower with the strapping insole, compared with the conventional insole (P=0.0048 and 0.005, respectively). However, no significant difference was detected in severe OA patients (grade 4) between the two types of insoles (P=0.4). CONCLUSIONS: Both lateral wedged insoles significantly reduced the peak medial compartment load during gait. The subtalar strapping insole had a greater effect than the conventional insole, particularly in patients with moderate medial knee OA.

Up-regulation of osteopontin, chemokines, adhesion molecule, and heat shock proteins in 1-hour biopsy from cardiac death donor kidneys.

AIMS: Since April 1979, 471 kidneys were retrieved from donors after cardiac death (DCD) using an in situ regional cooling technique, with excellent renal function and good long-term graft survival. However, the precise cascade of events following transplantation of DCD kidneys and the influence of ischemia-reperfusion injury remain unclear. In this study, we performed gene expression profiling using 1-hour biopsy samples from DCD kidneys versus those from living sources. METHODS: All kidney grafts were procured at our center using an in situ regional cooling technique from DCD. Living donor kidneys (LD) were harvested by open nephrectomy. All graft biopsies were performed 1 hour after reperfusion (DCD n = 8, LD n = 9). We analyzed the expression profile of 20,173 genes. RESULTS: One hundred seventy eight genes were up-regulated (>2-fold difference and DCD/LD > 1.5) and 120 down-regulated (<1/2-fold and LD/DCD > 1.5) in DCD kidneys. Expression of osteopontin (22.5 +/- 2.6-fold DCD vs 7.7 +/- 1.7 LD; P < .001), chemokines (CCL4 4.4 +/- 0.7 vs 2.5 +/- 0.3; P < .01), (CCL2 6.0 +/- 1.3 vs 2.8 +/- 0.5), CXCL1 (9.5 +/- 0.4 vs 2.0 +/- 0.2), and CXCL2 (16.7 +/- 5.3 vs 4.8 +/- 1.3; P < .05), adhesion molecule (ICAM-1 4.7 +/- 0.7 vs 2.5 +/- 0.4; P < .05), and heat shock proteins (HSPA1L 6.7 +/- 0.7 vs 1.6 +/- 0.3, HSPA1A 17.7 +/- 2.6 vs 2.4 +/- 0.5, HSPA1B 13.3 +/- 0.2 vs 3.0 +/- 0.7, HSPA5 6.7 +/- 0.8 vs 3.2 +/- 0.3, HSPB1 2.9 +/- 0.2 vs 1.0 +/- 0.1, and HSPH1 19.4 +/- 3.0 vs 5.9 +/- 1.1; P < .001) were up-regulated in the kidneys from DCD. CONCLUSION: This report analyzed global gene expression using 1-hour biopsy samples from DCD kidneys. These results may provide new insight into the identification of novel target genes for the development of therapeutic approaches and for determining graft viability of kidneys from DCD.

Gene expression profile in rat renal isografts from brain dead donors.

BACKGROUND: Brain death (BD) and the subsequent ischemia/reperfusion (I/R) injury have cardinal implications for the pathogenesis of kidney transplantation (Tx). However, the precise mechanistic pathway of BD and the subsequent I/R injury are unknown. In this study, we performed genome-wide analysis for differential gene expression in kidney isografts from BD donors. Their gene expressions were compared with those from living sources. METHODS: Kidneys from BD rats were engrafted and their gene expressions were compared with those from living controls. Donors were intubated, and mechanically ventilated for 6 hours. Grafts were harvested 6 hours after BD, and 1 hour after engraftment. The expression profile of approximately 20,500 genes was analyzed. RESULTS: Gene expression of chemokines (Scya2 and Gro1), cytokines (IL-1 and -6) and adhesion molecules (E- and P-selectin and ICAM-1) were upregulated in the BD kidneys and 1 hour after engraftment. An antiapoptotic gene (Birc2), IkappaB-zeta, and protective gene (HO-1) were also upregulated. Other upregulated genes included oncogenes (lipocalin2, Bcl3, and CCAAT/enhancer binding protein delta), Calgranulin B, DEXRAS1, insulin-like growth factor binding protein-1, inhibin beta-B-subunit gene, IgG Fc receptor, and FK 506 binding protein 5. We also observed downregulation of the genes Amphiphsin, Jagged 1, Pace 4, Slc15a2, Kcnn2, and gap junction membrane channel protein alpha5 only in kidneys from BD donors. CONCLUSIONS: This is the first demonstration of global gene expression analysis using the rat brain-death isograft model. These results provide new insights for the detection of novel target genes for treatment and prognosis of grafts from brain-dead and extended marginal donors.

Hyaluronic acid and silver sulfadiazine-impregnated polyurethane foams for wound dressing application.

Five different kinds of PU foam wound dressings were prepared to investigate their wound healing capability. They include (i) PU+silver sulfadiazine (AgSD), (ii) PU+alginate (Al), (iii) PU+Al+AgSD, (i.v.) PU+hyaluronic acid (HA), and (v) PU+HA+AgSD. Physical properties and in vitro behaviors of AgSD release and fibroblast adhesion on those dressings were evaluated. From the drug release and fibroblast adhesion studies, it was observed that PU foam impregnated with both HA and AgSD shows good drug release behavior and low adhesion of the cells. Furthermore, the HA and AgSD-containing PU foam showed excellent wound healing effect without any inflammation or yellow cluster. The wound size decreased around 77% after 1 week application of that foam dressing onto a rat skin defect.

Tissue-engineered product: allogeneic cultured dermal substitute composed of spongy collagen with fibroblasts.

Recently, various types of allogeneic skin substitutes including cultured epidermal substitute (CES), cultured dermal substitute (CDS), and cultured skin substitute (CSS), which are composed of keratinocytes and/or fibroblasts as the cellular component(s), have been used as biological wound dressings. In our study, the allogeneic CDS was prepared by plating fibroblasts on a spongy collagen. The clinical evaluation was conducted using fresh or cryopreserved allogeneic CDS. In 145 of our clinical cases, 95% (138/145) of various wounds were evaluated as achieving good or excellent results, including 96% (22/23) of deep dermal burns (DDB) and dermal burns (DB), 100% (53/53) of partial-thickness donor wounds, 91% (21/23) of traumatic skin defects, 100% (5/5) of pressure ulcers, 82% (9/11) of chronic skin ulcers, 100% (6/6) of coverage for debrided DB, and 92% (22/24) of coverage for autologous meshed graft. The results obtained in our study suggest that the allogeneic CDS is able to provide an effective therapy for patients with partial and/or full-thickness skin defects.

Clinical evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix.

We have developed an allogeneic cultured dermal substitute (CDS) that was prepared by plating fibroblasts on to a spongy collagen matrix and culturing them for 7 to 10 days. The matrix was freeze-dried from a 1% aqueous solution of bovine-hide atelocollagen. This study was designed to evaluate the efficacy of promoting epithelialisation clinically on 26 donor-site wounds for split-thickness skin grafts. One half of a wound was covered with an allogeneic CDS and the other half side was covered with a commercially-available freeze-dried porcine dermis (FPD). Both macroscopically and histologically the epithelialisation on the area of the donor site that was covered with allogeneic CDS was more rapid than that covered with FPD. In a representative donor-site wound covered with allogeneic CDS, there was a stratified structure of epithelial cells on the underlying connective tissue on day 5, and the epithelium had matured by day 12. When covered with FPD a stratified structure of epithelial cells was noted on day 8, and the epithelium had matured by day 15. We conclude that allogeneic CDS provides a good environment for epithelialisation.

Allogeneic cultured dermal substitute composed of spongy collagen containing fibroblasts: evaluation in animal test.

The authors developed a cultured dermal substitute (CDS) composed of a spongy collagen containing cultured fibroblasts. The cultured fibroblasts derived from Sprague Dawley rat skin were seeded on a spongy collagen at a density of 5 x 10(5) cells cm(-2) and cultured for 7 days. This CDS was applied to the debrided wound of full-thickness burn which was inflicted experimentally on the dorsum of Wister rat, and then the wound conditions were observed over a period of 2 weeks. The comparative study was conducted using an acellular spongy collagen as well as a commercially available temporary wound dressing, Biobrane, since a different type of cultured dermal substitute, Dermagraft-TC, is composed of Biobrane, whose inner site is combined with cultured fibroblasts. Each covering material was applied on the debrided wound area and exchanged by new one 1 week later. When the debrided wound was covered with Biobrane, a small portion of necrotic tissue was observed 1 week after application, and the granulation tissue formation was greatly delayed. This wound area showed a poor granulation tissue even 2 weeks later. On the contrary, when covered with an acellular spongy collagen, no necrotic tissue was observed. This wound area showed a more or less irregular granulation tissue at 1 week and then a healthy granulation tissue 2 weeks later. This preliminary comparative study suggests that an acellular spongy collagen is able to function as a more suitable matrix for CDS, compared with Biobrane. The wound area covered with a CDS assumed a moist, shiny, and hyperaemic appearance 1 week after application showing a healthy granulation tissue. The macroscopic evaluations indicate that the CDS is able to prepare a healthy granulation tissue at an earlier stage, compared with the acellular spongy collagen. In addition, the histologic views demonstrate that the CDS is able to prepare a thicker and denser granulation tissue, compared with the acellular spongy collagen. Although the fate of cultured fibroblasts in the CDS on the wound surface within 1 week is not clear, these findings suggest that fibroblasts in CDS are able to provide excellent conditions for wound healing.

Effect of a collagen matrix containing epidermal growth factor on wound contraction.

Excessive wound contraction is known to lead to pathological wound contracture. Using a rabbit model, we applied a bovine type I collagen matrix sponge as a dermal substitute and human epidermal growth factor to full-thickness excisional wounds. Wound contraction was assessed 14 and 28 days after wounding. It was found that both collagen matrix and epidermal growth factor significantly inhibited wound contraction (p < 0.001) in all wounds treated with collagen matrix alone or treated with 0.1 and 1 microg of epidermal growth factor 28 days after wounding. Interestingly, the combination of collagen matrix with epidermal growth factor strongly inhibited wound contraction over matrix alone (p < 0.01 on day 28). Histological analyses showed a regular horizontal arrangement of collagen fibers in the dermis under wounds treated with these substances but not under untreated wounds. Furthermore, using a fibroblast-populated collagen gel, the direct inhibitory effect of epidermal growth factor on gel contraction by fibroblasts was also observed. Collagen gels without stimulation contracted to 29.5 +/- 0. 6% of their original size, as determined 6 days after culturing. At 3 days or more, epidermal growth factor inhibited collagen gel contraction by fibroblasts (after 6 days: 34.2 +/- 1.8%, p > 0.05; 36.5 +/- 2.8%, p < 0.05; and 39.8 +/- 2.1%, p < 0.001 at 1, 10, and 100 ng/ml of epidermal growth factor, respectively). In conclusion, collagen matrix and epidermal growth factor, particularly in combination, may be useful in the prevention of wound contracture.

Development of new wound dressing composed of spongy collagen sheet containing dibutyryl cyclic AMP.

Although cyclic AMP has been considered to regulate cell proliferation, the mechanism of this function is largely unknown. Recent studies suggest that cyclic AMP promotes the proliferation of skin cells in a dose-dependent manner. An ointment containing dibutyryl cyclic AMP has been used in the treatment of skin ulcers and found to be effective in promoting tissue repair. To search more efficacious wound management, the authors developed a new wound dressing composed of a spongy atelo-collagen sheet containing dibutyryl cyclic AMP. This wound dressing was evaluated in two types of animal tests. One is the application of the wound dressing to a full-thickness skin defect in order to evaluate the granulation tissue formation and the wound size reduction. The wound dressing was found to promote the granulation tissue formation and naturally reduce the wound size. The other test was the application of the wound dressing to the full-thickness skin defect, leaving behind a skin island in a central portion, in order to evaluate the epithelialization. This skin island left in a full-thickness skin defect was extremely enlarged. The enlargement of the skin island seems to be related to the epithelialization from the margin of the skin island as well as by the expansion of a skin island induced by contraction of the developed granulation tissue in the surrounding wound area. These results suggest that an atelo-collagen spongy sheet containing dibutyryl cyclic AMP is effective in promoting the granulation tissue formation and epithelialization.

Cytotoxicity tests for antimicrobial agents using cultured skin substitutes fixed at interface of air and culture medium.

The present study is focused on a new cytotoxicity test using cultured dermal and epidermal sheets, which are fixed at the air and medium interface as a wound surface model. The cultured dermal sheet is composed of human fibroblasts and a collagen matrix, and the cultured epidermal sheet is composed of human keratinocytes and a collagen matrix. Each cultured sheet was fixed at the air and medium interface, over which a piece of test specimen was placed. The in vitro system created, provides a mimetic wound surface since during wound repair, fibroblasts are embedded in an extracellular matrix, while keratinocytes migrate and proliferate on provisional granulation tissue. The results thus obtained in this cytotoxicity test are useful for determining the efficacious amount of antimicrobial agent used in clinical cases.

Evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix as a wound dressing.

The purpose of this study was to examine whether epithelialisation is promoted when an allogeneic cultured dermal substitute is used as a biological wound dressing. The dermal substitute was prepared by plating fibroblasts on to a spongy collagen matrix, and then culturing them for 7 to 10 days. A new animal wound model was designed to measure re-epithelialisation quantitatively. A full thickness skin defect was made on the dorsum of each of 33 rats; the skin was excised, leaving a layer of pannicular carnosus with an island of intact skin in the central portion of the skin defect. In the first group of rats (n = 13), a piece of cultured dermal substitute was applied to the wound, and a medicated covering material was placed over it. Re-epithelialisation from the island of intact skin was monitored over a period of 7 days. In the second group of rats (n = 10), the wound was covered with an acellular collagen matrix in conjunction with the medicated covering material, and in the third group of rats (n = 10), the wound was covered with the medicated covering material alone. Both the macroscopic and histological findings indicated that the epithelial migration of the first group of rats was far more rapid than that in the other two groups. It comes to the conclusion that the application of this new fibroblastic cultured dermal substitute provided a good environment for the promotion of wound healing.

Development of a new wound dressing with antimicrobial delivery capability.

A bilaminar wound dressing composed of an outer membrane and an inner three-dimensional matrix of a fabric or a sponge may be considered to constitute an ideal structure that promotes wound healing: the outer membrane prevents body fluid loss, controls water evaporation, and protects the wound surface from bacterial invasion, and the inner matrix encourages adherence by tissue growth into the matrix. Using this concept, we developed a biosynthetic wound dressing with a drug delivery capability. This medicated wound dressing is composed of a spongy sheet of a chitosane derivative and collagen mixture that is laminated to an antimicrobial-impregnated polyurethane membrane. In this study, a gentamycin sulfate-impregnated wound dressing was prepared and evaluated. The antimicrobial efficacy of this wound dressing was examined on an agar plate seeded with Pseudomonas aeruginosa. Also, the cytotoxicity of an antimicrobial released from this wound dressing was examined in an in vitro system with cultured skin substitutes. Both in vitro tests have shown that this wound dressing is capable of suppressing bacterial growth and minimizing cellular damage. In addition, in the treatment of wounds inflicted on rats and rabbits, this wound dressing was shown to be efficacious in covering full-thickness and split-thickness skin defects. Finally, the efficacy of this wound dressing was evaluated in a nonrandomized open-label study of 31 clinical cases. In 31 cases treated with this wound dressing, good or excellent wound healing was achieved.

A cultured skin substitute composed of fibroblasts and keratinocytes with a collagen matrix: preliminary results of clinical trials.

The cultured skin substitute was created through successive cultivation of fibroblasts and keratinocytes that were combined within a collagen matrix. This collagen matrix was composed of a collagen spongy sheet and a collagen gel. The collagen spongy sheet was designed to produce a honeycomb structure having many holes in which all holes through the sheet were filled with collagen gel. This specific structure thereby allows for the nourishment of the cultured keratinocytes on the surface of the matrix when placed on the graft bed. In this study, autologous cultured skin substitute was applied to a 51-year-old man who had sustained a burn injury. Three sheets of the cultured skin substitute (6 x 9.5 cm) were grafted onto the full-thickness excised wound in the right anterior chest wall. One week after grafting most of the matrix disappeared and stratified keratinocytes were seen to have firmly attached to the underlying tissue. Five weeks after grafting a cornified epidermal layer was seen. Ten months after grafting a mature epidermis and a well-differentiated papillary and reticular dermis replacement were observed. The physical properties and color of this grafted area resemble those of normal skin. In the second test case, autologous cultured skin substitute was applied to a 30-year-old man with a scar remaining after tattoo removal. Eight sheets of the cultured skin substitute (10 x 18 cm) were applied on an excised wound (thickness, 0.02-0.025 in.) of both the fore- and upper arms. The histological appearance of a biopsied skin specimen from the grafted area at 3 months after grafting showed a mature epidermis and a well-differentiated reticular dermis replacement. The regenerated skin at 14 months after grafting showed an excellent result.

[Experimental reconstruction of the urinary bladder using atelocollagen sponge].

An experimental reconstruction of the urinary bladder using specially designed bovine cross linked atelocollagen sponge was performed after partial cystectomy in 12 male rabbits. Bladder reconstruction with hybrid type biomaterials using both atelocollage sponge and cultured autologous cells were performed in 5 rabbits (the 1st group). The autologous cells were collected from the mucosa and muscular layer of the urinary bladder and were seeded on the atelocollagen sponge before reconstruction. Reconstruction using atelocollagen sponge without autologous cells were performed in the other 7 rabbits (the 2nd group). In 9 of these 12 rabbits, atelocollagen sponge was successfully implanted in the native urinary bladder. Histopathological findings revealed that seeded autologous cells and growth of surrounding host cells could be seen similarly in the atelocollagen sponge but these could not be differentiated by these could not be differentiated by routine histopathological techniques. In conclusion, these results showed a possibility of construction of artificial urinary tract using both atelocollagen sponge and autologous cells.

A silver-sulfadiazine-impregnated synthetic wound dressing composed of poly-L-leucine spongy matrix: an evaluation of clinical cases.

The management of severe burns requires the suppression of bacterial growth, particularly when eschar and damaged tissue are present. For such cases, silver sulfadiazine (AgSD) cream has been traditionally applied. This antibacterial cream, however, cannot be used in conjunction with a temporary wound dressing that is needed to promote healing. The authors developed a synthetic wound dressing with drug delivery capability for clinical use by impregnating a poly-L-leucine spongy matrix with AgSD, which is released in a controlled, sustained fashion. In general, the dressing adhered firmly to the wound in the case of superficial second-degree burns, and during the healing process it separated spontaneously from the re-epithelialized surface. In the management of deep second-degree burns where eschar and damaged tissue were present, the dressing had to be changed at intervals of 3 to 5 days until it adhered firmly to the wound. Once the dressing had firmly attached to the wound, it was left in place until it separated spontaneously from the re-epithelialized surface. Dressing changes were fewer than with other treatments and the pain was effectively reduced. Cleansed wounds were effectively protected from bacterial contamination. Of 52 cases treated with this wound dressing, 93% (14/15) of superficial second-degree burns, 75% (3/4) of deep second-degree burns, 85% (6/7) of superficial and deep second-degree burns, and 75% (12/16) of split-thickness skin donor sites were evaluated as achieving good or excellent results.

Evaluation of a synthetic wound dressing capable of releasing silver sulfadiazine.

A silver sulfadiazine-impregnated poly-L-leucine wound dressing, AgSD-medicated wound dressing, was evaluated for antibacterial capacity against Pseudomonas aeruginosa and cytotoxicity to human fibroblasts and human epidermal keratinocytes. This wound dressing contained 0.4 mg AgSD/cm2. Antibacterial capacity was examined on experimentally infected wound surfaces (3.4 x 10(4) P. aeruginosa organisms/gm) on the dorsum of mice. The AgSD-medicated wound dressing showed effective bacterial control. Cytotoxicity was examined on a monolayer of cells formed in culture dishes. Cellular damage was reduced by the controlled release of AgSD from the hydrophobic poly-L-leucine sponge matrix of the AgSD-medicated wound dressing. Cytotoxicity of the AgSD-medicated wound dressing was much lower than that of 1% AgSD cream.

Argon laser-assisted anastomoses in medium-size vessels: one-year follow-up.

Laser-assisted anastomosis of medium-size vessels can be performed with satisfactory short-term patency. This study was undertaken to evaluate patency and structural integrity up to 1 year. An argon laser was used to make bilateral femoral arteriovenous anastomoses in 12 dogs compared to conventional suture method in another 8 dogs. These anastomoses were evaluated for patency and aneurysm formation at 1 hour; 1, 2, 4, and 8 weeks; and 12 months after surgery. All anastomotic sites were patent and without aneurysmal change or luminal narrowing at all harvesting intervals. Histologic examination revealed that within 1 month laser anastomotic sites were almost completely healed and without intimal hyperplasia. In suture anastomoses, foreign-body reaction remained evident up to 1 year. Use of the argon laser for medium size vessel anastomoses resulted in excellent patency without aneurysm formation or intimal hyperplasia even in the long term. These data suggest promising clinical applications.