Kotaro Yoshimura

Kotaro YoshimuraJichi Medical University · Plastic Surgery

· M.D.
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  • About
    Current institution
    Jichi Medical University
    Current position
    • Professor and Chair
    Top co-authors
    Projects (1)
    Archived project
    The role of adipose-derived stem cells in wound healing.
    Research Items (197)
    Chronic changes following radiotherapy include alterations in tissue-resident stem cells and vasculatures, which can lead to impaired wound healing. In this study, novel recombinant human collagen peptide (rhCP) scaffolds were evaluated as a biomaterial carrier for cellular regenerative therapy. Human adipose-derived stem cells (hASCs) were successfully cultured on rhCP scaffolds. By hASC culture on rhCP, microarray assay indicated that expression of genes related to cell proliferation and extracellular matrix production was upregulated. Pathway analyses revealed that signaling pathways related to inflammatory suppression and cell growth promotion were activated as well as signaling pathways consistent with some growth factors including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor beta (TGF-β1), although gene expression of these growth factors was not upregulated. These findings suggest the rhCP scaffold showed similar biological actions to cytokines regulating cell growth and immunity. In subsequent impaired wound healing experiments using a locally irradiated (20 Gray) mouse, wound treatment with rhCP sponges combined with cultured hASCs and human umbilical vein endothelial cells accelerated wound closure compared to wounds treated with rhCP with hASCs alone, rhCP only, and control (dressing alone), with better healing observed according to this order. These results indicating the therapeutic value of rhCP scaffolds as a topical biomaterial dressing and a biocarrier of stem cells and vascular endothelial cells for regenerating therapies. The combination of rhCP and functional cells was suggested to be a potential tool for revitalizing stem cell-depleted conditions such as radiation tissue damage.
    For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 10⁶ ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20–100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.
    Background:. Multidirectional cranial distraction osteogenesis (MCDO) is a procedure of ours developed earlier for treating craniosynostosis. However, the numerous bone flaps led to prolonged operative time and occasional bone detachment from dura. We have since simplified the osteotomy design. In treating sagittal synostosis, required bone flaps have been reduced to 11 (from ~20). Methods:. In a 2-year period (2014–2015), 5 boys with sagittal synostosis underwent MCDO using our simplified and fixed-form osteotomy. Mean age at surgery was 9.4 months (range, 8–11 months). Pre- and postoperative cranial morphology was assessed by cephalic index and by mid-sagittal vector analysis. Results:. Improved cranial shape was confirmed by 3-dimensional CT scans and by mid-sagittal vector index. Mean preoperative cephalic index (68.7) progressively increased to means of 78.5 immediately after distraction device removal, 75.2 at postoperative month 6, and 75.1 at 1 year postoperatively. There were no major complications, although transient cerebrospinal fluid leakage and loosening of anchor pins occurred in 1 patient. Conclusions:. Simplified MCDO has a number of advantages over conventional distraction procedures such as discretionary reshaping/expansion of cranium and predictable osteogenesis and is a valid treatment option for patients with sagittal synostosis.
    Background: Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study is to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting. Methods: Lipoaspirates were harvested from six adult females and preserved using two cryopreservation methods: 1) simple cooling to -80˚C (cryo-1); or 2) programmed cooling to -190˚C (cryo-2). Fresh fat, cryo-1 fat, and cryo-2 fat were analyzed both in vitro and in vivo. Results: Immunohistochemistry of both types of cryopreserved adipose tissue revealed that most adipocytes were necrotic. The cell number and viability of stromal vascular fraction (SVF) cells were significantly decreased in cryo-1 fat (1.7×10 cells, 42.6% viable) and cryo-2 fat (2.0×10 cells, 55.4% viable), compared to fresh fat (3.9×10 cells, 90.6% viable). Although adipose-derived stem cells (ASCs) successfully were cultured from all fats, functional ASCs from cryopreserved fats were much smaller in number with comparable multi-lineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, three months after transplantation, all of the cryopreserved fats maintained their volume to some extent; however, the cryopreserved fats were mostly filled with dead tissue and produced significantly lower engraftment scores than fresh fat. Conclusions: Most of adipocytes were killed in the process of cryopreservation and thawing. ASCs were isolated from cryopreserved fats, but the number of functional ASCs was very limited in both cryopreservation methods. After grafting, cryopreserved fat retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.
    Background: Although the charting of normal intracranial volume (ICV) is fundamental for managing craniosynostosis, Asian norms in this regard are unknown. The purpose of this study was to establish a growth curve for ICVs in a large series of normal Asian children, providing reference values to guide corrective surgery. Methods: A total of 124 normal children (male, 63; female, 61) and 41 children diagnosed with craniosynostoses were analyzed. Patients aged 0-8 years presenting to the emergency room and subjected to computed tomography (CT) for head trauma served as the reference cohort. Axial CT head scan data were obtained from radiographic archives at Jichi Medical University. Imaging was done on a Siemens CT scanner (5-mm slice thickness), using a DICOM viewer to measure ICVs. Results: ICVs were plotted against age, and best-fit logarithmic curves for normal subjects were generated, without and with gender stratification. Male and female growth curves were similar in shape but diverged past the age of 1 year (male > female). ICVs of patients with craniosynostoses were plotted to male and female growth curves by disease subset, revealing the following: sagittal synostosis, near normal (or marginally larger); metopic synostosis, below normal; other non-syndromic synostoses (unilateral, bilateral, and lambdoidal) and Crouzon syndrome, near normal; Apert syndrome, above normal; and Pfeiffer syndrome, variable. Conclusion: ICVs of early childhood were investigated in Asian subjects, creating growth curves that set criteria for timing, planning and goalsetting in surgical correction of craniosynostosis.
    Background: The deep inferior epigastric perforator (DIEP) flap, which is a modification of the muscle-sparing free transverse rectus abdominis musculocutaneous (msTRAM) flap, is being more frequently used in an effort to reduce postoperative abdominal morbidity. However, there is no consensus as to which of these flaps is superior. We aimed to compare quantitative measurements of abdominal function obtained with an isokinetic dynamometer after DIEP and msTRAM flap elevation. Methods: Patients who underwent unilateral single-pedicled DIEP (n = 42) or msTRAM flap (n = 36) breast reconstruction performed by a single surgeon were included in this study. Pre- and postoperative trunk flexion parameters were measured prospectively using an isokinetic dynamometer in all patients. The occurrence of postoperative pain, stiffness, and bulging along with patient activity level were also investigated. Results: At 3 months postoperatively, abdominal functions were decreased in both groups, with a larger decline in the msTRAM group. However, at 6 months postoperatively, abdominal muscle function recovered to preoperative levels in both groups. These findings were consistent with the absence of a statistically significant difference in patient postoperative abdominal pain and stiffness, activity level, and the incidence of bulging between the two groups at 6 months postoperatively. Conclusions: From these results, we propose that the surgeon can select the msTRAM flap, without hesitation or concern regarding abdominal morbidity, when a thick and reliable perforator does not exist and multiple thin perforators must be incorporated.
    Anteromedial maxillectomy is typically performed in conjunction with low-dose radiotherapy and intraarterial chemotherapy. In doing so, the extent of surgical defects is reduced. However, nasal deviation and oral incompetence may ensue, due to cicatricial contracture of wounds, and may be distressing to these patients. Herein, we report a series of eight free perforator flap procedures (anterolateral thigh [ALT] flap, 6; thoracodorsal artery perforator [TAP] flap, 2) used to correct such deformities. The TAP flap was combined with scapular tip [ST] osseous flap in patients with added zygomatic prominence defects. Three adipocutaneous parts developed from each perforator flap were applied as follows: two to reconstruct nasal lining and oral vestibule, and one to augment cheek volume. All aesthetic results proved satisfactory, although orbital dystopia and contracture of mimic muscles were not resolved completely. These secondary interventions are suitable for sequelae of simple anteromedial maxillectomy. Immediate reconstruction should be considered if orbital floor and mimic muscles are involved.
    Background: The proximal ends of internal mammary (IM) vessels are now the most common recipient vessels for breast reconstruction. On the other hand, bilateral deep inferior epigastric artery perforator (DIEP) flaps are often needed according to the territory and the volume required for reconstruction. The usefulness of retrograde IM vessels as second recipients has recently been reported, but there are very few quantitative studies on the hydrodynamics of the retrograde IM vessels. Because the flow is dependent on the pressure differential, the blood pressures of the antegrade IM artery (AIMA), antegrade IM vein (AIMV), retrograde IM artery (RIMA), retrograde IM vein (RIMV), and recirculated intraflap vein (FV) were investigated to solve this question and to confirm the reliability and usefulness of the retrograde IM vessels. Methods: Ten free flap breast reconstructions were included in this study. The IM vessels were exposed, and the pressures were measured. After recirculation, the FV pressures were measured when the flap was not ischemic or congestive. Systemic blood pressure was also recorded during the whole measurement period. Results: The AIMA and RIMA pressures were 70.4 ± 8.2 mmHg and 54.0 ± 8.6 mmHg (p = 0.000003), respectively, while the systemic pressure was 65.1 ± 10.0 mmHg. The AIMV pressure was always smaller than the RIMV pressure; the mean AIMV pressure was 5.3 ± 1.6 mmHg. In addition, the FV pressure was greater (p = 0.03) than the RIMV pressure (17.7 ± 9.9 mmHg), while the RIMV pressure was 8.7 ± 2.0 mmHg. Conclusions: Both the RIMA and RIMV are useful and reliable as second recipients for bipedicled free flap transfers. This is a great benefit because it would provide two recipients in one surgical site and would be especially useful in thin patients or patients with previous abdominal scars requiring double pedicled DIEP flaps. Level of evidence: Therapeutic Study, Level IV.
    Background Keloids are a dermal fibroproliferative scar of unknown etiology. There is no good animal model for the study of keloids, which hinders the development and assessment of treatments for keloids. Methods Human keratinocytes and dermal fibroblasts were isolated from 3 human skin tissues: normal skin, white scars, and keloids. A mixed-cell slurry containing keratinocytes and dermal fibroblasts was poured into a double chamber implanted on the back of NOD/Shi-scid/IL-2Rγnull mice. After 12 weeks, the recipient mice had developed reconstituted human skin tissues on their backs. These were harvested for histological studies. Results Macroscopically, the reconstituted skins derived from both normal skin and white scars were similar to normal skin and white scars in humans, respectively. Keloid-derived reconstituted skins exhibited keloid-like hypertrophic nodules. Histological findings and immunohistochemical staining confirmed that the reconstituted skin tissues were of human origin and the keloid-derived reconstituted skin had the typical features of human keloids such as a hypertrophic dermal nodule, collagen type composition, orientation of collagen fibers, and versican expression. Conclusion The mouse model with humanized keloid tissue presented here should be a useful tool for future keloid research.
    Background: There is no standard method to ensure survival of random-pattern skin flaps. The authors developed a rat anemia model to observe survival of random-pattern skin flaps after blood transfusion and hemodilution. Methods: Anemia was induced by withdrawal of 35 percent blood volume followed by compensation with the same amount of blood (blood transfusion model) or plasma equivalent (normovolemic hemodilution). Control rats were subjected to a sham procedure. Subsequently, a random-pattern skin flap (1.5 × 6 cm) was elevated on the back of each rat. Physiologic assessments of flap vascularity/viability were performed using laser Doppler spectrophotometry before and after flap elevation. Results: The normovolemic hemodilution group showed anemia (hemoglobin, 9.5 ± 0.8 g/dl) but less flow occlusion and greater flap survival (72.8 ± 8.6 percent) compared with control (57.4 ± 9.6 percent; p < 0.01) and blood transfusion (62.1 ± 6.5 percent; p < 0.089) groups. In control and blood transfusion groups but not the normovolemic hemodilution group, blood flow was decreased and relative quantity of hemoglobin was increased toward the flap tip, indicating congestion. In control and blood transfusion groups, blood flow and tissue oxygen saturation dropped after flap elevation, but recovered by day 7; congestion gradually improved by day 7. Conclusions: The authors determined that congestion promoted necrosis and hemodilution reduced microcirculatory occlusion and increased blood flow and oxygenation in skin flaps. It was suggested that perioperative hemodilution is superior to blood transfusion in any flap operations unless there is a critical systemic need for blood transfusion.
    Background: The pathophysiology of lymphedema is poorly understood. Current treatment options include compression therapy, resection, liposuction, and lymphatic microsurgery, but determining the optimal treatment approach for each patient remains challenging. Objectives: We characterized skin and adipose tissue alterations in the setting of secondary lymphedema. Methods: Morphologic and histopathologic evaluations were conducted for 70 specimens collected from 26 female patients with lower extremity secondary lymphedema following surgical intervention for gynecologic cancers. Indocyanine green lymphography was performed for each patient to assess lymphedema severity. Results: Macroscopic and ultrasound findings revealed that lymphedema adipose tissue had larger lobules of adipose tissue, with these lobules surrounded by thick collagen fibers and interstitial lymphatic fluid. In lymphedema specimens, adipocytes displayed hypertrophic changes and more collagen fiber deposits when examined using electromicroscopy, whole mount staining, and immunohistochemistry. The number of capillary lymphatic channels was also found to be increased in the dermis of lymphedema limbs. Crown-like structures (dead adipocytes surrounded by M1 macrophages) were less frequently seen in lymphedema samples. Flow cytometry revealed that, among the cellular components of adipose tissue, adipose-derived stem/stromal cells (ASCs) and M2 macrophages were decreased in number in lymphedema adipose tissue compared to normal controls. Conclusions: These findings suggest that long-term lymphatic volume overload can induce chronic tissue inflammation, progressive fibrosis, impaired homeostasis, altered remodeling of adipose tissue, impaired regenerative capacity, and immunologic dysfunction. Further elucidation of the pathophysiologic mechanisms underlying lymphedema will lead to more reliable therapeutic strategies. This article is protected by copyright. All rights reserved.
    Serious lip injuries can occur during orthognathic surgery. Although an Angle Wider device, which is commonly used during orthognathic surgery, provides some lip protection, it leaves more than half of the lip exposed to surgical instruments. Here, we describe a novel technique to protect the entire upper and lower lips during orthognathic surgery using a minilaparotomy wound edge protector (Lap-Protector). We used this method in 60 patients who have undergone orthognathic surgeries such as sagittal split ramus osteotomy and Le Fort I osteotomy since 2009, and no lip injuries have occurred. Although this technique can be somewhat challenging at first and creates some difficulty in exposing the surgical field on the lateral side, we believe that using a wound edge protector minimizes the risk of lip injury during orthognathic surgery.
    Immediate reconstruction of orbitomaxillary defects is challenging for head and neck reconstructive surgeons. The primary goals of orbitomaxillary reconstruction are to cover the skin and mucosal defects, fill the defect space, and reconstruct the natural facial contour. In this report, two patients who underwent extended orbitomaxillectomy and immediate reconstruction using a combined latissimus dorsi musculocutaneous and scapular angle osseous free flap (LD-SA flap) are described. The LD-SA flap has substantial advantages, such as providing structural support to the malar prominence, filling the large soft tissue defect, and preventing postoperative drooping of the cheek. The surgical technique is relatively simple, requiring a shorter operative time and producing less blood loss than other reconstruction approaches. The LD-SA flap is a useful option for extended orbitomaxillary defect reconstruction.
    A deep burn wound is a critical condition that generally necessitates vascularized tissue coverage. We performed the injection of platelet-derived factor concentrates combined with non–cross-linked hyaluronic acid scaffolds for 2 patients with critical burn wounds with bone and tendon exposure and achieved successful healing. Hyaluronic acid was considered to have served as a controlled-release carrier of platelet-derived factors, being clinically effective for the treatment of deep burn wounds.
    Introduction: Condensation of grafted fat has been considered a key for achieving better outcomes after fat grafting. The purpose of this study was to investigate the therapeutic potentials of two mechanical tissue micronizing procedures: squeeze and emulsification. Materials and methods: Human aspirated fat was centrifuged (centrifuged fat: CF) and fragmented with an automated slicer (squeezed fat: SF). Alternatively, CF was emulsified by repeated transfer between two syringes through a small-hole connecter, then separated by mesh filtration into two portions: residual tissue of emulsified fat (REF) and filtrated fluid of emulsified fat (FEF). The four products were examined for cellular components. Results: Histological and electron microscopic analyses revealed that SF and REF contained broken adipocytes and fragmented capillaries. Compared to CF, the SF and REF products exhibited increased specific gravity and increased numbers of adipose-derived stem/stromal cells (ASCs) and endothelial cells (ECs) per volume, suggesting successful cell/tissue condensation in both SF and REF. Although cell number and viability in the stromal vascular fraction (SVF) were well maintained in both SF and REF, SVF culture assay showed that ASCs were relatively damaged in REF but not in SF. By contrast, no ASCs were cultured from FEF. Conclusion: Our results demonstrated that mechanical micronization is easily conducted as a minimal manipulation procedure, which can condense the tissue by selectively removing adipocytes without damaging key components, such as ASCs and ECs. Depending on the extent of adipocyte removal, the product may be a useful therapeutic tool for efficient tissue volumization or therapeutic revitalization/fertilization.
    Background and aim: The reduced incidence of donor site morbidity after deep inferior epigastric perforator (DIEP) flap is because the rectus muscle and its fascia are preserved. However, no study has proved that trunk flexion recovers not by the compensatory effect of the contralateral rectus muscle but by reinnervation of the ipsilateral rectus muscle. We hypothesized that if sufficient reinnervation occurs, patients who undergo single-pedicled DIEP (S-DIEP) flap or double-pedicled DIEP (D-DIEP) flap breast reconstruction would have similar levels of preoperative trunk flexion. To determine this, we investigated perioperative changes in trunk flexor muscle ability quantitatively using an isokinetic dynamometer in patients who had received S-DIEP or D-DIEP. Methods: Patients who underwent breast reconstruction with S-DIEP (n = 37) and D-DIEP (n = 30) were included in this study. Pre- and postoperative trunk flexor muscle ability was measured prospectively by an isokinetic dynamometer in all patients. Postoperative abdominal pain and stiffness, patients' activity, and incidence of bulging were also investigated. Results: Six months after surgery, the trunk flexor muscle ability recovered and did not significantly decrease subsequently in either group. This finding was consistent with the result that patients' activities and the incidence of bulging were similar between the two groups. Conclusions: Our results show that reinnervation of the rectus muscle can be confirmed at 6 months after DIEP flap elevation. Thus, we recommend D-DIEP flap without concern for abdominal wall weakness, especially in patients with multiple abdominal scars and who require breast tissue exceeding the amount of tissue that can be transferred with S-DIEP flap.
    Autologous fat graft is now established as a useful method for breast reconstruction, but the unpredictable rate of graft survival has proven problematic. We have previously introduced our method of Cell Assisted Lipotransfer (CAL) to improve graft survival rate. In this report, we describe a novel method using multiple CAL sessions to completely reconstruct postmastectomy breasts in stages. We have named this method Step-CAL. In Step-CAL, we inserted a tissue expander underneath the pectoral muscle of the postmastecto-my patient prior to the CAL session. After the skin flap was extended, the first fat graft was implanted into the subcutaneous tissue and pectoral muscle, and then the amount of water in the tissue expander was reduced. After repeating these procedures two or three times at 3-month intervals and removing the tissue expander, the breast eventually was reconstructed with only the patient's own fat. This method is suitable for the patient with enough fat for several liposuctions, enough thickness of the breast skin flap after mastectomy, and a small opposite breast. After reconstruction with Step-CAL, the breast is soft and smooth with minimal scarring. We think this method could be a novel option for reconstruction of the breast post-mastectomy.
    Autologous free fat grafting and lipoinjection have a history of 120 years for the treatment of deformity. The change of transplantation techniques from excised adipose tissue grafting to lipoinjection, development of techniques of liposuction and lipoinjection, and discovery of adipose-derived stem and stromal cells have provided major advancements in the clinical outcomes of fat grafting. In this decade, lipoinjection has been widely used worldwide. Although clinical results largely depend on the surgeons' techniques, experiences and knowledge of lipoinjection, severe complications, such as huge oil cysts and lipogranulomas, can be avoidable with careful procedures based on the following principles: 1) atraumatic liposuction, 2) removal of unwanted components from the suctioned fat, and 3) multiple injections of small amounts of fat tissue into multiple subcutaneous tunnels. Although there remain several concerns such as how to process fat tissue for the further improvement of surgical outcomes, the indications for lipoinjection can be expanded to include treatments for skin texture, pigmentation, and cicatricial contracture.
    Although various injection techniques of hyaluronic acid (HA) filler for facial rejuvenation have been developed, correction of deep wrinkles/grooves, such as the nasolabial fold (NLF), with intradermal or subdermal injections remains difficult. We tested the intradermal HA injection method to place multiple HA struts by (1) inserting a small needle perpendicularly to the wrinkle and (2) injecting HA as intradermal struts with the skin fully stretched by the practitioner's fingers. The results of both NLFs in 10 patients suggest that this technique improves NLFs and maintain the effects more consistently than conventional techniques, although the effects of both methods were almost lost after 6 months. Selective and/or combined application of this technique may enhance the current approach to facial rejuvenation with dermal fillers.
    Unlabelled: Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage. Significance: This study shows the therapeutic value of human adipose-derived stem cell spheroids prepared in hyarulonic acid gel. The spheroids have various benefits as an injectable cellular product and show therapeutic potential to the stem cell-depleted conditions such as diabetic chronic skin ulcer.
    Background: Despite the clinical potential of adipose-derived stem/stromal cells (ASCs), there are some clinical difficulties due to the regulation of cell therapies. Materials & methods: Micronized cellular adipose matrix (MCAM) injectable was prepared through selective extraction of connective tissue fractions in fat tissue only through mechanical minimal manipulation procedures. Results: It retained some capillaries and ASCs, but most adipocytes were removed. The presence of viable ASCs, vascular endothelial cells was confirmed and ASCs of MCAM kept intact mesenchymal differentiation capacity. In diabetic mice, skin wounds treated with MCAM showed significantly accelerated healing compared with phosphate-buffered saline-treated ones. Conclusion: The proven potential of MCAM to accelerate healing in ischemic diabetic ulcers may offer a simple, safe and minimally invasive means for tissue repair and revitalization.
    Ionizing radiation is often used to treat progressive neoplasms. However, the consequences of long-term radiation exposure to healthy skin tissue are poorly understood. We aimed to evaluate the short- and long-term radiation damage to healthy skin of the same irradiation given either as single or fractional doses. C57BL/J6 mice were randomly assigned to one of three groups: a control and two exposure groups (5 Gy ×2 or 10 Gy ×1). The inguinal area was irradiated (6-MeV beam) 1 week after depilation in the treatment groups. Skin samples were evaluated macroscopically and histologically for up to 6 months after the final exposure. After anagen hair follicle injury by irradiation, hair cycling resumed in both groups, but hair graying was observed in the 10 Gy ×1 group but not in the 5 Gy ×2 group, suggesting the dose of each fractional exposure is more relevant to melanocyte stem cell damage than the total dose. On the other hand, in the long term, the fractional double exposures induced more severe atrophy and capillary reduction in the dermis and subcutis, suggesting fractional exposure may cause more depletion of tissue stem cells and endothelial cells in the tissue. Thus, our results indicated that there were differences between the degrees of damage that occurred as a result of a single exposure compared with fractional exposures to ionizing radiation: the former induces more severe acute injury to the skin with irreversible depigmentation of hairs, while the latter induces long-term damage to the dermis and subcutis.
    Recent technical and scientific advances in fat grafting procedures and concepts have improved predictability of fat grafting. Large-volume fat injection is gaining much attention as an attracting procedure for body contouring and reconstruction, but an increasing number of complications also has been recognized over the world. In this article, typical complications after fat grafting are described, as well as an explanation of how and why they occur, and how surgeons can avoid and treat complications.
    The demand for minimally invasive procedures has increased over the years in the field of cosmetic surgery, and we here introduce a novel facial rejuvenation procedure which uses pneumatic injection of hyaluronic acid and hypertonic glucose solution. We mixed 1 mL non-crosslinked hyaluronic acid (HA) and 9 mL 20% glucose solution, and injected the mixture into the subcutaneous layer; injected sites were distributed along the temporal hairlines and at the malar prominence. Thirty Japanese patients underwent three treatment sessions at 4-week intervals and were evaluated at 1- and 6-month follow-up. Mild to moderate aesthetic changes were observed, especially in the jawline and the marionette lines. More than half of the patients felt improvement at 1- and even at 6-month, although partial loss of correction was observed. No serious complications occurred. In conclusion, pneumatic injection of HA and hypertonic solution could be a potent non-invasive facial rejuvenation treatment, although further studies are needed.
    Autologous fat grafting is an exciting part of plastic and reconstructive surgery. Fat serves as a filler and its role in tissue regeneration will likely play a more important role in our specialty. As we learn more about the basic science of fat grafting and the standardized techniques and instruments used for fat grafting, this procedure alone or in conjunction with invasive procedures may be able to replace many operations that we perform currently. Its minimally invasive nature will benefit greatly our cosmetic and reconstructive patients, and may even achieve better clinical outcomes. Copyright © 2015 Elsevier Inc. All rights reserved.
    Deep-tissue injury (DTI) is a unique type of pressure ulcer (PU) in which deep-tissue damage expands outwards to the superficial skin. DTI progresses rapidly into a severe PU, despite initially appearing as only a bruise or darkened tissue in the superficial skin. Although some DTI detection methods are available, there is currently no strategy for treating deteriorating DTI. This study investigated the efficacy of vibration therapy for preventing DTI deterioration through down-regulation of the hypoxia-inducible factor-1 (HIF-1)-matrix metalloproteinase (MMP) axis in rats. We prepared a conventional PU rat model (PU group) and a DTI deterioration rat model (DTI group). The DTI group was further divided into two groups subjected to vibration and control treatments, respectively. Macroscopic and histological features, hypoxia, oxidative stress, apoptosis, and MMP2 and MMP9 activities in compressed skin were analyzed. Hypoxia, oxidative stress, and MMP activity were enhanced in the DTI group compared with the PU group. Vibration remarkably inhibited DTI deterioration, hypoxia, and the expression/activities of MMP2 and MMP9. These results suggest that vibration therapy can effectively attenuate deterioration of DTI. This report provides the first evidence for a therapeutic treatment for deteriorating DTI. This article is protected by copyright. All rights reserved. © 2015 by the Wound Healing Society.
    Recent research indicates that the adipose tissue of non-vascularized grafts is completely remodeled within 3 months, although origins of next-generation cells are unclear. Inguinal fat pads of GFP mice and wild-type mice were cross-transplanted beneath the scalp. At 1, 2, 4, and 12 weeks post-transplantation, grafted fat was harvested, weighed and analyzed through immunohistochemistry, whole-mount staining, and flow cytometry of cell isolates. Bone marrow of GFP mice was transplanted to wild-type mice (after irradiation). Eight weeks later, these mice also received fat grafts, which were analyzed as well. The majority of host-derived cells detected during remodeling of grafted fat were macrophages (>90% at early stage; 60% at 12 weeks). Cell origins were analyzed at 12 weeks (i.e., when completely regenerated). At this point, mature adipocytes largely were derived from adipose stem/stromal cells (ASCs) of grafts. Although vascular wall constituents chiefly were graft-derived, vascular endothelial cells originated equally from graft and host bone marrow. ASCs of regenerated fat were an admixture of grafted, host non-bone marrow, and host bone marrow cells CONCLUSIONS:: Above findings underscore the importance of ASCs in the grafted fat for adipocyte regeneration. Host bone marrow and local tissues contributed substantially to capillary networks and provided new ASCs. An appreciation of mechanisms that are operant in this setting stands to improve clinical outcomes of fat grafting and cell-based therapies.
    Aspirated fat contains unnecessary components such as water, oil, and blood cells. For better outcomes, tissue purification and condensation are useful, especially when injection volume to the recipient site is limited. Because aspirated fat is relatively poor in adipose-derived stem/stromal cells (ASCs), ASC condensation seems important for obtaining better regeneration and retention. Reducing tissue volume by removing some adipocytes or supplementation of stromal vascular fraction or ASCs can increase the ASC/adipocyte ratio in the graft. Clinical results of ASC supplementation remain controversial, but ASC condensation seems to lead to expanding applications of fat grafting into revitalization of stem cell-depleted tissue. Copyright © 2015 Elsevier Inc. All rights reserved.
    Autologous fat grafting has become an important procedure for volumization and revitalization, although clinical outcomes depend greatly on technique. It was revealed recently how grafted fat tissue survives, regenerates, or dies. Experimental results provided the underlying mechanism and clinical implications for therapeutic strategies to maximize the effects of fat grafting, minimize necrosis, and avoid oil cyst formation. Copyright © 2015 Elsevier Inc. All rights reserved.
    Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3(+) cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues. ©AlphaMed Press.
    Autologous fat grafting is a common and safe method of treatment for volume and contour defects in plastic surgery. In this study, cell-assisted lipotransfer (CAL) was used to treat 18 patients (age range, 16 to 60 years) with chest wall deformity or mammary hypoplasia from 2006 to 2012. Included in this series were 3 Poland's syndrome, 2 unilateral mammary hypoplasia, and 12 pectus excavatum (PE) patients with of the 2 PE cases having received previous thoracic surgical treatment. CAL was used to treat breast and chest deformities in 14 patients, and CAL in association with silicone breast implants in 4 cases of amastia. In CAL, stromal vascular fraction containing adipose derived stem/stromal cells are used in combination with lipoinjection. It is useful in adding volume for correction of contour defects such as subclavicular hollow, margin irregularities of the breast implant and absence of the anterior axillary fold. There are limitations in the survival rates of grafted fat and potential injection volume in a single session, which creates the need for multiple fat grafting procedures in some cases. With assessment for surgical indication and appropriate procedure, autologous fat grafting can be a simple and reliable alternative for the treatment of chest wall deformity and mammary hypoplasia.
    Background: Fat grafting is a promising modality for soft-tissue augmentation/reconstruction. However, grafted fat tissue is not initially perfused and relies on plasmatic diffusion from the recipient bed until revascularization occurs. The authors evaluated the therapeutic effects of normobaric hyperoxygenation for enhancing fat graft retention. Methods: Aspirated human fat tissue was cultured under tissue hypoxia (1% oxygen), normoxia (6%), and hyperoxia (20%) levels, and evaluated for adipocyte viability. Inguinal fat pads were autografted under mouse scalps (n=36), and mice were housed in either 20% (control) or 60% (normobaric hyperoxygenation) atmospheric oxygen for the first 3 days, and then returned to normoxia. Samples harvested at 0, 1, 2, 4, 8, and 12 weeks were analyzed immunohistochemically for adipocyte viability and regeneration. Results: Organ culture adipocytes died more quickly under lower oxygen tensions; thus, hyperoxygenation of recipient tissues may delay adipocyte death after fat grafting. Autografted mouse adipose tissue underwent dynamic remodeling, from ischemic degeneration to partial regeneration, over 12 weeks. Normobaric hyperoxygenation grafted samples showed significantly larger survival zones and engraftment scores (calculated using sample weight and adipocyte viability) at 1 and 12 weeks, respectively, than control samples. In addition, adipocyte regeneration (number of perilipin-positive preadipocytes), which peaked at 4 weeks, was significantly increased in normobaric hyperoxygenation samples. Conclusion: The normobaric hyperoxygenation protocol using 60% oxygen can be safely applied to enhance adipocyte survival, regeneration, and final engraftment after fat grafting.
    Autologous fat injection into the breast has been performed widely for breast augmentation and reconstruction because of recent technical and scientific advancements. However, it is important to learn what occurs and how problematic it can be if fat grafting is not conducted appropriately. Oil cysts were explanted from three subjects who underwent cosmetic fat grafting to the breast 2, 4, and 6 years previously. The oil cyst samples were examined histopathologically. Computed tomographic, magnetic resonance imaging, and mammographic images obtained sequentially after fat grafting were also analyzed. The cyst wall consisted of innermost and outermost fibrous layers and intermediate tissue that contained the regular adipose portion, a degenerated adipose portion, and a fibrous area. Eggshell-like macrocalcifications were seen in the inner surface. Numerous inflammatory cells, mainly MAC2/CD206 anti-inflammatory M2 macrophages, were observed in the degenerated adipose portion. Oil cysts with a longer history showed more calcifications in the innermost layer and a larger fibrous area adjacent to the degenerated fat portion than those with a shorter history. These histopathologic findings and clinical computed tomographic images revealed that oil cysts continued to be inflammatory and calcifications continued to develop over several years. After fat necrosis, long-term chronic inflammation persists and calcification seems to progress without limits. Oil cysts are the worst outcome of fat grafting and must be avoided by standardizing meticulous injection techniques. Therapeutic, V.
    Background: Hyperbaric oxygenation has been used for various purposes, but its clinical application is limited due to its pulmonary toxicity. We evaluated the therapeutic value of normobaric hyperoxygenation (NBO) for vascularized and nonvascularized tissue transplantation. Methods: Tissue oxygen partial pressure (PtO2) was measured for various organs in mice under inspiratory oxygen of 20%, 60%, or 100%. A rectangular skin flap (1 × 4 cm) or a composite skin graft (2 × 2 cm) was made on the back of mice, which were housed under 20% or 60% oxygen for the first 3 days after surgery. Cell survival was also examined in organ culture skin samples. Results: PtO2 varied among tissues/organs, but increased depending on inspiratory oxygen concentration in all tissues/organs. Although NBO with 100% O2 was toxic, NBO with 60% O2 was safe even when used continuously for a long period. NBO did not significantly improve survival of the rectangular skin flap. On the other hand, in the composite skin graft model, the engraftment area increased significantly (52 ± 10 at 20% vs 68 ± 5.1 at 60%) and contraction decreased significantly (42 ± 8.0 at 20% vs 27 ± 5.7 at 60%). Organ culture of a composite skin sample showed significant cell death under lower oxygen concentrations, supporting the data in vivo. Conclusions: The composite graft was maintained until revascularization by plasmatic diffusion from surrounding tissues, in which PtO2 was improved by NBO. NBO may be an effective adjunct therapy that can be performed readily after nonvascularized tissue grafting.
    Fat grafting is promising, but clinical outcomes are not always predictable. The mechanisms of tissue revascularization/regeneration, and tissue necrosis and subsequent absorption/fibrosis of the graft, are poorly understood. An autologous inguinal fat pad was transplanted under the scalp of mice, and detailed cellular events during the first 3 months were investigated with immunohistochemistry. Except for the most superficial surviving zone, death of all adipocytes was confirmed at 1 week. Perilipin-positive small new adipocytes appeared at 1 week and peaked in number at 4 weeks in the regenerating zone (the second zone). In the most central necrotizing zone, adipogenesis did not occur and many inflammatory cells were observed after 2 weeks. CD34/Ki67 proliferating adipose stem/progenitor cells were seen at 1 to 4 weeks, but the majority of proliferating cells were MAC2 monocytes/macrophages. Although CD206 M1 macrophages surrounded oil droplets for phagocytosis, CD206 M2 macrophages appeared in areas where adipocyte replacement failed and formed multiple layers for cicatrization of oil drop spaces. Adipogenesis was complete by 12 weeks, but stabilization of nonregenerated areas was still ongoing at that time. Lipid droplets derived from dead adipocytes were absorbed slowly and thus aided adipose remodeling by maintaining the space until adipocyte regeneration. Dynamic remodeling after fat grafting was confirmed. Adipocyte fate differed, depending on the microenvironment: intact survival, replacement with a new adipocyte, or replacement with cicatrization/oil cyst. This detailed understanding will help refine surgical grafting procedures and postoperative evaluation.
    Adipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells. We applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells. The column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples. Our results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.
    The heterogeneous stromal vascular fraction (SVF), containing adipose-derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell-processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side-effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell-based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice-level cell processing area. Copyright © 2012 John Wiley & Sons, Ltd.
    The cauliflower ear is a thickened ear deformity seen after repeated traumatic hematoma in the auricle, most commonly occurs during sporting activities, such as wrestling and rugby. This hypertrophy of the ear is considered to be fibrosis and neocartilage resulting from scarring and stimulation of mesenchymal cells in the perichondrium. However, we have operated an adult male with cauliflower ear resulting from rugby play, and the histopathological manifestation of the excised hyperplastic tissues showed rigid born formation, as well as fibro-neocartilaginous tissue formation. Our findings suggest the cauliflower ear condition may provide valuable insight into efforts to clarify the osteogenic potential in the perichondrium, which could help in developing a new treatment for bone/cartilage regeneration.
    Distinct B cell populations, designated regulatory B (Breg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified Breg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue Breg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support Breg cell function, and Breg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue Breg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that Breg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.
    The mammalian hair follicle epithelial component contains various lineages of keratinocytes as well as their progenitor/stem cells. To characterize the subpopulations contained within this component and assess their functional capacity, the development of a feasible method to isolate them is greatly needed. In this chapter we describe a standard method by which a small subset of human follicular keratinocytes can be isolated for subsequent analysis. Detailed methods for enzymatic digestion of fresh human scalp, flow cytometry, cultivation of sorted cells and a colony forming assay are described.
    The aim of this study was to review diagnostic/therapeutic strategies of umbilical endometriosis managed in our department and evaluate the effectiveness of these strategies. Medical records for patients with diagnosis of endometriosis managed from 1999 through 2011 in the University of Tokyo Hospital were retrospectively reviewed. Cases with diagnosis of umbilical endometriosis were identified. Clinical information of age, gravida, parity, histories of surgery and oral contraceptive (OC), management for the disease prior to the first visit, symptoms, patients' desire for pregnancy, diagnostic/therapeutic methods and prognosis were reviewed and summarized. During the period, 2530 patients with diagnosis of endometriosis were identified. Seven patients had diagnosis of umbilical endometriosis, giving an incidence of 0.29% of all endometriosis cases and 5.6% of extragenital endometriosis cases. A definitive diagnosis was made by histological examination following a biopsy (two cases) or a resection (three cases). A clinical diagnosis was made by empirical treatment with OC (one case) or dienogest (one case). With regard to therapy, three patients chose expectant management and did not require therapeutic intervention. Three patients began OC and symptoms were well controlled in all patients. One patient who wished to conceive chose a wide resection followed by umbilical reconstruction. She became pregnant afterwards and recurrence was not reported. There are various options of diagnostic/therapeutic strategies, such as empirical treatments and OC that can provide individualized management of umbilical endometriosis, congruent with the severity of patient symptoms, age and desire for pregnancy.
    Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. The cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, and the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than did the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.
    Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 μm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristic of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34+/CD31- and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies involving each vessel wall-resident stromal population.
    Deep tissue injuries (DTIs) can become significant problems because of their rapid deterioration into deep pressure ulcers. Presently, no animal model of DTI deterioration has been developed. By concentrating pressure and shear stress in deep tissues while minimising pressure and shear stress in the overlying skin, we produced an effective rat model of DTI deterioration. Two-dimensional finite element method (FEM) simulated the distribution of pressure and shear stress under several pressure-loading conditions. FEM showed that concentrated shear stress in deep tissue with minimum shear stress in the overlying skin could be created by using a prominence and a cushion, respectively. On the basis of the results of FEM analysis, we selected suitable conditions for testing the rat DTI deterioration model. The compressed area was macroscopically observed until day 13, and histopathologic analysis via haematoxylin and eosin (H&E) staining was performed on days 3, 7 and 13. H&E staining showed that the distribution of tissue damage was similar to the predicted FEM results. Deep ulceration and tissue damage extending from deep tissues to the overlying skin and surrounding tissues were observed in the DTI deterioration model, which are similar to the clinical manifestations of DTI deterioration. In conclusion, a representative DTI deterioration model was established by concentrating high shear stress in deep tissues while minimising shear stress in the overlying skin. This model will allow a better understanding of the mechanisms behind DTI deterioration and the development of preventative strategies.
    Background aims: Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Methods: Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. Results: In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. Conclusions: The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.
    Background: Hyaluronic acid (HA) fillers have become the most popular tool for wrinkle treatment and volumization, although HA is generally absorbed within 6-12 months and requires repeated treatments to maintain the effects. Methods: HA was injected onto the bone for volumization with a small 30-gauge needle to examine the long-lasting effects. Of the 63 Japanese patients with 97 treated sites followed up more than 12 months, 51 had HA injections for cosmetic purposes and twelve were treated for reconstructive volumization of facial deformity such as localized scleroderma and postsurgical bony deformity. Treated sites included the forehead, temple, nasal root, mentum, tear trough, and infraorbital sulcus. Results: After long-term follow up (12 to 93 months, mean= 21.6), persistent volumizing effects were observed in most patients. In fact, 86.6% of the treated sites showed >50% volume retention and 49.5% showed >75% retention. Magnetic resonance imaging (MRI) analyses revealed that the injected space was well maintained, capsulated, and filled with heterogeneous content. MRI quantitative T2 maps indicated that much of the injected HA was replaced with other materials. Together with clinical inspection, these findings suggest that onlay injection of HA on the bone induced formation of capsule, fibrosis, and/or calcification, which contributed to persistent volumization. Conclusions: Semi-permanent volumizing effects can be achieved by HA injection if the target area has an underlying bony floor. Periosteal stem cells may be activated by HA injection and may contribute to persistent volumizing effects. This treatment may be a much less invasive alternative to fat or bone grafting.
    The skin stem cells are located at two distinct locations, the basement of epidermis and the hair follicle bulge. The bulge stem cell is considered to be of higher hierarchy in terms of the stemness than the epidermal stem cell. Recently, hair follicle bulge cells can be successfully isolated using fluorescence-activated cell sorting (FACS) by labeling of a specific combination of surface antigens. In this chapter, we describe a standard method by which the bulge subset in the human follicle can be isolated for subsequent functional analyses.
    Because wound exudate includes secreted proteins that affect wound healing, its biochemical analysis is useful for objective assessment of chronic wounds. Wound blotting allows for collection of fresh exudate by attaching a nitrocellulose membrane onto the wound surface. To determine its applicability for several analysis methods and its executability in clinical wound assessment, this study comprised an animal experiment and clinical case reports. In the animal experiment, full-thickness wounds were created on the dorsal skin of mice, and exudate samples were collected daily by a conventional method and by wound blotting. Extremely small but adequate volumes of exudate were collected by wound blotting for subsequent analysis in the animal experiments. Immunostaining showed the concentration and distribution of tumor necrosis factor (TNF) α. The activity of alkaline phosphatase was visualized by reaction with chemiluminescent substrate. The TNF distribution analysis indicated three different patterns: wound edge distribution, wound bed distribution, and a mostly negative pattern in both the animal and clinical studies, suggesting association between the TNF distribution pattern and wound healing. Our results indicate that wound blotting is a convenient method for biochemical analysis of exudate and a candidate tool with which to predict the healing/deterioration of chronic ulcers.
    Breast reconstruction with tissue expanders and silicone breast implant has become a common and reliable method in breast reconstruction after mastectomy. But sometimes contour deformities occur such as step deformity around an implant and implant rippling over a mastectomy skin flap. Nowadays autologous fat graft has become a common technique for the management of soft tissue deformity, even in the case of revisional breast surgery. In this series we describe our method of Cell Assisted Lipotransfer (CAL), and our experience of CAL for the correction of acquired breast deformities. In CAL, a stromal vascular fraction (SVF) containing adipose derived stem/stromal cells (ASCs) is used in combination with lipoinjection. It is helpful in improving contour, volume, breast shape, symmetry and skin softness. This method is a less invasive, simple and effective tool for secondary breast reconstruction with silicone breast implant. It also may have applicability to cosmetic breast augmentation after explanation of problematic prostheses. Possible complications would be the formation of small oil cysts and microcalcification as a result of fat necrosis, but they are less problematic. With refinements of surgical technique and the novel technologies of stem cells, fat grafting will continue to evolve.
    Adipose tissue (AT) is composed of mature adipocytes and stromal vascular fraction (SVF) cells including adipose stem/stromal cells (ASC). We characterized hematopoietic cells residing in human non-obese AT by analyzing the SVF isolated from human lipoaspirates and peripheral blood (PB). Flow cytometry revealed that AT-resident hematopoietic cells consisted of AT-resident macrophages (ATM) or lymphocytes with a negligible number of granulocytes. AT-resident lymphocytes were composed of helper T cells and NK cells. Almost no B cells and few cytotoxic T cells were observed in non-obese AT. More than 90% of ATMs were M2 state CD206+ macrophages (CD45+/CD14+) that were located in the periendothelium or interstitial spaces between adipocytes. We also discovered a novel subpopulation of CD34+/CD206+ ATMs (11.1% of CD206+ATMs) that localized in the perivascular region. Microarray of non-cultured CD34+/CD206+ ATMs, CD34-/CD206+ ATMs, CD45-/CD31-/CD34+ ASCs, and PB-derived circulating monocytes revealed that CD34+/CD206+ ATMs shared characteristics with ASCs and circulating monocytes. Unlike CD34-/CD206+ ATMs, CD34+/CD206+ ATMs could grow in adherent culture and were capable of differentiating into multiple mesenchymal (adipogenic, osteogenic, and chondrogenic) lineages, similar to ASCs. CD34+/CD206+ ATMs grew rapidly and lost expression of CD45, CD14, and CD206 by passage 3, which resulted in a similar expression profile to ASCs. Thus, this novel ATM subpopulation (CD45+/CD14+/CD34+/CD206+) showed distinct biological properties from other ATMs and circulating monocytes/macrophages. The CD34+/CD206+ ATMs possessed characteristics similar to ASCs including adherence, localization, morphology, and mesenchymal multipotency. This AT-resident subpopulation may have migrated from the bone marrow and may be important to tissue maintenance and remolding.
    Aging is accelerated, at least in part, by pathological condition such as metabolic syndrome (MetS), and various molecular pathways such as oxidative stress are common mediators of aging and MetS. We previously developed the aging-like skin model by single ultraviolet (UV) irradiation on the MetS model mice. Recent studies revealed that mineralocorticoid receptor (MR) signaling plays a pivotal role for various tissue inflammation and damages in MetS. Although previous studies reported that MR is expressed in the skin and that overexpression of MR in the skin resulted in the skin atrophy, the physiological or pathological functions of MR in the skin are not fully elucidated. Here, we show the involvement of MR signaling in the aging-like skin changes in our own model. Elevations of oxidative stress and inflammation markers were observed in the MetS mice, and the UV evoked aging-like skin damages were attenuated by topical antioxidant. MR expression was higher in the MetS mouse skin, and notably, expression of its effecter gene Sgk1 was significantly upregulated in the aging-like skin in the UV irradiated MetS mice. Furthermore, topical application of MR antagonist spironolactone suppressed Sgk1 expression, oxidative stress, inflammation and the aging-like changes in the skin. The two week UV onto the non-MetS mice, the more usual photoaging model, resulted in the skin damages mostly equivalent to the MetS mice with single UV, but they were not associated with upregulation of MR signaling. Our studies suggested an unexpected role of MR signaling in the skin aging in MetS status. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
    Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.
    An animal model is needed to study the pathophysiology of wound infections; however, an animal model that is reproducible and clinically relevant has not previously been available. In addition, an animal model of wound colonization generated in a manner similar to the wound infection model would be useful. Here, we describe new animal models of the wound infection continuum for the characterization of essential host-pathogen relationships. We determined the conditions needed to establish rat models of stable wound colonization and infection, without the use of disturbing factors (e.g., foreign bodies or induction of diabetes mellitus). We found that the age of the rats, bacterial inoculum size, and wound location were important elements in generating reproducible, obvious, spreading wound infections. We inoculated approximately 6-month-old rats with 2.06 × 10(9) or 4.12 × 10(9) colony-forming units of Pseudomonas aeruginosa to generate the wound colonization and wound infection models, respectively. Wounds were made 2 cm cranial to the greater trochanter. These clinically relevant and highly reproducible animal models can be used to investigate the mechanisms of wound infection and monitor the effect of therapeutic agents in vivo.
    Dermal papilla cells (DPCs) interact with epithelial stem cells and induce hair folliculogenesis. Cell-based therapies using expanded DPCs for hair regeneration have been unsuccessful in humans. Two major challenges remain: first, expanded DPCs obtained from adult hair follicles have functional limitations; second, a clinically applicable method is needed for transplanting DPCs. This study aimed to identify an efficient, minimally invasive and economical DPC transplantation procedure for use in clinical settings. Five clinically applicable transplantation procedures were tested, termed the Pinhole, Laser, Slit, Non-vascularized sandwich (NVS) and Hemi-vascularized sandwich (HVS) methods. Labelled rat dermal papilla tissue was transplanted into rat sole skin, and hair follicle regeneration was evaluated histologically. Regenerated follicles and labelled DPCs were detected for all methods, although some follicles showed abnormal growth, i.e. a cystic or inverted appearance. The HVS method, pioneered here, resulted in significantly larger number of regenerated follicles that were more mature and regular than those observed using the other methods. Moreover, hair growth was detected after expanded adult-derived DPC transplantation using the HVS method. These results suggest that direct contact of epithelial and dermal components and better vascularization/oxygenation of the recipient site are critical for hair regeneration in cell-based therapies.
    : One issue in the adoption of autologous fat transfer to the breast is concern over mammographic changes that may obscure cancer detection. The authors compared mammographic changes following fat grafting to the breast with changes seen after breast reduction. : Twenty-seven women who had normal preoperative mammograms were treated with fat grafting to the breast, including admixing of autologous adipose stem cells with the fat graft, for cosmetic augmentation. Repeated mammograms were performed 12 months after surgery. As a control group, postsurgical mammograms from 23 reduction mammaplasty patients were compared. Eight academic breast imaging radiologists reviewed each mammogram in a blinded fashion. Outcomes analysis accounting for individual radiologist's tendencies was performed using generalized estimating equations. : The average volume of fat injected per patient was 526.5 cc. Fifty mammograms (27 lipotransfer, 23 breast reduction) were assessed. Differences in interpretation among individual radiologists were consistently observed (p < 0.10). Differences in abnormality rates were nonsignificant for oil cysts, benign calcifications, and calcifications warranting biopsy. Scarring (p < 0.001) and masses requiring biopsy (p < 0.001) were more common in the reduction cohort. Breast Imaging Reporting and Data System scores were higher after breast reduction (p < 0.001). Significant differences in the recommended follow-up time were also seen (p < 0.01). : Compared with reduction mammaplasty, a widely accepted procedure, fat grafting to the breast produces fewer radiographic abnormalities with a more favorable Breast Imaging Reporting and Data System score and less aggressive follow-up recommendations by breast radiologists. : Therapeutic, III.
    Clinical outcomes following fat grafting are variable and technique dependent, and it is unknown how the graft is revascularized. The authors recently observed that living and dead adipocytes can be differentiated not with hematoxylin and eosin staining but with immunohistochemistry for perilipin. The viability of cellular components (adipocytes, adipose stem/stromal/progenitor cells, vascular endothelial cells, and hematopoietic cells) in human adipose tissue was evaluated using (1) stored lipoaspirates, (2) cultured cells, and (3) organ-cultured adipose tissue. In addition, the groin fat pad (150 to 200 mg) in mice was transplanted under the scalp, and the graft was stained at 0, 1, 2, 3, 5, 7, or 14 days. In vitro studies revealed that adipocytes are most susceptible to death under ischemic conditions, although adipose-derived stromal cells can remain viable for 3 days. The in vivo study indicated that most adipocytes in the graft began to die on day 1, and only some of the adipocytes located within 300 μm of the tissue edge survived. The number of proliferating cells increased from day 3, and an increase in viable adipocyte area was detected from day 7, suggesting that repair/regeneration of the dead tissue had begun. The authors show convincing evidence of very dynamic remodeling of adipose tissue after nonvascularized grafting. The authors observed three zones from the periphery to the center of the graft: the surviving area (adipocytes survived), the regenerating area (adipocytes died, adipose-derived stromal cells survived, and dead adipocytes were replaced with new ones), and the necrotic area (both adipocytes and adipose-derived stromal cells died).
    Recent progress in microsurgical techniques has improved the results of finger reconstruction using free partial toe transfers and vascularized nail flaps especially in fingertips and nail reconstruction with minimal invasion. However, surgical techniques such as the method of harvesting the toe skin flaps, design of skin flaps, whether or not thin peripheral bone is included, the length of the vessel pedicle, the level and number of vessel anastomosis all make little differences in outcome for the reconstructed fingers. Methods: Selection for flaps is different in each case: Vascularized onychocutaneous flap for nail loss, trimmed great or second toe tip transfer for claw nail deformity of thumb, thin osteo-onychocutaneous flap from great toe for thumb distal phalanx loss, total second toe distal phalanx transfers for distal phalanx loss excluding thumb. Conclusions: Esthetic fingertip reconstructions including nail are possible with custom-made toe tip transfers.
    The purpose of this study was to test the hypothesis that obese diabetic mice exhibit marked skin fragility, which is caused by increased oxidative stress and increased matrix metalloproteinase (MMP) gene expression in the subcutaneous adipose tissue. Scanning electron microscopy of skin samples from Tsumura-Suzuki obese diabetic (TSOD) mice revealed thinner collagen bundles, and decreased density and convolution of the collagen fibres. Furthermore, skin tensile strength measurements confirmed that the dorsal skin of TSOD mice was more fragile to tensile force than that of non-obese mice. The mRNA expressions of heme oxygenase 1 (Hmox1), a marker of oxidative stress, Mmp2 and Mmp14 were increased in the adipose tissue of TSOD mice. Antioxidant experiments were subsequently performed to determine whether the changes in collagen fibres and skin fragility were caused by oxidative stress. Strikingly, oral administration of the antioxidant dl-α-tocopherol acetate (vitamin E) decreased Hmox1, Mmp2 and Mmp14 mRNA expressions, and improved the skin tensile strength and structure of collagen fibres in TSOD mice. These findings suggest that the skin fragility in TSOD mice is associated with dermal collagen damage and weakened tensile strength, and that oxidative stress and MMP overexpression in the subcutaneous adipose tissue may, at least in part, affect dermal fragility via a paracrine pathway. These observations may contribute to novel clinical interventions, such as dietary supplementation with antioxidants or application of skin cream containing antioxidants, which may overcome skin fragility in obese patients with diabetes.
    Many features of adipose stem/progenitor cells, including their physiological functions and localization, have been clarified in the past decade. Adipose tissue turns over very slowly, with perivascular progenitor cells differentiating into new adipocytes to replace dead adipocytes. A number of clinical trials using freshly isolated or cultured adipose-derived stromal cells containing adipose progenitor/stem cells are ongoing. Therapeutic use of adipose stem/progenitor cells has been shown to promote angiogenesis and adipose tissue regeneration. Identification of adipocyte-releasing factors upon apoptosis/necrosis would be a breakthrough and could lead to the next stage for adipose tissue regeneration. Activation of precursors in perichondrium and periosteum shows a dramatic neogenesis by simple injection and is an ideal example of in situ tissue engineering. The 'hit and catch' strategy using a mobilizer of bone-marrow stem/progenitor cells (hit) and attractants to lead the cells to proper homing into the target tissue (catch) may be the future of stem cell manipulation. Careful design of the microenvironment, cell delivery protocol to avoid unexpected behavior and induce maximal potential, and selection of target diseases, will be critical to the success of clinical applications of adipose-derived stromal cells.
    Wound infection is a form of host damage resulting from an imbalance in pathogen virulence and the host immune response. However, at present, diagnosis is based solely on bacterial numbers or inflammatory signs and is therefore not precise. Thus, infection diagnosis requires indicators of both of these factors. We focused on wound fluid because it includes both bacteria and host cells. The purpose of this study was to establish biomarkers that reflect both bacterial and host factors using the reverse transcription-polymerase chain reaction method on the centrifugal precipitation of wound fluids (wound fluid RT-PCR). We created full thickness wounds in animal models of the three groups: control, colonization and infection, which were conditioned by administration of different concentrations of Pseudomonas aeruginosa dispersion. Messenger RNA expression in bacteria and host cells was analysed. Expression of bacterial housekeeping genes was detected in the samples in the colonization and infection groups. Expression of host housekeeping genes was detected in all samples from the three groups. Expression of toxA, encoding the virulence factor exotoxin A, was detected in 90% of samples in the infection group only. Expression of Foxp3, encoding the transcription factor forkhead box P3, was detected in 100% of samples only in the colonization group. These results revealed that wound fluid RT-PCR analysis reflected both bacterial virulence and the host immune status, and we determined the combination of novel biomarkers that can discriminate these three groups. We anticipate that wound fluid RT-PCR could be applied in the future to diagnose wound infection.
    Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230-270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.
    Although hypertrophic scars (HTSs) and keloids are challenging problems, their pathogenesis is not well understood, making therapy difficult. We showed that matrix metalloproteinase (MMP)-1 expression was downregulated in HTS compared with normal skin from the same patients, whereas type 1 and 3 collagen and transforming growth factor-β (TGF-β) were upregulated. These differences, however, were not seen in cultured fibroblasts, suggesting the involvement of microenvironmental factors in the pathogenesis of HTS. Fibroblast growth factor-2 (FGF-2) highly upregulated the expression of MMP-1 and hepatocyte growth factor (HGF) in both HTS-derived and control fibroblasts; the upregulation was reversed by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors. An animal study using human HTS tissue implanted into nude mice indicated that controlled-release FGF-2 resulted in significantly less weight and decreased hydroxyproline content in HTS. Degradation of collagen fibers in FGF-2-treated HTS was also confirmed histologically. Western blotting showed that FGF-2-treated HTS expressed significantly higher MMP-1 protein than control. Decreased MMP-1 expression may be an important transcriptional change in HTS, and its reversal as well as upregulation of HGF by FGF-2 could be a new therapeutic approach for HTS.
    Lipoinjection is a promising treatment, but is currently limited by unpredictable outcomes and a low rate of graft survival due to partial necrosis. To address these problems we developed a novel strategy called cell-assisted lipotransfer (CAL) in which autologous adipose-derived stem (stromal) cell (ASC) supplementation is used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is isolated from half of an aspirated fat sample and is recombined with the remaining half of the aspirated fat sample. This process converts relatively progenitor-poor aspirated fat to progenitor-rich fat. Our experience with the CAL technique showed that by transplanting the ASC-enriched fat tissue postoperative atrophy of transplanted fat grafts was minimal and satisfactory clinical results were generally achieved without any major complications, suggesting that ASC supplementation is both effective and safe. Further studies with longer follow-up are necessary to establish the value of this technique. Continued improvements in the technique could make autologous tissue transfer the first choice for breast augmentation in the future.
    Obesity is recognized as a risk factor for delayed cutaneous wound healing. The authors hypothesized that the secretion of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) from subcutaneous adipose tissue correlates with disorder of the healing process in obese subjects. Findings from previous studies on the expression of MMPs and TIMPs in obese adipose tissue are inconsistent. Since these conflicting results could be due to the effect of several intrinsic factors, the authors conducted a simple in vitro experiment to clarify the change in profile of MMPs and TIMPs in excessively matured adipocytes. The authors cultured the induced adipocytes under conditions of high or low glucose and with or without insulin supplementation. Oil red O staining and its dye extraction assay revealed excessive lipid accumulation in high glucose and insulin-supplemented adipocytes. Additionally, there was altered expression of adipokines, similar to the change in adipose tissue in obese subjects. Under these conditions, the expression/activity of MMP8 was promoted and the expression of MMP3 and TIMP3 was inhibited. Further studies to determine the effect of other obesity-related factors, such as insulin resistance, on MMPs and TIMPs are required.
    Skin maceration is recognized as a risk factor for the development of certain skin lesions. In health care settings, incontinence-associated skin maceration is highly prevalent in the elderly. However, the effect of senescence on maceration has not been fully elucidated. To reveal the enhancement of the maceration-induced ultrastructural alteration and barrier function of the epidermis by aging. Skin maceration was reproduced by exposure to agarose gel in human and rat. The ultrastructural alterations in human and rat tissue were observed by transmission electron microscopy. The skin barrier function was evaluated by noninvasive methods in human, and by the transdermal penetration of small- and large-fluorescent molecules in rat. In order to reveal the effect of aging on the skin maceration, we compared these parameters between young and aged rats. In macerated skin, we observed expansion of the interstices of the stratum corneum, spinosum, and basale of the epidermis; disruption of the intercellular lipid structure in the stratum corneum; a decreased number of cell processes in the stratum spinosum and basale. The transdermal penetration test in the rat using two types of fluorescein indicated that maceration disrupted skin barrier function. Furthermore, senescence-enhanced ultrastructural and functional alterations were revealed in the rodent studies. This study demonstrates that aging enhances skin maceration. Considering that maceration is a risk factor for the skin damage, the development of technology to promote skin barrier recovery after maceration in the elderly is warranted.
    We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.
    Based on the analysis of exudates from injured adipose tissue, we prepared a mixture containing the injury-associated growth factors at the same proportion as the exudates, named adipose injury cocktail (AIC). We hypothesized that AIC induces a series of regenerating and angiogenic processes without actual wounding. The purpose of this study is to elucidate the therapeutic potentials of AIC. AIC preferentially activated adipose-derived stem/progenitor/stromal cells (ASCs) to proliferate, migrate, and form networks compared with vascular endothelial cells, whereas vascular endothelial growth factor did not induce mitogenesis or chemotaxis in human ASCs. Each component growth factor of AIC was differently responsible for the ASC activation. AIC-treated ASCs tended to differentiate into adipocytes or vessel-constituting cells rather than into other cell types. In ischemic adipose tissues of mice, induced by either a surgical intervention or diabetes, AIC administration enhanced proliferation, especially of CD31(-)/CD34(+) ASCs, and mitigated tissue hypoxia by increasing capillary density and reducing fibrogenesis. These results suggest that AIC may have therapeutic potentials for various ischemic/hypoxic conditions by inducing adipose remodeling and neovascularization through activation of ASCs and other cells. Treatment with AIC has many advantages over cell-based therapies regarding morbidity, cost, and physical risks and may be used as an alternative therapy for improving tissue oxygen.
    Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90  ml and time ≤2  h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9)  cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.
    Postinflammatory hyperpigmentation (PIH) is the most common skin complication in Asians after invasive cosmetic treatments. To determine whether oral tranexamic acid (TA) reduces the incidence of PIH after Q-switched ruby laser (QSRL) treatment. Thirty-two Japanese women underwent QSRL treatment for senile lentigines on the face. They were randomly divided into two groups that did (n=15) and did not (n=17) receive oral TA treatment (750 mg/d) for the first 4 weeks after QSRL treatment. Nineteen participants had melasma-like maculae at baseline. Clinical and colorimetric assessments were performed at baseline and 2 and 4 weeks later. Pigmentation was effectively treated using QSRL at 2 weeks, but PIH was frequently seen at 4 weeks. There was no significant difference in the incidence of PIH between participants who received oral TA and those who did not. The presence of melasma did not influence the effectiveness of the treatment. Although oral TA has been reported to have depigmentation effects, it may not be effective for preventing PIH after QSRL. Considering the dosage and duration of treatment, an optimal protocol may be needed to induce the efficacy of this treatment to achieve the PIH-preventing effect of oral TA.
    Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes. Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs. Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes. Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes.
    Thread lifting has become popular as a minimally-invasive suspension procedure, but there is little basic and clinical evidence in the literature on the long-term effects. The authors investigate the effects of two types of lifting threads in a rat model over the course of seven months. The dorsal skin of 18 Wistar rats was implanted with a 20-mm fragment of one of three types of thread: nonabsorbable monofilament cog, pure gold (24 karat) with no cog, and pure gold-coated cog. Six rats were in each group. Tissue samples were harvested and histologically evaluated at one, three, and seven months. Histological assessment indicated (1) acute tissue reactions to the regular cog thread involving myofibroblasts and (2) delayed tissue reactions to the pure gold thread involving giant cells. The gold-coated cog thread showed a combination of the histological reactions associated with the cog thread and the pure gold thread, including faint early reactions, strong delayed reactions, and long-lasting capsule formation. Notably, the gold coating gradually came loose from the thread surface, suggesting that the release of tiny gold particles may promote longer-lasting tissue reactions. The combination of cog structure and pure gold coating was evaluated for the first time in this study and results suggest that the gold-coated cog thread has clinical potential.
    Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3Kγ) in inflammatory states, little is known about the role of PI3Kγ in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3Kγ in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110γ (Pik3cg(-/-)), the catalytic subunit of PI3Kγ, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg(-/-) mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3Kγ also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3Kγ plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3Kγ can be a therapeutic target for type 2 diabetes.
    Although double eyelid plasty, levator aponeurotic surgery, and epicanthoplasty are well-accepted cosmetic treatments for Asian eyes, some patients are incompletely satisfied with the outcomes and request further surgery. Although lower eyelid descent is generally recognized as a symptom of aging or a complication after blepharoplasty, the authors propose a perceptional change: a lowering the lower eyelid procedure to vertically enlarge the palpebral aperture in selected Asian patients. A total of 125 Japanese patients underwent the lowering the lower eyelid procedure between 2005 and 2009. The main indications were patients with vertically narrow palpebral aperture or an up-slanting appearance. The lowering the lower eyelid procedure is performed by a combination of the removal of approximately 4 to 6 mm of the subciliary skin (usually the lateral one-third to two-thirds of the lower eyelids) and static shortening of the lower eyelid retractors (posterior lamella) through a transconjunctival approach. The middle lamella was not touched during the procedure. The up-slanting lower eyelid margin was lowered and the lateral part of the palpebral aperture was enlarged by the procedure in all cases. Cosmetic outcomes were encouraging and satisfying to most patients. Three complications occurred (2.4 percent): lagophthalmos in one patient (0.8 percent) and entropion in two patients (1.6 percent). These minor complications resolved within 1 month. Eight revision operations were required for undercorrection. The lowering the lower eyelid procedure offers Asian patients desiring large oval eyes a novel surgical option. The procedure proved to be a reliable and consistent technique that provided satisfactory results in carefully selected patients.
    Following various types of plastic surgery, such as adipose grafting and flap elevation, adipose tissue undergoes ischemia, leading to hypoxia and nutrient depletion. However, few studies have examined ischemic and/or hypoxic changes in adipose tissue. The authors established surgically induced ischemia models by severing blood vessels supplying the inguinal fat pads in mice. The partial pressure of oxygen in adipose tissue was measured with an oxygen monitor, and ischemic changes were analyzed by whole-mount staining, immunohistochemistry, flow cytometry, and Western blotting. The authors also examined cell survival under a hypoxic condition in vitro. Models for three degrees (mild, intermediate, and severe) of ischemia showed approximately 75, 55, and 20 percent of the partial pressure of oxygen level in normal adipose tissue (50.5±1.3 mm Hg), respectively. Adipose tissue atrophy with substantial fibrosis on day 28 was seen, depending on the severity of ischemia. Intermediate and severe ischemia induced elevated expression of hypoxia-inducible factor 1α and fibroblast growth factor 2 on day 1 and degenerative changes (i.e., apoptosis, necrosis, and macrophage infiltration and phagocytosis) in adipose tissue. Dead cells included adipocytes, vascular endothelial cells, and blood-derived cells, but not adipose-derived stem/progenitor cells. Subsequent to degenerative changes, regenerative changes were seen, including angiogenesis, adipogenesis, and proliferation of cells (adipose-derived stem/progenitor cells, vascular endothelial cells, and blood cells). The authors found that, in vitro, the experimentally differentiated adipocytes underwent apoptosis and/or necrosis under severe hypoxia, but adipose-derived stem/progenitor cells remained viable. Severe ischemia/hypoxia induces degenerative changes in adipose tissue and subsequent adaptive tissue remodeling. Adipocytes die easily under ischemic conditions, whereas adipose-derived stem/progenitor cells are activated and contribute to adipose tissue repair.
    Five familial cases exhibited ephelides-like multiple lentigines, and we examined three of them, a mother and two sons. All three patients presented with small dark-brown maculae on the face and neck and electrocardiographic abnormalities. These findings sufficed to fulfill the criteria for LEOPARD syndrome (multiple lentigines syndrome), although they lacked five of seven major clinical features. However, the family members presented with a webbed neck and pectus excavatum, which are more frequently seen in Turner or Noonan syndrome. Histological examination of the lentigines revealed slightly elongated rete ridges, a hyperpigmented basal layer, and melanophages in the papillary dermis. Direct sequencing of the patients' genomic DNA revealed that all three had a consistent missense mutation [c.1403C > T (p.T468M)] in the PTPN11 gene, confirming LEOPARD syndrome with an atypical phenotype. It was suggested that LEOPARD syndrome shows a diverse phenotype but its diagnosis can be verified by mutation analysis.
    Lipoinjection is a promising treatment, but is currently limited by unpredictable outcomes and a low rate of graft survival due to partial necrosis. To address these problems we developed a novel strategy called Cell-Assisted Lipotransfer (CAL) in which autologous adipose-derived stem (stromal) cell (ASC) supplementation is used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is isolated from half of an aspirated fat sample and is recombined with the remaining half of the aspirated fat sample. This process converts relatively progenitor-poor aspirated fat to progenitor-rich fat. Our experience with the CAL technique showed that by transplanting the ASC-enriched fat tissue post-operative atrophy of transplanted fat grafts was minimal and satisfactory clinical results were generally achieved without any major complications, suggesting that ASC supplementation is both effective and safe. Further studies with longer follow up are necessary to establish the value of this technique. Continued improvements in the technique could make autologous tissue transfer the first choice for breast augmentation in the future.
    Various kinds of tissue expansion have been performed clinically with internal devices, but external expansion has not been previously investigated. We applied continuous external force on skin tissue in a mouse model. Four weeks of external suspension caused enlargement of the subcutaneous tissue, particularly adipose tissue, although the enlargement was reversible. We found that the enlarged tissue volume could be adequately sustained with controlled release of basic fibroblast growth factor (bFGF), administered at the time the device was removed. Ki67+ proliferating cells, perilipin+ small adipocytes, lectin+ capillaries, and glycerol-3-phosphate dehydrogenase activity in the tissue increased during the expansion process, indicating dynamic adipose remodeling with adipogenesis and angiogenesis. Histological analyses revealed that vessels had elongated in the direction of the external force. Adipose-resident progenitor cells (adipose-derived stem/stromal cells) were the primary proliferating cell population involved in the remodeling process, particularly in the superficial layer. Treatment with bFGF did not enhance the small adipocyte number, but promoted angiogenesis; this mechanism may contribute to the preservation of enlarged tissue. Our results suggested that external tissue suspension induced adipose tissue enlargement by activating resident progenitor cells and that this external suspension approach, combined with controlled release of bFGF, has therapeutic potential for soft tissue engineering.
    Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor-supplemented adipose tissue (cell-assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 +/- 50 mL (Right; mean +/- SD) and 143 +/- 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants.
    Background: Adipose tissue is an easily accessible tissue for use as a soft-tissue filler and source of adult multipotent cells, called adipose-derived stem/stromal/progenitor cells. However, many aspects of its anatomy remain unclear because of the fragile structure of adipocytes and adipose tissue. Methods: Aspirated (n = 15) or intact (n = 9) subcutaneous adipose tissue samples were evaluated by (1) whole-mount histology with triple-fluorescence staining for three-dimensional visualization of living adipose tissue, (2) glycerol-3-phosphate dehydrogenase assay, (3) multicolor flow cytometry (CD34, CD31, and CD45), and (4) adherent cell culture of stromal vascular fraction cells for viable adipose-derived stromal cell yield. Results: Whole-mount histology revealed the presence of a capillary network running alongside adipocytes, which was partly disrupted in aspirated adipose tissues. Aspirated adipose tissue also lacked large vasculature structures and showed significantly larger numbers of small lipid droplets (ruptured adipocytes) (p = 0.00016) and dead cells (p = 0.0038) compared with excised adipose tissue. Adipocyte number was less than 20 percent of total cellularity, and vasculature-associated cells, including endothelial cells and adipose-derived stromal cells, constituted over one-half of total cells in both intact and aspirated adipose tissue. Glycerol-3-phosphate dehydrogenase assay suggested that greater than 30 percent and 5 percent of adipocytes were ruptured in aspirated and excised adipose tissue, respectively (p = 0.032). Multicolor flow cytometric analysis indicated a much higher percentage of blood-derived cells (CD45+) contaminated in aspirated adipose tissue (p = 0.0038), and adipose-derived stromal cell yield in aspirated adipose tissue was approximately one-half that in excised tissue (p = 0.011). Conclusion: The authors’ results indicate the differential structure and cellular composition of the two tissues, and significant tissue damage and progenitor yield deficiency in aspirated adipose tissue.
    Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals. In particular, obese adipose tissue shows features characteristic of active local inflammation. At present, however, little is known about the sequence of events that comprises the inflammatory cascade or the mechanism by which inflammation develops. We found that large numbers of CD8(+) effector T cells infiltrated obese epididymal adipose tissue in mice fed a high-fat diet, whereas the numbers of CD4(+) helper and regulatory T cells were diminished. The infiltration by CD8(+) T cells preceded the accumulation of macrophages, and immunological and genetic depletion of CD8(+) T cells lowered macrophage infiltration and adipose tissue inflammation and ameliorated systemic insulin resistance. Conversely, adoptive transfer of CD8(+) T cells to CD8-deficient mice aggravated adipose inflammation. Coculture and other in vitro experiments revealed a vicious cycle of interactions between CD8(+) T cells, macrophages and adipose tissue. Our findings suggest that obese adipose tissue activates CD8(+) T cells, which, in turn, promote the recruitment and activation of macrophages in this tissue. These results support the notion that CD8(+) T cells have an essential role in the initiation and propagation of adipose inflammation.
    The concept of deep tissue injury under intact skin helps us understand the pathogenesis of pressure ulcers, but the best method for detecting and evaluating deep tissue injury remains to be established. Intermediate-frequency (10-MHz) ultrasonography was performed to evaluate deep tissue injury. The authors analyzed 12 patients (nine male patients and three female patients aged 16 to 92 years) who showed deep tissue injury-related abnormal findings on ultrasonography at the first examination and were followed up until the pressure ulcer reached a final stage. The stage of ulcer worsened in six of 12 cases compared with baseline, and healed in the remaining six patients. The authors recognized four types of abnormal signs unique to deep tissue damage in ultrasonography: unclear layered structure, hypoechoic lesion, discontinuous fascia, and heterogeneous hypoechoic area. Unclear layered structure, hypoechoic lesion, discontinuous fascia, and heterogeneous hypoechoic area were detected at the first examination in 12, 10, seven, and five patients, respectively. Unclear layered structure and hypoechoic lesion were more commonly seen in pressure ulcers in deep tissue injury than the other features, but the follow-up study suggested that discontinuous fascia and heterogeneous hypoechoic area are more reliable predictors of future progression of pressure ulcers. The use of intermediate-frequency ultrasound reliably identified deep tissue injury and was believed to contribute to prevention and treatment of pressure-related ulcers. The results suggest that specific ultrasonographic characteristics may predict which pressure ulcers will progress.
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