Human Adipose-Derived Endothelial Progenitor Cells Accelerate Epithelialization of Radiation Ulcers in Nude Mice.

BACKGROUND: Cotransplantation of adipose-derived stem cells (ASCs) and endothelial progenitor cells has shown superior angiogenic effects compared with ASCs alone in recent animal studies. However, endothelial progenitor cells could only be collected from blood vessels or bone marrow. Thus, the authors have established a method for purifying adipose-derived endothelial progenitor cells (AEPCs). The authors hypothesized that AEPCs would enhance the therapeutic effect of ASCs on radiation ulcers. METHODS: Seven-week-old male nude mice (BALB/cAJcl-nu/nu) were irradiated on the dorsal skin (total 40 Gy); 12 weeks later, 6-mm-diameter wounds were created. The mice were then treated with subcutaneous injection of human ASCs [1 × 10 5 ( n = 4)], human AEPCs [2 × 10 5 or 5 × 10 5 ( n = 5)], combinations of those [ASCs 1 × 10 5 plus AEPCs 2 × 10 5 ( n = 4) or 5 × 10 5 ( n = 5)], or only vehicle ( n = 7). The nonirradiated group was also prepared as a control ( n = 6). The days required for macroscopic epithelialization was compared, and immunostaining for human-derived cells and vascular endothelial cells was performed at day 28. RESULTS: AEPC-ASC combination-treated groups healed faster than the ASC-treated group (14 ± 0 days versus 17 ± 2 days; P < 0.01). Engraftment of the injected cells could not be confirmed. Only the nonirradiated mice had significantly higher vascular density (0.988 ± 0.183 × 10 -5 /µm -2 versus 0.474 ± 0.092 × 10 -5 /µm 2 ; P = 0.02). CONCLUSION: The results suggested therapeutic potentials of AEPCs and an enhanced effect of combination with ASCs. This study is a xenogenic transplantation model, and further validation in an autologous transplantation model is needed. CLINICAL RELEVANCE STATEMENT: Human AEPCs and their combination with ASCs accelerated epithelialization of radiation ulcers in nude mice. The authors suggest that administration of humoral factors secreted from AEPCs (eg, treatment with culture-conditioned media) could be used for the same purpose.

Irradiation affects adipose-derived stem cells and wound healing depending on radiation dose and frequency.

BACKGROUND: Radiation therapies are often associated with permanent devitalization in the surrounding tissue. We hypothesized that stem cells are damaged depending on each irradiation dose and frequency of fractionated radiotherapies, which results in impaired tissue function including wound healing capacity. METHODS: To test the hypothesis, susceptibility of human adipose-derived stem cells (ASCs) to a single irradiation (0-10 Gy) was assessed in vitro. In vivo chronic radiation effects were also assessed on the mouse dorsal skin (N=4-5) for 6 months after a total of 40 Gy irradiation (0 Gy as control) using one of three fractionated protocols (2 Gy daily for 20 days, 10 Gy weekly for 4 weeks, or 10 Gy monthly for 4 months). Oxygen partial pressure, oxygen saturation of hemoglobin, and dorsal skin viscoelasticity were periodically measured, and wound healing and tissue immunohistology were compared at 6 months. RESULTS: A single irradiation of cultured human ASCs resulted in a dose-dependent increase in cell death up to 2 Gy but with no further increases between 2 and 10 Gy. Most of the apoptotic ASCs were in the proliferation phase. Among the three in vivo irradiation protocols, the 2 Gy×20 group had the most severe chronic tissue damage (i.e., skin dysfunction, subcutaneous atrophy, and depletion of CD34+ stem cells) 6 months after the irradiation. Wound healing was also impaired most significantly in the 2 Gy×20 group. CONCLUSIONS: These results have important clinical implications for surgeons and radiotherapists such as the timing of surgical interventions and the optimization of fractionation protocols.Clinical Relevance Statement: Irradiation damages stem cells depending on the radiation dose and frequency. Using the ultimately optimized protocol, we can minimize the long-term functional deficits of radiated tissue without losing anti-cancer efficacy of radiation therapy.

Genetic and cytometric analyses of subcutaneous adipose tissue in patients with hemophilia and HIV-associated lipodystrophy.

BACKGROUND: The authors recently performed plastic surgeries for a small number of patients with hemophilia, HIV infection, and morphologic evidence of lipodystrophy. Because the pathophysiological mechanism of HIV-associated lipodystrophy remains to be elucidated, we analyzed subcutaneous adipose tissues from the patients. METHODS: All six patients had previously been treated with older nucleoside analogue reverse-transcriptase inhibitors (NRTIs; stavudine, didanosine or zidovudine). Abdominal and inguinal subcutaneous fat samples were obtained from the HIV+ patients with hemophilia and HIV- healthy volunteers (n = 6 per group), and analyzed via DNA microarray, real-time PCR, flow cytometry and immunohistochemistry. RESULTS: The time from initial NRTI treatment to collecting samples were 21.7 years in average. Cytometric analysis revealed infiltration of inflammatory M1 macrophages into HIV-infected adipose tissue and depletion of adipose-derived stem cells, possibly due to exhaustion following sustained adipocyte death. Genetic analysis revealed that adipose tissue from HIV+ group had increased immune activation, mitochondrial toxicity, chronic inflammation, progressive fibrosis and adipocyte dysfunction (e.g. insulin resistance, inhibited adipocyte differentiation and accelerated apoptosis). Of note, both triglyceride synthesis and lipolysis were inhibited in adipose tissue from patients with HIV. CONCLUSIONS: Our findings provide important insights into the pathogenesis of HIV-associated lipodystrophy, suggesting that fat redistribution may critically depend on adipocytes' sensitivity to drug-induced mitochondrial toxicity, which may lead either to atrophy or metabolic complications.

Purification and characterization of human adipose-resident microvascular endothelial progenitor cells.

Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs). However, no standardized method has been established for the isolation and purification of adipose-derived EPCs (AEPCs). The aim of this study was to establish a method for the isolation and purification of AEPCs. The stromal vascular fraction (SVF) was extracted from human lipoaspirates, and the CD45-CD31+ fraction of the SVF was collected by magnetic-activated cell sorting (MACS). The CD45-CD31+ fraction was cultured for 4.5 days, followed by a second MACS separation to collect the CD31+ fraction. Purified AEPCs were expanded without being overwhelmed by proliferating ASCs, indicating that a high level (> 95%) of AEPC purification is a key factor for their successful isolation and expansion. AEPCs exhibited typical endothelial markers, including CD31, von Willebrand factor, and the isolectin-B4 binding capacity. AEPCs formed colonies, comparable to cultured human umbilical vein endothelial cells (HUVECs). Both AEPCs and HUVECs formed capillary-like networks in the tube formation assay, with no significant difference in network lengths. We are the first to establish a purification and expansion method to isolate these cells. Because adipose tissue is a clinically accessible and abundant tissue, AEPCs may have potential advantages as a therapeutic tool for regenerative medicine.

Selective Proliferation of Highly Functional Adipose-Derived Stem Cells in Microgravity Culture with Stirred Microspheres.

Therapeutic effects of adult stem-cell transplantations are limited by poor cell-retention in target organs, and a reduced potential for optimal cell differentiation compared to embryonic stem cells. However, contemporary studies have indicated heterogeneity within adult stem-cell pools, and a novel culturing technique may address these limitations by selecting those for cell proliferation which are highly functional. Here, we report the preservation of stemness in human adipose-derived stem cells (hASCs) by using microgravity conditions combined with microspheres in a stirred suspension. The cells were bound to microspheres (100-300 µm) and cultured using a wave-stirring shaker. One-week cultures using polystyrene and collagen microspheres increased the proportions of SSEA-3(+) hASCs 4.4- and 4.3-fold (2.7- and 2.9-fold increases in their numbers), respectively, compared to normal culture conditions. These cultured hASCs expressed higher levels of pluripotent markers (OCT4, SOX2, NANOG, MYC, and KLF), and had improved abilities for proliferation, colony formation, network formation, and multiple-mesenchymal differentiation. We believe that this novel culturing method may further enhance regenerative therapies using hASCs.

Sodium Alginate as a Potential Therapeutic Filler: An In Vivo Study in Rats.

Filler injection demand is increasing worldwide, but no ideal filler with safety and longevity currently exists. Sodium alginate (SA) is the sodium salt of alginic acid, which is a polymeric polysaccharide obtained by linear polymerization of two types of uronic acid, d-mannuronic acid (M) and l-guluronic acid (G). This study aimed to evaluate the therapeutic value of SA. Nine SA types with different M/G ratios and viscosities were tested and compared with a commercially available sodium hyaluronate (SH) filler. Three injection modes (onto the periosteum, intradermally, or subcutaneously) were used in six rats for each substance, and the animals were sacrificed at 4 or 24 weeks. Changes in the diameter and volume were measured macroscopically and by computed tomography, and histopathological evaluations were performed. SA with a low M/G ratio generally maintained skin uplift. The bulge gradually decreased over time but slightly increased at 4 weeks in some samples. No capsule formation was observed around SA. However, granulomatous reactions, including macrophage recruitment, were observed 4 weeks after SA implantation, although fewer macrophages and granulomatous reactions were observed at 24 weeks. The long-term volumizing effects and degree of granulomatous reactions differed depending on the M/G ratio and viscosity. By contrast, SH showed capsule formation but with minimal granulomatous reactions. The beneficial and adverse effects of SA as a filler differed according to the viscosity or M/G ratio, suggesting a better long-term volumizing effect than SH with relatively low immunogenicity.

Hair Regeneration Potential of Human Dermal Sheath Cells Cultured Under Physiological Oxygen.

We investigated the effect of oxygen tension on the proliferation and hair-inductive capacity of human dermal papilla cells (DPCs) and dermal sheath cells (DSCs). DPCs and DSCs were separately obtained from human hair follicles and each cultured under atmospheric/hyperoxic (20% O2), physiological/normoxic (6% O2), or hypoxic (1% O2) conditions. Proliferation of DPCs and DSCs was highest under normoxia. Compared with hyperoxia, hypoxia inhibited proliferation of DPCs, but enhanced that of DSCs. In DPCs, hypoxia downregulated the expression of hair-inductive capacity-related genes, including BMP4, LEF1, SOX2, and VCAN. In DSCs, both normoxia and hypoxia upregulated SOX2 expression, whereas hypoxia downregulated BMP4 expression. Microarray analysis revealed that normoxia increased the expression of pluripotency-related genes, including SPRY, NR0B1, MSX2, IFITM1, and DAZL, compared with hyperoxia. In an in vivo hair follicle reconstitution assay, cultured DPCs and DSCs were transplanted with newborn mouse epidermal keratinocytes into nude mice using a chamber method. In this experiment, normoxia resulted in the most efficient induction of DPC hair follicles, whereas hypoxia caused the most efficient induction and maturation of DSC hair follicles. These results suggest that application of physiological/hypoxic oxygen tension to cultured human DSCs enhances proliferation and maintenance of hair inductivity for skin engineering and clinical applications. Impact statement Dermal sheath cells (DSCs) and dermal papilla cells (DPCs) are useful cell sources for cell-based regenerative therapy. This is the first report to describe that low-oxygen conditions are better for DSCs. Normoxic and hypoxic culture of DSCs is beneficial for expanding these hair follicular cells and advancing development of cell-based therapy for both wound healing and hair regeneration. The current study supports that optimized oxygen tension can be applied to use expanded human DPCs and DSCs for skin engineering and clinical applications.

Contact Dermatitis Caused by Dermabond Advanced Use.

BACKGROUND: Dermabond Advanced (DBA) has been widely used globally; however, severe contact dermatitis (CD) can be a serious adverse effect of DBA use. In this study, we investigated the characterization and incidence rate of CD after using DBA and the safe use of DBA. METHODS: One hundred consecutive patients who underwent skin closure with DBA were investigated. All patients were women undergoing breast reconstruction. DBA was applied to their trunk and limbs following reconstruction. RESULTS: Seven patients (7%) presented with CD. Of these, 4 patients exhibited CD after the second DBA use; sensitization influence by the first DBA use was considered. One of 3 patients presenting with CD after the first DBA use was allergic to cosmetic glue, and the influence of immunological cross-reaction of acrylates was suggested. CONCLUSION: We consider that DBA use is inadequate for wounds with an improper margin and in dry and low-skin barrier areas such as the trunk and limbs because it may induce irritant CD and sensitization of DBA and subsequent allergic CD. Frequent use can also induce sensitization. If patients have a history of acrylate allergies, DBA use should be avoided because immunological cross-reaction from acetylates could result.

Tumoral Calcinosis: A Giant Tumoral Mass in the Shoulder Joint of a Dialysis Patient.

Tumoral calcinosis is a rare condition in which a calcified mass grows around a large joint, and can occur in patients undergoing renal dialysis. Here, we report the case of a 64-year-old man with a long history of dialysis who presented with a giant, painless mass in his right shoulder joint. A near-complete surgical resection is performed without muscle function loss and with no sign of recurrence after 1 year.

Therapeutic Effects of Human Adipose-Derived Products on Impaired Wound Healing in Irradiated Tissue.

BACKGROUND: Clinical sequelae of irradiation result in tissue devitalization (e.g., ischemia, fibrosis, and atrophy) where wound healing capacity is impaired. Fat-derived products may work to treat such pathology. METHODS: Nonlethal irradiation at various doses (5, 10, and 15 Gy) and frequencies (one to three times on sequential days) was delivered to dorsal skin of nude mice, and subsequent gross and microscopic changes were evaluated for up to 4 weeks. Cutaneous punch wounds were then created to compare wound healing in irradiated and nonirradiated states. Wounds were also locally injected with vehicle, cultured adipose-derived stem cells, centrifuged fat tissue, or micronized cellular adipose matrix, and the therapeutic impact was monitored for up to 15 days. RESULTS: Nude mice given total doses greater than 15 Gy spontaneously developed skin ulcers, and radiation damage was dose-dependent; however, a fractionated irradiation protocol was able to reduce the damage. Histologic assessment revealed dose-dependent dermal fibrosis/thickening and subcutaneous atrophy. Dose-dependent (5 to 15 Gy) impairment of wound healing was also evident. At the highest dosage (15 Gy three times), open wounds persisted on day 15. However, wounds injected with cultured adipose-derived stem cells were nearly healed on day 12, and those treated with injection of centrifuged fat or micronized tissue healed faster than untreated controls (p < 0.05). There was no significant differences between treated groups. CONCLUSIONS: Tissue devitalization by irradiation was dose-dependent, although fractionated protocols helped to reduce it. Adipose-derived stem cells and other fat-derived products harboring adipose-derived stem cells successfully revitalized irradiated tissues and accelerated wound healing.

Therapeutic effects of a recombinant human collagen peptide bioscaffold with human adipose-derived stem cells on impaired wound healing after radiotherapy.

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, hepatocyte growth factor, and transforming growth factor beta, 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 with 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.

Biological Properties and Therapeutic Value of Cryopreserved Fat Tissue.

BACKGROUND: Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study was to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting. METHODS: Lipoaspirates were harvested from six women and preserved using two cryopreservation methods: (1) simple cooling to -80°C (cryo-1); or (2) programmed cooling to -196°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 cells were significantly decreased in cryo-1 fat (1.7 × 10 cells, 42.6 percent viable) and cryo-2 fat (2.0 × 10 cells, 55.4 percent viable), compared with fresh fat (3.9 × 10 cells, 90.6 percent viable). Although adipose-derived stem cells were cultured successfully from all fats, functional adipose-derived stem cells from cryopreserved fats were much fewer, with comparable multilineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, 3 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 adipocytes were killed in the process of cryopreservation and thawing. Adipose-derived stem cells were isolated from cryopreserved fat, but the number of functional adipose-derived stem cells was very limited in both cryopreservation methods. After grafting, cryopreserved fat was retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.

Prompt Procedures Have a Great Impact on the Consequences of Asiatic Black Bear Mauling.

INTRODUCTION: Although bear-inflicted human injuries are rare and the mortality rate of the injuries is usually not high, the chances of bear-human interactions have been increasing, with fatal cases being reported in Japan every year. The aim of this study was to report a series of bear-inflicted injuries and discuss their management and severity. MATERIALS AND METHODS: A retrospective study was performed at the emergency and critical care center of Aizu Chuo Hospital, a tertiary care center in Japan, from May 2013 to September 2015. During this period, we encountered 14 black bear injury patients (12 men and 2 women). RESULTS: Six victims were attacked in dense forests while collecting wild vegetables; 4 victims were attacked near their houses. Lacerations of the scalp and face were the most frequent injuries, affecting 13 patients. Three patients developed hemorrhagic shock. Injury severity scores ranged from 2 to 12 (median value, 6). None of the injuries was fatal. Thirteen patients were transported by the local fire department's ambulances, 9 of whom were transferred to doctor ambulances. CONCLUSIONS: Although the severity and mortality rate following bear-inflicted injuries are not high, these encounters usually take place in remote rural areas, so a delay in rescue and proper care can lead to the incident becoming fatal. Hence, early decisions and arrangements for patient transportation to a tertiary care center and prompt measures to save lives will have a positive impact on the consequences of such incidents.

Multidirectional Cranial Distraction Osteogenesis with Simplified Modifications for Treating Sagittal Synostosis.

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.

Growth curves for intracranial volume in normal Asian children fortify management of craniosynostosis.

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.

Reconstitution of Human Keloids in Mouse Skin.

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.

An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells.

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.