Three-dimensional imaging of palatal muscles in the human embryo and fetus: Development of levator veli palatini and clinical importance of the lesser palatine nerve.

BACKGROUND: After palatoplasty, incomplete velopharyngeal closure in speech articulation sometimes persists, despite restoration of deglutition function. The levator veli palatini (LVP) is believed to be significantly involved with velopharyngeal function in articulation; however, the development and innervation of LVP remain obscure. The development of LVP in human embryos and fetuses has not been systematically analyzed using the Carnegie stage (CS) to standardize documentation of development. RESULTS: The anlage of LVP starts to develop at CS 21 beneath the aperture of the auditory tube (AT) to the pharynx. At CS 23, LVP runs along AT over its full length, as evidenced by three-dimensional image reconstruction. In the fetal stage, the lesser palatine nerve (LPN) is in contact with LVP. CONCLUSIONS: The positional relationship between LVP and AT three-dimensionally, suggesting that LVP might be derived from the second branchial arch. Based on histological evidence, we hypothesize that motor components from the facial nerve may run along LPN, believed to be purely sensory. The multiple innervation of LVP by LPN and pharyngeal plexus may explain the postpalatoplasty discrepancy between the partial impairment in articulation vs. the functional restoration of deglutition. That is, the contribution of LPN is greater in articulation than in deglutition.

Preparation of Partial-Thickness Burn Wounds in Rodents Using a New Experimental Burning Device.

OBJECTIVE: The manual application of hot water or hot metal to an animal's skin surface is often used to prepare burn wound models. However, manual burn creation is subject to human variability. We developed a new device that can control the temperature, time, and pressure of contact to produce precise and reproducible animal burn wounds and investigated the conditions required to prepare various burn wounds using our new device. METHODS: We prepared burn wounds on F344 rats using 3 contact times 2, 4, and 10 seconds using a stamp heated to 80°C. We observed the wound-healing process macroscopically and histologically and evaluated the burn depth using a laser speckle contrast-imaging device, which evaluated the blood flow of the wound. RESULTS: The changes in the burned area over time, tissue perfusion of the burn wounds, histological evaluation of the burn depth by hematoxylin-eosin and azocarmine and aniline blue staining, and the epithelialization rate (the ratio of the epithelialized area to the wound length) were evaluated on histological sections. Results indicated that the burn wounds prepared with contact times of 2, 4, and 10 seconds corresponded to superficial dermal burns, deep dermal burns, and full-thickness burns, respectively. CONCLUSIONS: We demonstrated that partial- and full-thickness burn wounds can be precisely and reproducibly created with our new automated burning device.

Biocompatibility and efficacy of collagen/gelatin sponge scaffold with sustained release of basic fibroblast growth factor on vocal fold fibroblasts in 3-dimensional culture.

OBJECTIVE: Treatment of vocal fold scarring remains challenging. We have previously reported the therapeutic effects of local injection of basic fibroblast growth factor (bFGF) in animal models and humans. A novel collagen/gelatin sponge (CGS) is capable of sustained release of bFGF, which compensates for its quick absorption in vivo, avoiding multiple injections. This study aimed to evaluate the biocompatibility and efficacy of the CGS in rat vocal fold fibroblasts prior to human trials. METHODS: Fibroblasts extracted from Sprague-Dawley rat vocal folds were seeded onto a CGS and then cultivated with bFGF at concentrations of 0, 10, and 100 ng/mL. Vocal fold fibroblast morphology, adhesion, proliferation, and gene expression were measured under these 3-dimensional conditions. RESULTS: Cells adhered to the CGS from day 1. Although no significant differences in cell morphology were detected, cell proliferation was accelerated by bFGF administration. Expression of endogenous bFGF and hepatocyte growth factor was significantly up-regulated at 10 ng/mL bFGF. The expression of procollagen I and procollagen III was significantly suppressed, whereas HAS-1 and HAS-2 were up-regulated at 10 and 100 ng/mL bFGF. CONCLUSION: The collagen/gelatin sponge is biocompatible with vocal fold fibroblasts and may be useful as a bFGF drug delivery system for the treatment of scarred vocal folds.

Treatment of acute temporomandibular joint dislocation using manipulation technique for disk displacement.

Temporomandibular joint dislocation is not frequently encountered, but it is often difficult to reduce the dislocation with conventional methods described in textbooks. The key points to success of reduction depend on the patient's position, route of approach, and timing of reducing each side. We apply a manipulation technique for disk displacement to the reduction that corresponds to these key points. Using our method, temporomandibular joint dislocation can be easily reduced, without using sedative or analgesics. This method is simple, convenient, and worth trying in place of the conventional method.

Treatment for infection of artificial dura mater using free fascia lata.

Synthetic artificial dura mater materials, such as expanded polytetrafluoroethylene sheets, are widely used in dura mater reconstruction in cases involving brain tumors or trauma surgery. In patients with postoperative infection related to the use of artificial dura mater, surgical debridement of the infected wound and removal of the artificial dura mater materials are necessary to control infection. In cases involving cerebrospinal fluid leakage, dura mater reconstruction must be performed immediately. Many useful techniques for performing dura mater reconstruction to treat postoperative infection have been reported; however, some have drawbacks with respect to the need for microvascular anastomosis or difficulties in obtaining watertight closure. We successfully treated 6 patients with postoperative artificial dura mater infection using free thigh fascia lata. Some surgeons believe that the use of free fascia in infected wounds is dangerous because free fascia is a non-vascularized tissue. However, performing complete debridement and covering such free fascia with well-vascularized tissue allow the fascia to become vascularized and tolerant of infection. Therefore, if the blood flow in the scalp is acceptable after a sufficient debridement, free fascia lata can be used for reconstruction in patients with postoperative infection of artificial dura mater. Furthermore, skull reconstruction can be performed safely and easily with solid-type artificial bone, sometimes combined with tissue expansion, thus resulting in good aesthetic outcomes.

An exploratory clinical study on the safety and efficacy of an autologous fibroblast-seeded artificial skin cultured with animal product-free medium in patients with diabetic foot ulcers.

Cultured dermal substitutes have been used for the treatment of chronic skin ulcers; however, the biological risks of animal-derived materials in the culture process such as foetal bovine serum (FBS) have been reported. In this study, we prepared an autologous fibroblast-seeded artificial dermis (AFD) using animal-product-free medium supplemented with 2% patient autologous serum and without any animal-derived materials such as trypsin in the culturing process. We applied the AFD in five patients with diabetic ulcers and investigated its safety and efficacy. As the primary endpoint, we defined 'wound bed improvement' according to the percentage of granulation area to the whole wound area on day 21, and 60% or higher was regarded as improved. The mean age of the patients was 60·6 years and the mean duration of the ulcer was 22·6 months. In the evaluation of the primary endpoint, the 'wound bed' was improved in all patients [proportion of improvement: 100%, 95% confidence interval (CI): 48% to 100%]. Three patients had complete wound healing within 12 weeks after application and two patients had >80% wound healing at 12 weeks. Side effects were not serious. Our AFD may be a safe and effective treatment of diabetic ulcers.

Treating a collagen scaffold with a low concentration of nicotine promoted angiogenesis and wound healing.

BACKGROUND: Nicotine, one of the major pharmacologically active agents of cigarette smoke, has various effects on cell proliferation, and it has recently been reported to have angiogenic effects. In our previous study, we showed that the topical administration of nicotine at a low concentration accelerated wound healing. This study aimed to evaluate the efficacy of nicotine and synergistic effects of combination treatment with nicotine and basic fibroblast growth factor (bFGF) in a murine excisional wound model treated with artificial dermis. METHODS: Full-thickness defects (8 mm in diameter) were created on the backs of mice, and artificial dermis was sutured to the defects. Phosphate-buffered saline (10 µL), nicotine (10(-3), 10(-4), or 10(-5) M), bFGF (0.5 µg), and both bFGF and 10(-4) M nicotine were topically administered to the artificial dermal tissue for 7 d. The mice were killed on day 14, and the wound area, neoepithelium length, and area of newly formed capillaries in the artificial dermis were evaluated. RESULTS: The wound areas treated with 10(-4) M nicotine, bFGF, or bFGF plus 10(-4) M nicotine were significantly smaller than those in the control group. In these three groups, the neoepithelium in the bFGF plus 10(-4) M nicotine group was significantly longer than that in the other groups. There was no significant difference between the neoepithelium lengths of the control and 10(-5) M nicotine groups. The 10(-3) M nicotine group displayed the least re-epithelization among the groups. CONCLUSIONS: In this study, 10(-4) M nicotine induced angiogenesis in, and accelerated the healing of, wounds treated with artificial dermis. bFGF and nicotine had synergistic effects, and the combined use of nicotine and bFGF is an effective wound healing method.

Autologous skin reconstruction by combining epidermis and acellular dermal matrix tissue derived from the skin of giant congenital melanocytic nevi.

Giant congenital melanocytic nevi (GCMN) are defined as nevi greater than 20 cm in diameter. It is difficult to completely remove GCMN because of the lack of available skin grafts for covering the resultant defects. This study examined whether it is possible to produce reconstructed skin by combining epidermal and acellular dermal matrix (ADM) tissue derived from excised GCMN. GCMN skin samples were obtained with the informed consent of volunteer patients. The abilities of hypertonic saline (1 N NaCl), 0.05% trypsin, 0.1% SDS (sodium dodecyl sulfate), and phosphate buffered saline (PBS) to decellularize GCMN tissue were compared. The specimens were incubated in one of the test solutions at 37 °C for 48 h, before being washed with PBS at 4 °C for 14 days. Residual nuclei, residual DNA, nevus tissue viability, and the structural integrity of the basement membrane and capillaries were evaluated before treatment, and after 48 h' treatment with or without 7 or 14 days' washing. We tried to produce reconstructed skin by combining the resultant ADM with enzymatically separated GCMN epidermal tissue. The histological structure of the reconstructed skin was examined after it had been cultured for 5 days. In the SDS group, most cells had been removed after 48 h, and the DNA content of the ADM was significantly lower than in the other groups. As for viability, no significant difference was detected among the groups. The basement membrane and capillaries remained intact in all groups. After 5 days' culturing, the epidermis had become attached to the ADM in all groups, except the SDS group. SDS displayed a superior decellularization ability compared with the other methods; however, it cannot be used to produce reconstructed skin because of its toxicity. In conclusion, we produced reconstructed skin that was devoid of nevus cells by combining GCMN epidermal tissue with GCMN-derived ADM produced with NaCl or trypsin. This is a promising treatment strategy for giant nevus.

Efficacy of novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor for dermis-like tissue regeneration.

We have developed collagen/gelatin sponges (CGS) with a gelatin concentration of 10 wt% to sustain the release of basic fibroblast growth factor (bFGF). The objective of this study is to elucidate the efficacy of CGS impregnated with different concentrations of bFGF, using mouse skin defects. CGSs impregnated with normal saline solution (NSS) or bFGF solution (1, 7, 14, or 50 µg/cm) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, and total area of newly formed capillaries in CGS were evaluated. The group of CGS with 7-µg/cm bFGF was significantly superior to the NSS group in all evaluated items. CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation 2 or 3 times earlier than existing artificial dermis. The combination of CGS and bFGF could solve the problem of the existing artificial dermis and be very promising for the treatment of skin defects.

Collagen-gelatin scaffold impregnated with bFGF accelerates palatal wound healing of palatal mucosa in dogs.

BACKGROUND: We have developed a collagen-gelatin sponge (CGS) as a scaffold capable of the sustained release of bFGF to improve the healing process of the existing collagen scaffold. The aim of this study was to evaluate the efficacy of CGS impregnated with basic fibroblast growth factor (bFGF) in palatal wound healing in beagles. MATERIALS AND METHODS: Four standardized 6 mm diameter full-thickness wounds were made in the palate of each dog and covered with CGS impregnated with normal saline or bFGF at concentrations of 1 µg/cm2, 7 µg/cm2 and 14 µg/cm2. One and 2 wk after surgery, the wound area, neoepithelium length, thickness, area of regenerated submucosal tissue, and the number and total area of neoformed capillaries were evaluated. RESULTS: Two weeks after implantation, wounds treated with bFGF 7 µg/cm2 and 14 µg/cm2 were completely epithelized, while the length of the neoformed epithelium was significantly longer in the 7 µg/cm2 group. Groups impregnated with bFGF 7 µg/cm2 and 14 µg/cm2 showed promoted regeneration of submucosal tissue 2 wk later. The number and area of neoformed capillaries were significantly higher in the bFGF 7 µg/cm2 group than in other groups. We conclude that palatal wound healing in the bFGF 7 µg/cm2 group was promoted with good neovascularization and showed less contracture than other groups. CONCLUSIONS: Our new collagen-gelatin scaffold, CGS, impregnated with bFGF, could be a promising treatment to accelerate the regeneration of palatal mucosa.

Comparisons of the postoperative rectus abdominis muscle thickness and the biomechanical properties of donor sites among different subtypes of muscle-sparing transverse rectus abdominis myocutaneous flaps (MS0, MS1, MS2, MS3): a rat model.

BACKGROUND: The primary presumed advantages of the deep inferior epigastric perforator flap over the other muscle-sparing (MS) transverse rectus abdominis myocutaneous flaps are the improved functional rectus abdominis muscle outcomes and decreased abdominal contour morbidities. The purpose of this study was to verify this viewpoint objectively and quantitatively using an animal model. METHODS: Six rats were used in the pilot study and 40 rats were equally divided into a control group and 4 experimental groups (MS0, MS1, MS2, and MS3) according to the amount of rectus abdominis muscles harvested. At 3 and 6 weeks, 4 rats in each group were killed, then, the percentage of residual rectus abdominis muscle thickness compared with the control side and the ultimate load at failure of the abdominal wall were recorded and compared. RESULTS: All of the flaps survived completely without complications. Regarding the percentage of residual rectus abdominis muscle thickness compared with the control side, the results suggested MS3 > MS2 ≈ MS1 > MS0; concerning the ultimate load at failure of the abdominal wall, the results showed MS3 ≈ MS2 > MS1 > MS0. (≈: P > 0.05; >: P < 0.05). CONCLUSIONS: According to the findings of this study, we advocate the use of the deep inferior epigastric perforator flap when possible, and the MS2-transverse rectus abdominis myocutaneous flap can serve as a backup when the perforator anatomy does not meet the flap safety criteria for preservation of rectus abdominis muscle function.

Safety of Silk-elastin Sponges in Patients with Chronic Skin Ulcers: A Phase I/II, Single-center, Open-label, Single-arm Clinical Trial.

BACKGROUND: Although traditional wound dressings such as collagen scaffolds promote granulation tissue formation, the efficacy of these dressings in chronic wounds is limited because of high susceptibility to bacterial growth. Biomaterials that can be applied to chronic wounds should have an anti-bacterial function. We previously reported that administering a silk-elastin solution that forms moisturizing hydrogels to wound surfaces of diabetic mice reduced bacterial growth and promoted granulation tissue formation compared with control or carboxymethyl cellulose hydrogels. We hypothesized that silk-elastin promotes wound healing in human chronic wounds by suppressing bacterial growth. METHODS: An open-label, clinical case series was conducted with a prospective, single-arm design at Kyoto University Hospital in Kyoto, Japan. In this study, 6 patients with chronic skin ulcers of any origin (2 < ulcer area (cm2) < 25) on their lower extremities were included; patients with critical ischemia were excluded. Silk-elastin sponges were applied and covered with a polyurethane film without changing the dressing for 14 days. Inflammation triggered treatment discontinuation due to fear of infection. The primary study endpoint was adverse events, including inflammation and infection. RESULTS: Poor hydrogel formation, possibly due to continuous exudation, was observed. No serious adverse events were noted. Two patients discontinued treatment on day 6 and day 7, respectively, due to inflammation, but they were not infected. The other 4 patients completed the 14-day silk-elastin sponge treatment without infection. CONCLUSION: Silk-elastin sponge is safe for chronic skin ulcers, and its ability to promote wound healing should be determined by confirmatory clinical trials.

The surgical strategy of Purpura fulminans triggered by pyothorax associated with lung cancer.

Purpura fulminans is a rare disease that usually causes sepsis and is accompanied by disseminated intravascular coagulation and symmetric gangrene of distal extremities. We had to consider the most appropriate surgery approach. The most important point was attempting to rescue the patient's ability to walk under his own power.

Glucocorticoids suppress fibroblast apoptosis in an in vitro thermal injury model.

The wounds of full- and deep partial-thickness burns result in hypertrophic scars and lead to skin contracture more severely than those of superficial partial-thickness burns. Therefore, preventing burn progression may help improve the aesthetic and functional outcomes after healing. Although a number of studies have focused on elucidating the underlying mechanisms of and preventing burn wound progression, it is still difficult to rescue burned dermis unless early tangential excision is performed. To investigate the underlying mechanisms of and prevent cell death of heat-injured fibroblasts, we developed an in vitro experimental model of heat-injured fibroblasts. We confirmed that heating at 55°C for 30s caused fibroblast necrosis immediately after heating, whereas heating at 46°C for 30s induced apoptosis 24h after heating. We also found that the supplementation of 100ng/ml betamethasone to the culture medium after heating decreased the number of apoptotic cells and increased that of live cells. Our studies suggest that glucocorticoids suppress apoptosis of heat-injured fibroblasts and may be useful for preventing burn wound progression.

The Utility of Silk-elastin Hydrogel as a New Material for Wound Healing.

Cutaneous ulcers are treated with dressing materials and/or ointments to keep the wound in an appropriately moist environment. However, chronic cutaneous ulcers commonly have bacterial colonization that can cause local infection in such an environment. Therefore, the dressing materials and/or ointments should have antibacterial potency to treat chronic ulcers. Acute cutaneous wounds, by contrast, heal rapidly without local infection. The aim of treating acute cutaneous wounds is therefore not only wound closure but also preventing scar contracture after wound healing. However, no dressing materials or ointments available at present are simultaneously effective for preventing infection in chronic ulcers and reducing wound contracture in acute ulcers. Silk-elastin is a recombinant protein polymer with repeating units of silk-like and elastin-like blocks. Silk-elastin solution can self-assemble from a liquid to a hydrogel. We preliminarily reported that silk-elastin hydrogels have the potential to accelerate wound healing in decubitus ulcers of diabetic mice, which are animal models of severe, intractable cutaneous ulcers. In the present study, we examined the effects of silk-elastin hydrogels in chronic and acute ulcer models in comparison with conventional products (carboxymethyl cellulose gel). Silk-elastin hydrogels resulted in significantly higher epithelialization rates than conventional hydrogels in both the chronic and acute ulcer models and significantly larger areas of granulation tissue in acute ulcer models. These results show that silk-elastin hydrogel is a promising material for promoting the healing of cutaneous wounds, including decubitus ulcers, chronic ulcers, and acute ulcers.

The development of a novel wound healing material, silk-elastin sponge.

Silk-elastin is a recombinant protein polymer with repeating units of silk and elastin blocks. This novel wound healing promoting material has the ability to self-assemble from a liquid to a gel. We have already reported that an aqueous solution of silk-elastin has the potential to accelerate wound healing; however, there are several problems in applying silk-elastin in the clinical setting. To solve these problems, we developed a silk-elastin sponge that is easy to use in the clinical setting. In the present study, we examined whether the wound healing effect of the silk-elastin sponge is equal to the aqueous solution of silk-elastin in vivo. The granulation tissue formation promoting effect of the silk-elastin sponge was equal to that of the aqueous solution the silk-elastin, as after application to the wound surface, the sponge was absorbed and dissolved by the exudate. At body temperature the silk-elastin then formed temperature gel. The silk-elastin gel that was obtained contained abundant cytokines from the exudate. We believe that silk-elastin sponge can be applied to various wounds that are difficult to treat with the aqueous solution.

The efficacy of a novel collagen-gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar.

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen-gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α-smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose-dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm2 ; CGS + bFGF group); or a sham-treated group. Vibratory examination revealed that the glottal gap was significantly reduced in the bFGF group and the two implanted groups, whereas the CGS + bFGF group showed higher mucosal wave amplitude. Histological examination revealed significantly restored hyaluronic acid and elastin redistribution in the CGS + bFGF group and reductions in dense collagen deposition. These results provide evidence that CGS and bFGF combination therapy may have therapeutic potential and could be a promising tool for treating vocal fold scar. Copyright © 2015 John Wiley & Sons, Ltd.

The Lesser Palatine Nerve Innervates the Levator Veli Palatini Muscle.

When the lesser palatine nerve (LPN) is supposed to be a branch of the trigeminal nerve and innervate sensation of the soft palate, whether the LPN contains motor fibers is unclear. In this study, we monitored the electromyogram of the levator veli palatini (LVP) muscle on stimulating the LPN during palatoplasty in 3 patients. The electromyogram of the muscles showed the myogenic potential induced by electrostimulation of the LPN. Taken together with the finding from our previous anatomical study that the motor fibers come from the facial nerve, this result supports the double innervation theory of the LVP, which posits that both the pharyngeal plexus and the facial nerve innervate it. Identifying and preserving the LPN during palatoplasty might improve postoperative speech results.

A retrospective analysis on the proper size of tissue expanders to treat scalp lesions.

BACKGROUND: Tissue expanders have become established instruments for scalp reconstruction. However, selection of the size of the expander has not been investigated systematically, and it generally depends on the experience of the surgeon. METHODS: We retrospectively analyzed 21 patients who had undergone treatment for scalp lesions using tissue expanders without any complications and measured 2 variables: the volume of the expanders per area of the excised lesions and the hypothetical stretched functional skin width relative to the width of the excised lesions. We also sought to evaluate the relationship between these 2 variables and the need for revision surgery during the postoperative course. RESULTS: The need for revision surgery was statistically higher in patients with a volume of 7 ml/cm(2) lesion or less and width of functional skin of less than 2.5 cm/cm lesion (P < 0.05). For scar repairs, the required size and volume of the expanders tended to be larger than those required for any other lesions. CONCLUSIONS: Expanders that generate functional skin at least more than 2.5 times the width of the lesion and have a volume more than 7 ml/cm(2) lesion are necessary to cover scalp lesions without complications.

Comparison of ultrasonography-assisted closed reduction with conventional closed reduction for the treatment of acute nasal fractures.

Ultrasonography has often been reported to be a useful tool in cases of nasal fracture, not only for diagnosing such fractures but also for intraoperatively assessing surgical outcomes. In this study, we examined the utility of ultrasonography for intraoperatively assessing the results of surgery for acute nasal fractures. In the conventional group, the outcome of each fracture reduction procedure was intraoperatively confirmed by visual inspection and palpation. In the ultrasound group, intraoperative ultrasonography was used to assess the condition of the fracture before and after closed reduction. The outcomes of the reduction procedures and the reoperation rate were compared between the two groups. According to computed tomography-based evaluations, there were no significant differences in the outcomes of the reduction procedures between the two groups (p > 0.05). As for the reoperation rate, two patients (2.8%) in the conventional group underwent reoperations, but no patient (0%) required reoperations in the ultrasound group. However, the difference in the reoperation rate between the two groups was not significant (p > 0.05). These results indicate that visual inspection and palpation are as reliable as ultrasonography for intraoperatively assessing the outcomes of surgery for acute nasal fractures. Surgeons should not depend on ultrasonography alone, but rather should use it in addition to visual inspection and palpation.