Transmalar Kirshner Wire Fixation Under Ultrasound Scanning is Useful for Unstable Zygomatic Arch Fracture Combined With Zygomatic Body Fracture.

Plate fixation to zygomatic arch fractures carries the risk of facial nerve palsy and scarring of the cheek; however, without plate fixation, bone deviation or displacement may reoccur after surgery. Furthermore, zygomatic arch fractures combined with zygomatic body fractures are more postoperatively unstable than single zygomatic arch fractures. Few reports have focused on this combined fracture type, and no consensus has been reached regarding treatment. Because plate fixation for slight deviation of the zygomatic body has little advantage for stabilization, the authors, usually opt for transmalar pinning alone instead of plate fixation at the hospital. This study is a retrospective case series of 7 patients, among 100 zygomatic fractures excluding isolated zygomatic arch fractures, treated using transmalar pinning under ultrasound scanning. The reduction was performed through the oral and temporal incision as a surgical procedure. Under ultrasound observation, a Kirshner wire was inserted into the zygomatic body from the unaffected side while maintaining the reduced position. The wire was removed at an outpatient visit 2 to 3 months following surgery. In all cases, the zygomatic body was of the laterally rotated type, and postoperative morphologic evaluation showed improvement without postoperative complications. Scores were higher in middle-aged and older than in young people. Correction of zygomatic rotation also scored higher than zygomatic arch morphology. Transmalar Kirshner wire fixation under ultrasound observation is a simple and minimally invasive method for zygomatic arch fractures, which avoids the possible complications related to plate fixation.

Long-term results of orbicularis oris muscle reconstruction in primary cleft lip repair using the "basket-weave method".

The basket-weave method is an orbicularis oris muscle reconstruction method used in primary unilateral cleft lip repair. We compared the long-term results of the basket-weave method with those of a conventional method. For primary unilateral cleft lip repair, we compared the long-term results of 7 cases in which the orbicularis oris muscle was reconstructed by use of the basket-weave method, and of 7 cases in which the reconstruction was performed by use of the conventional method. The average postoperative follow-up period was 12 years and 7 months for the basket-weave method, and 11 years and 9 months for the conventional method. Using photographs of the front and elevation angle views, we evaluated the results as good if the philtrum ridge was formed on the fissure side and was almost symmetrical in height; as fair if the philtrum ridge was lower than the normal side; and as poor if the philtrum ridge had disappeared. For the basket-weave method, the results were good in 6 cases (85.7%), fair in 1 case (14.3%), and poor in 0 cases. For the conventional method, the results were good in 2 cases (28.6%), fair in 4 cases (57.1%), and poor in 1 case (14.3%). A significant difference was found between the 2 groups (Mann-Whitney U test, P = 0.0417). The philtrum ridge shape could be reconstructed by use of the basket-weave method, which gave better results in the long-term than did the conventional method for orbicularis oris muscle reconstruction in primary unilateral cleft lip repair.

Hydrocolloid wound dressing for sealing periwound with poor normal skin: negative pressure wound therapy for deep limb burns with extensive burns.

BACKGROUND: Negative pressure wound therapy (NPWT) is effective for wounds with exposed bones and tendons, but when the wound is accompanied by extensive burns, sealing is difficult. We performed sealing with a hydrocolloid wound dressing on limb burns. CASE REPORT: A 61-year-old woman was burned in a fire at her home. Split-thickness skin grafting was performed 14 and 35 days post injury, but exposure of the right patella and patellar tendon became apparent. The hydrocolloid wound dressing was wrapped around the proximal and distal aspects of a deep wound. The limb was sandwiched from the front and back surfaces and sealed with 2 film dressings, including the hydrocolloid, according to the sandwich method. Using this method, NPWT could be performed without leakage, the exposed tendons and bones were covered with granulation, and skin grafts were performed on day 88 after injury. CONCLUSION: Our method allows NPWT to be easily and effectively performed for deep limb burns with poor normal skin periwound area.

Therapeutic Potential of Adipose-Derived Stem Cell-Conditioned Medium and Extracellular Vesicles in an In Vitro Radiation-Induced Skin Injury Model.

Radiotherapy (RT) is one of three major treatments for malignant tumors, and one of its most common side effects is skin and soft tissue injury. However, the treatment of these remains challenging. Several studies have shown that mesenchymal stem cell (MSC) treatment enhances skin wound healing. In this study, we extracted human dermal fibroblasts (HDFs) and adipose-derived stem cells (ADSCs) from patients and generated an in vitro radiation-induced skin injury model with HDFs to verify the effect of conditioned medium derived from adipose-derived stem cells (ADSC-CM) and extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) on the healing of radiation-induced skin injury. The results showed that collagen synthesis was significantly increased in wounds treated with ADSC-CM or ADSC-EVs compared with the control group, which promoted the expression of collagen-related genes and suppressed the expression of inflammation-related genes. These findings indicated that treatment with ADSC-CM or ADSC-EVs suppressed inflammation and promoted extracellular matrix deposition; treatment with ADSC-EVs also promoted fibroblast proliferation. In conclusion, these results demonstrate the effectiveness of ADSC-CM and ADSC-EVs in the healing of radiation-induced skin injury.

A Novel Model of Perifascial Areolar Tissue Transplant in Rats.

Background Perifascial areolar tissue (PAT) transplant is a method of transplanting loose connective tissue harvested in a sheet form from above the fascia to the wound bed and is effective for wounds with exposed ischemic tissue. However, the engraftment mechanism is unknown, and no animal models of PAT transplant for wound healing exist. Methods In this study, we harvested connective tissue from the backs of Wistar rats in a sheet form to simulate a human PAT transplant. The PAT was affixed to exposed bone of the head. Results In the PAT(+) group, the wound areas gradually decreased due to epithelialization and contraction. The wound area of the PAT(+) group was significantly smaller than that of the PAT(-) group. Conclusions This clinically relevant rat model is useful for elucidating the mechanism of the PAT transplant and establishing a reliable surgical method.

Accessory columellas: A case series on surgical method and short-term postoperative course.

PURPOSE: Accessory columellas are rare congenital anomalies characterized by skin appendage in the columella of the nostril. Case reports are scattered, but there are few descriptions about the clinical features and surgical course. METHOD: In this study, 3 patients with 4 lesions were identified (2013-2020). They were morphologically classified, and the accompanying nose deformity, surgical procedure, and postoperative course were examined. RESULTS: According to the morphologic classification, 1 lesion was of the sessile-lobed type, 2 lesions were of the sessile-nodular type, and 1 lesion was of the pedunculated-ovoid type. In terms of accompanying nose deformities, 1 lesion had a wide nasal columella, and 1 lesion had an enlarged left nostril due to a depression at the base of the lesion. Simple ablations were performed in 2 of the lesions, and plastic procedures were performed in the 2 lesions with an accompanying nose deformity. CONCLUSION: As in our cases, accessory columellas may have a variety of appearances and accompanying deformities. The surgical procedure must be considered according to the case. In addition, any changes due to growth must be observed and taken into consideration when they are reoperated.

Therapeutic Potential of Adipose Stem Cell-Derived Conditioned Medium on Scar Contraction Model.

Scars are composed of stiff collagen fibers, which contract strongly owing to the action of myofibroblasts. To explore the substances that modulate scar contracture, the fibroblast-populated collagen lattice (FPCL) model has been used. However, the molecular signature of the patient-derived FPCL model has not been verified. Here, we examined whether the patient-derived keloid FPCL model reflects scar contraction, analyzing detailed gene expression changes using comprehensive RNA sequencing and histological morphology, and revealed that these models are consistent with the changes during human scar contracture. Moreover, we examined whether conditioned media derived from adipose stem cells (ASC-CM) suppress the scar contracture of the collagen disc. Detailed time-series measurements of changes in disc area showed that the addition of ASC-CM significantly inhibited the shrinkage of collagen discs. In addition, a deep sequencing data analysis revealed that ASC-CM suppressed inflammation-related gene expression in the early phase of contraction; in the later phase, this suppression was gradually replaced by extracellular matrix (ECM)-related gene expression. These lines of data suggested the effectiveness of ASC-CM in suppressing scar contractures. Therefore, the molecular analysis of the ASC-CM actions found in this study will contribute to solving medical problems regarding pathological scarring in wound prognosis.

Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing.

Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (Npas2)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates Npas2 activity. The hit compound (Dwn1) suppressed circadian Npas2 expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgfß1, and α-smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.

Combination of Three Different Negative Pressure Wound Therapy Applications and Free Flap for Open Elbow Joint Injury With Extensive Burns.

Negative pressure wound therapy (NPWT) for treating burns has a variety of therapeutic applications. Here, we present a case of a 53-year-old woman with self-inflicted burn injuries in whom NPWT was applied for three different purposes. The injured sites were the anterior neck, bilateral arms from the wrists upwards to the chest, and back. The left arm was deeply injured, and the elbow joint cavity was opened during treatment. First, NPWT was used for bridge to skin grafting on the entire upper left limb. Second, NPWT was used as a bolster dressing for the autograft after skin grafting was performed on the left arm except the open part of the joint. Third, NPWT over flap was used on the subsequent flap surgical site to address prolonged exudate from the flap margin. The exudate resolved after about a week. Good results were obtained using NPWT during the perioperative period of free flap transplantation for extensive open elbow joint burns. The use of NPWT is an effective option in the treatment of burns.

In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.

Background: The core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring. Materials and methods: To test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 µM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays. Results: Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa. Conclusion: Dwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.

Nasal molding prevents relapse of nasal deformity after primary rhinoplasty in patients with unilateral complete cleft lip: An outcomes-based comparative study of palatal plate alone versus nasoalveolar molding.

OBJECTIVES: In recent years, many studies have reported that the presurgical nasoalveolar molding method improves the nose morphology; however, the reason for its effectiveness after surgery has never been understood. We evaluated the effect of nasoalveolar molding by comparing it with a passive orthopedic method without a nasal stent and focusing on the nostril morphology after primary cheiloplasty using various measurement methods. We then analyzed the essential factors. MATERIALS AND METHODS: The patients involved were 31 infants with unilateral complete cleft lip and palate treated with primary cheiloplasty at the University of Tsukuba Hospital from 2004 to 2011. Of the 31 infants, 16 received nasoalveolar molding treatment and 15 received passive orthopedic treatment as controls. Photographic facial measurements were performed for all patients immediately and 7 months after primary cheiloplasty. The esthetics of the nostrils were assessed according to the left-right nostril symmetry, as measured by the Hausdorff distance, area ratio, perimeter ratio, and aspect a/u (the aspect ratio of the affected side)/(the aspect ratio of the unaffected side) ratio. In addition, the inclination of the nasal ridge was assessed using anthropometric measurements (Grc-Grn∠midline and midline∠columellar axis). RESULTS: The area ratio, perimeter ratio, and Grc-Grn∠midline were significantly greater in the nasoalveolar molding group immediately after surgery (p = 0.00062, 0.016, and 0.048, respectively) than in the control group. However, the Hausdorff distance and aspect a/u ratio were more favorable (p = 0.0018 and 0.0039, respectively) in the nasoalveolar molding group after 7 months. CONCLUSIONS: The results of our study suggested that using nasoalveolar molding as a presurgical orthopedic treatment could improve the shape of the nasal cartilage with surgeon's corrections.

A Chemotactic Functional Scaffold with VEGF-Releasing Peptide Amphiphiles Facilitates Bone Regeneration by BMP-2 in a Large-Scale Rodent Cranial Defect Model.

BACKGROUND: Current common techniques for repairing calvarial defects by autologous bone grafting and alloplastic implants have significant limitations. In this study, the authors investigated a novel alternative approach to bone repair based on peptide amphiphile nanofiber gels that are engineered to control the release of vascular endothelial growth factor (VEGF) to recruit circulating stem cells to a site of bone regeneration and facilitate bone healing by bone morphogenetic protein-2 (BMP-2). METHODS: VEGF release kinetics from peptide amphiphile gels were evaluated. Chemotactic functional scaffolds were fabricated by combining collagen sponges with peptide amphiphile gels containing VEGF. The in vitro and in vivo chemotactic activities of the scaffolds were evaluated by measuring mesenchymal stem cell migration, and angiogenic capability of the scaffolds was also evaluated. Large-scale rodent cranial bone defects were created to evaluate bone regeneration after implanting the scaffolds and other control materials. RESULTS: VEGF was released from peptide amphiphile in a controlled-release manner. In vitro migration of mesenchymal stem cells was significantly greater when exposed to chemotactic functional scaffolds compared to control scaffolds. In vivo chemotaxis was evidenced by migration of tracer-labeled mesenchymal stem cells to the chemotactic functional scaffolds. Chemotactic functional scaffolds showed significantly increased angiogenesis in vivo. Successful bone regeneration was noted in the defects treated with chemotactic functional scaffolds and BMP-2. CONCLUSIONS: The authors' observations suggest that this bioengineered construct successfully acts as a chemoattractant for circulating mesenchymal stem cells because of controlled release of VEGF from the peptide amphiphile gels. The chemotactic functional scaffolds may play a role in the future design of clinically relevant bone graft substitutes for large-scale bone defects.

Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations.

BACKGROUND: Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. METHODS: We obtained levator veli palatini muscle fragments (3-5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. RESULTS: Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. CONCLUSION: Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Exposure to small molecule cocktails allows induction of neural crest lineage cells from human adipose-derived mesenchymal stem cells.

Neural crest cells (NCCs) are a promising source for cell therapy and regenerative medicine owing to their multipotency, self-renewability, and capability to secrete various trophic factors. However, isolating NCCs from adult organs is challenging, because NCCs are broadly distributed throughout the body. Hence, we attempted to directly induce NCCs from human adipose-derived mesenchymal stem cells (ADSCs), which can be isolated easily, using small molecule cocktails. We established a controlled induction protocol with two-step application of small molecule cocktails for 6 days. The induction efficiency was evaluated based on mRNA and protein expression of neural crest markers, such as nerve growth factor receptor (NGFR) and sex-determining region Y-box 10 (SOX10). We also found that various trophic factors were significantly upregulated following treatment with the small molecule cocktails. Therefore, we performed global profiling of cell surface makers and identified distinctly upregulated markers, including the neural crest-specific cell surface markers CD271 and CD57. These results indicate that our chemical treatment can direct human ADSCs to developing into the neural crest lineage. This offers a promising experimental platform to study human NCCs for applications in cell therapy and regenerative medicine.

A small number of residual teeth after the mandibular resection of oral cancer is associated with titanium reconstruction plate exposure.

Objective: Reconstruction plates are used to treat patients with a segmental mandibular defect after oral cancer surgery. Reconstruction plate failure analysis has rarely focused on occlusion, which conducts a mechanical force to the mandible and the plate. To determine the prognostic factors, we retrospectively evaluated patients who underwent reconstruction of a mandibular segmental defect with a reconstruction plate and assessed the number of residual paired teeth. Material and Methods: From among 390 patients with oral cancer who visited University of Tsukuba Hospital (Tsukuba, Japan) between 2007 and 2017, we selected and analyzed the data of 37 patients who underwent segmental resection of the mandible and reconstruction with reconstruction plates. Prognostic factors evaluated were patient age, sex, TNM classification, plate manufacturer, treatment with radiotherapy or chemotherapy, whether the patient had diabetes or smoked, and whether the patient had a small number of residual paired teeth, plate length, and use of a fibular-free flap. Among these 37 patients, eight reconstruction plates had intraoral or extraoral exposure and were removed in 5 years. Results: Kaplan-Meier and log-rank analyses revealed that the prognosis for the 5-year plate exposure-free rate was significantly poorer for patients with a small number of residual teeth than for patients with no teeth or those with a large number of residual teeth (.01). Univariate Cox regression analysis revealed that a small number of residual teeth was a significant prognostic factor in the loss of a reconstruction plate (hazard ratio: 5.63; 95% confidence interval [1.10, 25.85]; .04). Conclusions: A small number of residual teeth after the segmental resection of oral cancer is significantly involved in reconstruction plate survival and may be important in predicting reconstruction plate prognosis.

End-to-Side Venous Anastomosis to a Ligated Vein Stump for Free Flap Transfer in Head and Neck Reconstruction.

INTRODUCTION: In head and neck reconstruction, use of a free flap paired with end-to-side anastomosis to a preserved vein is generally performed. However, it is frequently difficult to select the recipient vein after a neck dissection in which there is only a ligated internal jugular vein/external jugular vein (IJV/EJV). Here, a new anastomosis technique using a ligated IJV/EJV stump is described. PATIENTS AND METHODS: End-to-side anastomoses to ligated vein stump surgeries for free flap transfer in head and neck reconstruction were performed at the Department of Plastic and Reconstructive Surgery, University of Tsukuba, from 2009 to 2016. RESULTS: The subject pool comprised 6 patients. All patients received a free flap transfer after head and neck tumor excision. The free flaps used were 1 free radial forearm flap, 1 free tensor fascia lata muscle perforator flap, and 4 free rectus abdominis musculocutaneous flaps. The cervical vessels used were 3 IJVs and 3 EJVs. All veins of the free flaps could be anastomosed end-to-side to ligated vein stumps without vein grafting. All flaps survived completely without complications. CONCLUSIONS: The end-to-side venous anastomosis to a ligated vein stump procedures were easy to perform and not dependent on the vessel diameters of the free flaps. No complications were observed in any patient owing to differences in vessel diameter, ease of anastomosis, and safety. Results suggest that this new technique is a simple and very useful option in head and neck reconstruction where the IJV/EJV cannot be preserved.

A simple, novel technique for fixing Penrose drains in minor surgeries, with advantages for remote outpatient clinics: a retrospective comparison with conventional drain fixation in North Ibaraki, Japan.

INTRODUCTON: In rural areas with few doctors, Penrose drains in minor surgeries for soft tissue trauma or small subcutaneous tumors are sometimes avoided, even though the drain would prevent hematoma, because of the limited availability of professional postsurgical care. The authors developed a simple fixation method for Penrose drains that can be used even in remote areas where a doctor is not present to remove the drain. A retrospective study was conducted to compare this new method of fixing Penrose drains with instances in which the Penrose drain was fixed to skin by conventional suturing. METHODS: The medical records of patients who underwent minor surgeries using Penrose drains were reviewed. The surgeries were performed from April 2012 to March 2015 in remote outpatient clinics in Ibaraki Prefecture, Japan. The cases were divided into two groups: those using the new method, in which the Penrose drains were sewn onto the wound dressings and could be automatically removed while changing the dressing, and those in which the Penrose drains were conventionally fixed to the skin and removed one or several days after surgery by another doctor at the outpatient clinic. The rates of drain-related complications and of automatic drain removal (ie removal without a doctor's assistance) between the two groups were compared. RESULTS: A total of 54 Penrose drains used for 48 lesions in 44 patients (25 men, 19 women) in the new-method group, and 36 Penrose drains for 25 lesions in 21 patients (12 men, 9 women) in the conventional-method (control) group were analyzed. All 54 Penrose drains in the new-method group were removed automatically, while none of the 36 drains in the control group were removed automatically. There were no drain-related complications, such as massive hematoma, retrograde infection, seroma, or drain breakage or straying, in any of the new-method or control cases. CONCLUSIONS: This new Penrose-drain fixation method is safe and is particularly suitable for minor surgeries in rural areas where there are no resident doctors. The wide use of this method for appropriate minor surgeries in doctorless rural areas has the potential to reduce surgical complications and the time burden for both patients and surgeons.

Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest-like precursors.

In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small-molecule cocktails in a step-by-step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time-lapse imaging of neural precursor-specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest-like precursor stage. Consistent with these results, the neural crest-like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.

A novel postoperative immobilization model for murine Achilles tendon sutures.

The body's motion and function are all in part effected by a vital tissue, the tendon. Tendon injury often results in limited functioning after postoperative procedures and even for a long time after rehabilitation. Although numerous studies have reported surgical procedures using animal models which have contributed to both basic and clinical research, modeling of tendon sutures or postoperative immobilizations has not been performed on small experimental animals, such as mice. In this study we have developed an easy Achilles tendon suture and postoperative ankle fixation model in a mouse. Right Achilles tendons were incised and 10-0 nylons were passed through the proximal and distal ends using a modified Kessler method. Subsequently, the right ankle was immobilized in a plantarflexed position with novel splints, which were made from readily available extension tubes. Restriction of the tendon using handmade splints reduced swelling, as opposed to fixating with the usual plaster of Paris. Using this method, the usage of the right Achilles tendons began on postoperative days 13.5 ± 4.6, which indicated healing within two weeks. Therefore our simple short-term murine Achilles tendon suture procedure is useful for studying immediate tendon repair mechanisms in various models, including genetically-modified mice.

Microsatellite instability in esophageal squamous cell carcinoma is not associated with hMLH1 promoter hypermethylation.

To test whether a subset of esophageal squamous cell carcinomas (SCC) develop through a deficiency in DNA mismatch repair, we examined microsatellite instability (MSI) using 11 microsatellite markers including BAT-26, hMLH1 protein expression by immunohistochemistry, and methylation status of the hMLH1 promoter by methylation-specific polymerase chain reaction (MSP). p53 mutations were also investigated. Microsatellite instability at one or more loci was observed in 40% (12/30) of esophageal SCC tumor samples, although only one of these tumors was categorized as high-frequency MSI (MSI-H) and none showed BAT-26 instability. While immunohistochemistry revealed decreased hMLH1 protein expression in 27% (8/30) of the tumors, hMLH1 promoter hypermethylation was not observed. Absence of hMLH1 protein expression was relatively common in well-differentiated (keratinizing-type) esophageal SCC, but was not associated with hMLH1 promoter hypermethylation. p53 mutation was detected in 37% (11/30) and loss of heterozygosity (LOH) in 90% (27/30) of esophageal SCC samples. Our results suggested that most esophageal SCC develop through defects in tumor suppressor genes (i.e. the suppressor pathway), and that MSI in esophageal SCC probably represent random replication errors rather than being associated with DNA mismatch repair deficiency.