Altered actin dynamics is possibly implicated in the inhibition of mechanical stimulation-induced dermal fibroblast differentiation into myofibroblasts.

The formation of hypertrophic scars and keloids is strongly associated with mechanical stimulation, and myofibroblasts are known to play a major role in abnormal scar formation. Wounds in patients with neurofibromatosis type 1 (NF1) become inconspicuous and lack the tendency to form abnormal scars. We hypothesized that there would be a unique response to mechanical stimulation and subsequent scar formation in NF1. To test this hypothesis, we investigated the molecular mechanisms of differentiation into myofibroblasts in NF1-derived fibroblasts and neurofibromin-depleted fibroblasts and examined actin dynamics, which is involved in fibroblast differentiation, with a focus on the pathway linking LIMK2/cofilin to actin dynamics. In normal fibroblasts, expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, significantly increased after mechanical stimulation, whereas in NF1-derived and neurofibromin-depleted fibroblasts, α-SMA expression did not change. Phosphorylation of cofilin and subsequent actin polymerization did not increase in NF1-derived and neurofibromin-depleted fibroblasts after mechanical stimulation. Finally, in normal fibroblasts treated with Jasplakinolide, an actin stabilizer, α-SMA expression did not change after mechanical stimulation. Therefore, when neurofibromin was dysfunctional or depleted, subsequent actin polymerization did not occur in response to mechanical stimulation, which may have led to the unchanged expression of α-SMA. We believe this molecular pathway can be a potential therapeutic target for the treatment of abnormal scars.

Surgical Correction of Nipple Hypertrophy after Nipple-sparing Mastectomy with Breast Reconstruction.

Nipple hypertrophy is a relatively common phenomenon, particularly in the Asian patient population. The incidence and prevalence or cause of nipple hypertrophy are not well defined in the literature. As survival rates for breast cancer patients continue to improve, there is an increasing emphasis on enhancing their quality of life. Treatment options, such as lumpectomy and radiation therapy or mastectomy, now prioritize preservation of the nipple-areolar complex (NAC) through techniques like nipple-sparing mastectomy (NSM). This approach has been shown to improve patient satisfaction and quality of life. However, it is important to note that NSM is associated with certain complications, including NAC necrosis, malposition, and local recurrence of the tumor. Among those complications, nipple hypertrophy is quite rare. In this report, we present a case of nipple hypertrophy that developed after breast reconstruction, using autologous tissue. The patient, a 48-year-old woman, underwent NSM for breast cancer, and had a tissue expander placed simultaneously. Following tissue expansion, breast reconstruction with a deep inferior epigastric perforator (DIEP) flap was performed. However, during the tissue expansion phase, the patient's nipple gradually enlarged, and the protrusion became more pronounced after the DIEP flap transfer. Nipple reduction surgery was subsequently performed for both diagnostic and cosmetic purposes. We present this case along with a review of relevant literature.

Three-Dimensional Craniofacial Landmark Detection in Series of CT Slices Using Multi-Phased Regression Networks.

Geometrical assessments of human skulls have been conducted based on anatomical landmarks. If developed, the automatic detection of these landmarks will yield both medical and anthropological benefits. In this study, an automated system with multi-phased deep learning networks was developed to predict the three-dimensional coordinate values of craniofacial landmarks. Computed tomography images of the craniofacial area were obtained from a publicly available database. They were digitally reconstructed into three-dimensional objects. Sixteen anatomical landmarks were plotted on each of the objects, and their coordinate values were recorded. Three-phased regression deep learning networks were trained using ninety training datasets. For the evaluation, 30 testing datasets were employed. The 3D error for the first phase, which tested 30 data, was 11.60 px on average (1 px = 500/512 mm). For the second phase, it was significantly improved to 4.66 px. For the third phase, it was further significantly reduced to 2.88. This was comparable to the gaps between the landmarks, as plotted by two experienced practitioners. Our proposed method of multi-phased prediction, which conducts coarse detection first and narrows down the detection area, may be a possible solution to prediction problems, taking into account the physical limitations of memory and computation.

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

One factor that can contribute to the development of hypertrophic scar contracture is mechanical stress. Mechanical cyclic stretch stimuli enhance the secretion of endothelin-1 (ET-1) from keratinocyte. Cyclical stretching of fibroblasts also increases the expression level of the transient receptor potential ion channel (TRPC3), which is known to couple with the endothelin receptor and induce intracellular Ca2+ signaling via the calcineurin/nuclear factor of activated T cells (NFAT) pathway. The aim of this study was to investigate the relationship between keratinocytes and fibroblasts when they are stretched. Methods: The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Results: Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. Conclusion: These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

Cutaneous fistula on the cheek associated with oculo-auriculo-vertebral spectrum.

A Japanese boy, presented with epibulbar dermoid and ipsilateral preauricular appendages, had a pit on his cheek of the same side. An atrial septal defect and vertebral fusions were also identified. He was diagnosed with a mild type of oculo-auriculo-vertebral spectrum (OAVS). At the age of 18 months, his cheek was swollen with a slight fever. An infected cyst and cutaneous fistula enveloped by the risorius muscle were extracted. It was assumed to be a remnant of the fissure between the maxillary and mandibular prominences. This was the first case of cutaneous fistula confirmed histologically with OAVS, although there seem to be more cases. The possibility of the mechanism of smiling cheek dimple is also discussed.

Reversible Color and Shape Changes of Nanostructured Fibers of a Macrocyclic π-Extended Thiophene Hexamer Promoted by Adsorption and Desorption of Organic Vapor.

A phenyl-substituted macrocyclic π-extended thiophene hexamer 1, composed of four thienylene-ethynylene and two thienylene-vinylene units, has a solid-state structure in which π-π, CH-π, and van der Waals interactions occur. Slow addition of acetone to a solution of 1 in CS2 produces a yellow nanostructured fiber 1-A containing a 1:1.5:1 ratio of 1, acetone, and CS2. Over a 2 min period at 25 °C, 1-A gradually changes to an orange fiber 1-B containing a 1:0.5:1 ratio of 1, acetone, and CS2. On exposure to acetone vapor, 1-B regenerates 1-A (vapochromism), and removal of all solvents from 1-A and 1-B generates a red-orange fiber 1-C, which upon brief immersion in acetone/CS2 produces 1-A. Furthermore, 1-C is converted to orange yellow fiber 1-D upon exposure to acetone vapor for 1 s at 25 °C. Analysis of the horizontal and vertical profiles of the X-ray diffraction (XRD) patterns shows that removal of solvent from 1-A reversibly creates 1-B in conjunction with a shape and size change along with arching.

Preparation, Spectroscopic Characterization and Theoretical Study of a Three-Dimensional Conjugated 70 π-Electron Thiophene 6-mer Radical Cation π-Dimer.

A radical cation, generated from an extended π-conjugated thiophene 6-mer composed of four ethynylene-thienylene and two vinylene-thienylene units, was observed to form a stable three-dimensional π-dimer containing 70 π-electrons. The π-dimer prepared in solution was investigated by using magnetic circular dichroism (MCD), ESR spectroscopy, and UV-vis-NIR absorption spectroscopy. Probing the individual NIR absorption bands showed that the MCD signals can be assigned to the pseudo Faraday A term, indicating that the absorption bands are comprised of nearly degenerate electronic transitions. X-ray crystallographic analysis revealed that the π-dimer has a three-dimensional face-to-face and continuous π-conjugated donutlike structure. Analysis of the UV-vis-NIR and ESR spectra of the π-dimer in the solid state confirmed that it possesses the dimer structure. The prediction made by using TD-DFT calculations that the dimer would have a 70 π-electron diatropic nature was confirmed by using solid state 1H NMR spectroscopy.

Analysis of 122 triplet and one quadruplet pregnancies after single embryo transfer in Japan.

RESEARCH QUESTION: What is the prevalence of triplet and quadruplet pregnancies after single embryo transfer (SET) in Japan. DESIGN: A retrospective observational study was conducted on 274,605 pregnancies after 937,848 SET cycles in registered assisted reproductive technology (ART) data from the Japanese ART national registry database between 2007 and 2014. A questionnaire survey of ART centres was also conducted. Data on pregnancies with embryo division into three or more after SET were analysed. RESULTS: According to the Japanese ART national registry database, SET resulted in 109 triplet pregnancies (0.04% of pregnancies), and the questionnaire reports from 31 centres revealed 33 triplet and one quadruplet pregnancies. After exclusion of 20 duplicated cases, 122 triplet and one quadruplet pregnancies included 46 monochorionic (one gestational sac [37.4%]), 18 dichorionic (two gestational sacs [14.6%]) and 59 trichorionic pregnancies (three gestational sacs [48.0%]). Compared with singleton pregnancies, patients with monozygotic triplet or quadruplet pregnancies were less frequently diagnosed with unexplained infertility (P = 0.004), more often received gonadotrophin injections for ovarian stimulation in 39 cases with information available (P = 0.021) and underwent more blastocyst transfers and assisted hatching (P = 0.002 and P < 0.001, respectively). The proportion of live birth, defined as at least one baby born, excluding induced abortion, was 64.6% (73/116 pregnancies) of monozygotic triplet or quadruplet pregnancies. CONCLUSIONS: Combined Japanese ART national registry and survey data revealed 122 triplet and one quadruplet pregnancies, the majority after cryopreserved embryo transfer. Most were conceived after blastocyst transfer and often after assisted hatching, which are potential risk factors for zygotic splitting.

Periostin is induced by IL-4/IL-13 in dermal fibroblasts and promotes RhoA/ROCK pathway-mediated TGF-ß1 secretion in abnormal scar formation.

Excess scar formation can occur after skin injurふy and lead to abnormal scar formation, such as keloids and hypertrophic scars, which are characterised by substantial deposition of extracellular matrix in the dermis. Periostin, an extracellular matrix protein that plays a crucial role in skin development and maintaining homeostasis, is also involved in skin disorders such as systemic/limited scleroderma, wound closure, and abnormal scar formation. However, the mechanism of periostin involvement in abnormal scar formation is not yet fully understood. In this study, we investigated the mechanism by which periostin is involved in abnormal scar formation. Treatment of human dermal fibroblasts (HDFs) with IL-4 and IL-13, which are cytokines of Th2 type immune responses that are up-regulated in abnormal scars, dramatically elevated the levels of periostin mRNA and protein, and also promoted the secretion of periostin by HDFs. Transforming growth factor-ß1 (TGF-ß1) had the same effect on HDFs as IL-4 and IL-13. Stimulation of HDFs with periostin promoted RhoA/ROCK pathway-mediated TGF-ß1 secretion from HDFs. Our results suggest that IL-4 and IL-13 induce periostin expression and secretion, and in turn, secreted periostin induces RhoA/ROCK pathway-mediated TGF-ß1 secretion. Secreted TGF-ß1 then induces further periostin production and secretion, thereby promoting abnormal scar formation.

Small Structural Changes in the Alkyl Substituents of Macrocyclic π-Extended Thiophene Oligomers Causes a Key Effect on Their Stacking and Functional Properties.

Three new macrocyclic π-extended thiophene hexamers composed of four thienylene-ethynylene and two thienylene-vinylene units with or without four alkyl substituents have been synthesized. Despite similar shape-persistent structures in solution, the alkyl substituents control the solid-state structures and morphologies. The unsubstituted hexamer exhibited a planar conformation with a theoretically predicted structure in the solid state; however, the planar hexamer with four ethyl substituents formed a closely stacked columnar crystal structure to exhibit π-π interactions. Interestingly, the hexamer with four butyl substituents adopted both planar and twisted conformations in the solid state, exhibiting polymorphism based on induced-fit stacking of molecules. Thus, the butyl-substituted hexamer produces a mixture of yellow, orange, and red single crystals from toluene/acetone, and X-ray analysis revealed six different conformations. Consequently, the small structural difference in the macrocycles causes a key effect on their functional properties in the solid state, and their morphology governs electrical conductivity and organic field-effect-transistor activity. The polymorphism of the hexamers was applied to the switching of film morphology.

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-ß1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population.

An Attempt to Make Platelet Rich Fibrin from Bone Marrow Aspirate.

INTRODUCTION: Platelet Rich Fibrin (PRF) was developed as a new generation of platelet concentration from peripheral blood. Method to make PRF is simple. It is easy to handle with its moderate firmness. Histologically, platelets and nucleated cells are packed along the yellow-red border. Bone marrow aspirate contains bone marrow cells that potentially work for tissue regeneration, and platelets which contain growth factors. The specific gravities of them are comparable. It implies that, if it is possible to make PRF from bone marrow aspirate, then high concentration of platelets and bone marrow cells can be obtained simultaneously by taking out yellow-red interface of it. AIM: To find out a method to make PRF from bone marrow aspirate. MATERIALS AND METHODS: Iliac crest of rabbits were punctured and aspirated with or without anti-coagulant, under general anaesthesia. The bone marrow aspirate was centrifuged in glass tubes. For the bone marrow aspirate taken with anti-coagulant, calcium chloride was added just before centrifugation. Products were taken out and observed grossly. The products were fixed with formaldehyde and observed histologically. RESULTS: Coagulated gels with two-toned colour were obtained by all methods. In the gels without anti-coagulant, interfaces between two colours were obscure. Histologically, platelets and nucleated cells scattered as clusters. Filtering caused haemolysis and reduced the yield of the product. With the aspirate taken with anti-coagulant, platelets and nucleated cells formed a band along the interface. CONCLUSION: PRF can be made from bone marrow aspirate by adding anti-coagulant in aspiration and reversed with calcium chloride just before centrifugation.

A new rabbit model of impaired wound healing in an X-ray-irradiated field.

Radiation is an important therapy for cancer with many benefits; however, its side effects, such as impaired wound healing, are a major problem. While many attempts have been made to overcome this particular disadvantage, there are few effective treatments for impaired wound healing in an X-ray-irradiated field. One reason for this deficiency is the lack of experimental models, especially animal models. We have previously reported a mouse model of impaired wound healing in which the irradiation area was restricted to the hindlimbs. In this mouse model, due to the size of the animal, a diameter of five millimeters was considered the largest wound size suitable for the model. In addition, the transplanted cells had to be harvested from other inbred animals. To investigate larger wounds and the impact of autologous specimen delivery, a rabbit model was developed. Rabbits were kept in a special apparatus to shield the body and hindlimbs while the irradiation field was exposed to radiation. Six weeks after irradiation, a 2 x 2 cm, full-thickness skin defect was made inside the irradiation field. Then, the wound area was observed over time. The wound area after irradiation was larger than that without irradiation at all time points. Both angiogenesis and collagen formation were reduced. For further study, as an example of using this model, the effect of autologous platelet-rich plasma (PRP) was observed. Autologous PRP from peripheral blood (pb-PRP) and bone marrow aspirate (bm-PRP) was processed and injected into the wounds in the irradiated field. Two weeks later, the wounds treated with bm-PRP were significantly smaller than those treated with phosphate buffer vehicle controls. In contrast, the wounds treated with pb-PRP were not significantly different from the controls. This rabbit model is useful for investigating the mechanism of impaired wound healing in an X-ray-irradiated field.

The Abnormal Architecture of Healed Diabetic Ulcers Is the Result of FAK Degradation by Calpain 1.

Delayed wound healing is a major complication of diabetes occurring in approximately 15% of chronic diabetic patients. It not only significantly affects patients' quality of life but also poses a major economic burden to the health care system. Most efforts have been focused on accelerating wound reepithelialization and closure. However, even after healing the quality of healed tissue in diabetics is abnormal and recurrence is common (50-75%). Thus, understanding how diabetes alters the ultimate mechanical properties of healed wounds will be important to develop more effective approaches for this condition. Focal adhesion kinase is an intracellular protein kinase that plays critical roles in cell migration, focal adhesion formation, and is an important component of cellular mechanotransduction. We have found that focal adhesion kinase expression is downregulated under a high glucose condition both in vitro and in vivo. This is secondary to increased activity of calpain 1, the primary enzyme responsible for focal adhesion kinase degradation, which becomes induced in hyperglycemia. We demonstrate that selective inhibition of calpain 1 activation improves wound healing and normalizes the mechanical properties of diabetic skin, suggesting a new therapeutic approach to prevent diabetic wound recurrence.

Endothelial cell-derived endothelin-1 is involved in abnormal scar formation by dermal fibroblasts through RhoA/Rho-kinase pathway.

Hypertrophic scars and keloids are characterized by excessive dermal deposition of extracellular matrix due to fibroblast-to-myofibroblast differentiation. Endothelin-1 (ET-1) is primarily produced by vascular endothelial cells and plays multiple roles in the wound-healing response and organ fibrogenesis. In this study, we investigated the pathophysiological significance of ET-1 and involvement of RhoA, a member of the Rho GTPases, in hypertrophic scar/keloid formation. We found that ET-1 expression on dermal microvascular endothelial cells (ECs) in hypertrophic scars and keloids was higher than that in normal skin and mature scars. We also confirmed that ET-1 induced myofibroblast differentiation and collagen synthesis in cultured human dermal fibroblasts through the RhoA/Rho-kinase pathway. Finally, since hypertrophic scar/keloid formation was most prominent in areas exposed to mechanical stretch, we examined how mechanical stretch affected ET-1 secretion in human dermal microvascular ECs, and found that mechanical stretch increased ET-1 gene expression and secretion from ECs. Taken together, these results suggest that dermal microvascular ECs release ET-1 in response to mechanical stretch, and thereby contribute to the formation of hypertrophic scars and keloids through the RhoA/Rho-kinase pathway.

Complete androgen insensitivity syndrome and anti-Müllerian hormone levels before and after laparoscopic gonadectomy.

We report cases of two sisters with complete androgen insensitivity syndrome (CAIS). A complete female appearance, blind-ending vagina, and testes in the pelvis are characteristics of CAIS. Prophylactic laparoscopic gonadectomy was performed in both cases. Anti-Müllerian hormone (AMH) level is known to be very high in patients with CAIS; AMH is secreted by Sertoli cells and testosterone suppresses the secretion. In our cases, serum AMH was very high before gonadectomy and dramatically decreased after gonadectomy. AMH could be the diagnostic feature for patients with CAIS.

Bilateral Maxillary Reconstruction Using Fibular Flap in Bisphosphonate-related Osteonecrosis.

Recent reports have shown successful transfer of vascularized fibular flap in bisphosphonate-induced mandibular osteonecrosis. We present a case of a 50-year-old patient who presented with bisphosphonate-related osteonecrosis of bilateral maxilla, which is reconstructed using a fibular flap.

An augmented reality system in lymphatico-venous anastomosis surgery.

Indocyanine green lymphography, displayed as infrared image, is very useful in identifying lymphatic vessels during surgeries. Surgeons refer the infrared image on the displays as they proceed the operation. Those displays are usually placed on the walls or besides the operation tables. The surgeons cannot watch the infrared image and the operation field simultaneously. They have to move their heads and visual lines. An augmented reality system was developed for simultaneous referring of the infrared image, overlaid on real operation field view. A surgeon wore a see-through eye-glasses type display during lymphatico-venous anastomosis surgery. Infrared image was transferred wirelessly to the display. The surgeon was able to recognize fluorescently shining lymphatic vessels projected on the glasses and dissect them out.

Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function.

Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-ß1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.

Growth Factor Measurement and Histological Analysis in Platelet Rich Fibrin: A Pilot Study.

OBJECTIVE: The aim of this study was to compare growth factor amount contained in platelet rich fibrin (PRF) and compare with that in platelet rich plasma (PRP), and in whole blood. And also to investigate distribution of growth factors and cellular components in PRF. MATERIALS AND METHODS: PRF and PRP were obtained from the same sample of peripheral blood. Extraction of proteins were done with lysis buffer, accompanied by freeze and thaw procedures. Concentration of two representative growth factors in platelets: platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-ß), were measured with enzyme-linked immunosorbent assay (ELISA). PRF was cut into three parts: (top, middle and bottom), and growth factor concentration was measured respectively. Paraffin embedded section of PRF was observed with Giemsa stain. Immuno-histochemical analysis with anti-PDGF and anti-TGF-ß antibodies was also conducted. RESULTS: The growth factor levels in PRF was higher than in peripheral blood and comparable to those in PRP. Growth factor levels in bottom part of PRF was much higher than in top and middle part. Microscopically, platelets and mono-nucleated cells were concentrated just above the yellow-red interface. Poly-nucleated cells were concentrated below the interface. CONCLUSION: The growth factors were surely concentrated in PRF. This result can support basis of good clinical outcomes. For effective application of PRF, the knowledge that growth factors and cells are not equally distributed in PRF should be utilized.