Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

Impact of Prevascularization on Immunological Environment and Early Engraftment in Subcutaneous Islet Transplantation.

BACKGROUND: The utilization of islet-like cells derived from pluripotent stem cells may resolve the scarcity of islet transplantation donors. The subcutaneous space is a promising transplantation site because of its capacity for graft observation and removal, thereby ensuring safety. To guarantee subcutaneous islet transplantation, physicians should ensure ample blood supply. Numerous methodologies, including prevascularization, have been investigated to augment blood flow, but the optimal approach remains undetermined. METHODS: From C57BL/6 mice, 500 syngeneic islets were transplanted into the prevascularized subcutaneous site of recipient mice by implanting agarose rods with basic fibroblast growth factor at 1 and 2 wk. Before transplantation, the blood glucose levels, cell infiltration, and cytokine levels at the transplant site were evaluated. Furthermore, we examined the impact of the extracellular matrix capsule on graft function and the inflammatory response. RESULTS: Compared with the 1-wk group, the 2-wk group exhibited improved glycemic control, indicating that longer prevascularization enhanced transplant success. Flow cytometry analysis detected immune cells, such as neutrophils and macrophages, in the extracellular matrix capsules, whereas cytometric bead array analysis indicated the release of inflammatory and proinflammatory cytokines. Treatment with antitumor necrosis factor and anti-interleukin-6R antibodies in the 1-wk group improved graft survival, similar to the 2-wk group. CONCLUSIONS: In early prevascularization before subcutaneous transplantation, neutrophil and macrophage accumulation prevented early engraftment owing to inflammatory cytokine production.

INVERSE KLIPPEL-TRENAUNAY SYNDROME.

OBJECTIVE: To report a rare case of inverse Kipplel-Trenaunay. CASE DESCRIPTION: A 16-year-old girl with a grayish-depressed plaque on her left thigh. Angioresonance showed a vascular malformation affecting the skin and subcutaneous tissue. COMMENTS: Inverse Klippel-Trenaunay is a Klippel-Trenaunay syndrome variation in which there are capillary and venous malformations associated to hypotrophy or shortening of the affected limb. Modifications on the limb's length or width result from alterations in bones, muscles, or subcutaneous tissues. It has few described cases. Further clinical and molecular studies must be performed for a proper understanding.

Intermittent normobaric oxygen inhalation enhances subcutaneous prevascularization for cell transplantation.

PURPOSE: Subcutaneous tissue is a promising site for cell transplantation; advantages include minimally invasive procedures and easy post-transplant monitoring. However, limited vascularity is the major known challenge. To address this challenge, a prevascularized graft bed is prepared in recipients. We aimed to establish an improved, clinically applicable approach to promote prevascularization of the subcutaneous graft bed prior to cell transplantation. METHODS: We applied a conventional prevascularization approach by subcutaneously implanting nylon discs into the backs of Lewis rats. After disc implantation, we treated rats with or without intermittent normobaric 100% oxygen inhalation (1 h, twice a day, for consecutive 7 days). We used histology to compare vascular density between the oxygen-treated or control groups. To assess the functional effects of prevascularization, we transplanted three hundred islets isolated from luciferase-transgenic Lewis rats into the oxygen-treated or control wild type Lewis recipients, then used bioluminescence imaging to track engraftment for 4 weeks. RESULTS: Oxygen treatment significantly augmented prevascularization in the subcutaneous site compared to controls. Islet transplantation into prevascularized graft beds demonstrated significant improvement in engraftment efficiency in oxygen-treated recipients compared to controls at 2-4 weeks post-transplantation. CONCLUSION: Combining intermittent normobaric 100% oxygen inhalation with a conventional vascularization approach promotes a functional vasculature within a week. A simple approach using normobaric oxygen has the potential for translation into clinical application in subcutaneous site cell transplantations.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation.

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

Vascular calcification in skin and subcutaneous tissue in patients with chronic and end-stage kidney disease.

BACKGROUND: Vascular calcification (VC) is well described in large- and medium-sized vessels in patients with chronic kidney disease (CKD), especially in those with end-stage kidney disease (ESKD) on dialysis. Medial calcification is particularly prevalent in this population and contributes to arterial stiffness and increased cardiovascular mortality and morbidity. Apart from in the setting of calciphylaxis, few studies have assessed skin and subcutaneous calcification and associations with abnormalities of bone and mineral metabolism in patients with CKD. METHODS: We performed a single-centre observational study to evaluate incisional skin tissue samples from three anatomical sites in patients with different stages of CKD undergoing elective surgery. We compared these samples to skin samples of a control cohort without CKD. Staining for calcification was performed with von Kossa method. A subgroup of skin samples were assessed by RT-PCR for upregulation of pro-calcific gene transcripts for tissue non-specific alkaline phosphatase (TNAP) and Runt-related transcription factor 2 (RUNX2). RESULTS: Forty-five patients were evaluated, 34 with CKD (including ESKD) and 11 control patients. VC was identified in 15 skin samples (13 CKD/ESKD and 2 controls). VC was present in the dermal and subcutaneous tissues of the neck, abdomen and arm samples. Two different histological types of VC were identified: speckled medial calcification and internal elastic lamina calcification. Presence of perieccrine calcification was identified in 14 samples, 10 with concurrent VC. There were no significant differences in serum parathyroid hormone, phosphate or calcium in patients with or without VC. Expression of TNAP or RUNX2 was not increased in samples from patients with ESKD or those with histological evidence of calcification. CONCLUSION: This study reports the novel finding of dermal and subcutaneous calcification in multiple anatomical locations in 38% of patients with advanced CKD/ESKD undergoing elective surgery but free from calciphylaxis.

Histological features of skin and subcutaneous tissue in patients with breast cancer who have received neoadjuvant chemotherapy and their relationship to post-treatment edema.

BACKGROUND: Lymphedema is a major complication of treatment for breast cancer. Although chemotherapy can cause lymphedema, there have been few reports about histological changes in skin and subcutaneous tissue after chemotherapy. The aim of our study was to determine whether chemotherapy affects blood and lymphatic vessels in the skin and subcutaneous fat and to investigate the relationship between these changes and extent of post-chemotherapy edema. METHODS: We compared histological findings in skin and subcutaneous fat of mastectomy specimens from 38 patients who had received NAC (neoadjuvant chemotherapy) and 56 who had not (non-NAC) attending our institution from 2007 to 2016. Patients whose tumor may have affected the area examined were excluded. Blood and lymphatic vessels were identified by CD31 and D2-40, respectively. We assessed microvessel density (MVD), lymphatic microvessel density (MLVD), lumen cross-sectional area (LA), and amount of endothelium (AE) in blood and lymphatic vessels. To minimize surgical effects, we measured edema, defined as ≥ 15% thicker dorsal subcutaneous tissue than baseline, on the contralateral side. RESULTS: MVD, LA, and AE of blood vessels were greater and MLVD not significantly different in the skin of NAC patients than in that of non-NAC patients. MVD was greater and AE of blood vessels less in subcutaneous fat of NAC patients than in that of non-NAC patients. Patients with edema had significantly less AE of blood vessels in skin than did those without it. CONCLUSIONS: These pathological findings can help to identify patients who will develop edema and improve their treatment.

Inverse klippel-trenaunay syndrome / Síndrome de klippel-trenaunay inverso

ABSTRACT Objective: To report a rare case of inverse Kipplel-Trenaunay. Case description: A 16-year-old girl with a grayish-depressed plaque on her left thigh. Angioresonance showed a vascular malformation affecting the skin and subcutaneous tissue. Comments: Inverse Klippel-Trenaunay is a Klippel-Trenaunay syndrome variation in which there are capillary and venous malformations associated to hypotrophy or shortening of the affected limb. Modifications on the limb's length or width result from alterations in bones, muscles, or subcutaneous tissues. It has few described cases. Further clinical and molecular studies must be performed for a proper understanding.

RESUMO Objetivo: Relatar um caso raro de Klippel-Trenaunay inverso. Descrição do caso: Menina de 16 anos com placa deprimida acinzentada na coxa esquerda, evidenciando-se, por meio de angioressonância, uma malformação vascular, acometendo a pele e tecidos subcutâneos. Comentários: Klippel-Trenaunay inverso é uma variante da síndrome de Klippel-Trenaunay em que há malformação capilar e venosa associada à hipotrofia ou encurtamento do membro afetado. Pode envolver acometimento ósseo, muscular ou subcutâneo, modificando o comprimento ou a circunferência do membro. Há poucos casos descritos, e mais estudos clínicos e moleculares precisam ser realizados para seu correto entendimento.

Elucidating the Mechanism of Absorption of Fast-Acting Insulin Aspart: The Role of Niacinamide.

PURPOSE: Fast-acting insulin aspart (faster aspart) is a novel formulation of insulin aspart containing two additional excipients: niacinamide, to increase early absorption, and L-arginine, to optimize stability. The aim of this study was to evaluate the impact of niacinamide on insulin aspart absorption and to investigate the mechanism of action underlying the accelerated absorption. METHODS: The impact of niacinamide was assessed in pharmacokinetic analyses in pigs and humans, small angle X-ray scattering experiments, trans-endothelial transport assays, vascular tension measurements, and subcutaneous blood flow imaging. RESULTS: Niacinamide increased the rate of early insulin aspart absorption in pigs, and pharmacokinetic modelling revealed this effect to be most pronounced up to ~30-40 min after injection in humans. Niacinamide increased the relative monomer fraction of insulin aspart by ~35%, and the apparent permeability of insulin aspart across an endothelial cell barrier by ~27%. Niacinamide also induced a concentration-dependent vasorelaxation of porcine arteries, and increased skin perfusion in pigs. CONCLUSION: Niacinamide mediates the acceleration of initial insulin aspart absorption, and the mechanism of action appears to be multifaceted. Niacinamide increases the initial abundance of insulin aspart monomers and transport of insulin aspart after subcutaneous administration, and also mediates a transient, local vasodilatory effect.

Autologous Vascularization: A Method to Enhance the Antibacterial Adhesion Properties of ePTFE.

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE), an ideal bioimplant material, is commonly used in surgical repair to treat soft tissue defects and deformities. However, the main disadvantage of ePTFE is that its distinctive porous ultrastructure is prone to bacterial adhesion that gives rise to infection and chronic inflammation, resulting in functional failure. Herein, a potentially promising approach to ePTFE autologous vascularization (AV-ePTFE) in vivo was established and developed to enhance the material's antibacterial properties. METHODS: Hematoxylin and eosin (H&E) staining and visual observation were performed to validate the intensity of the inflammatory response and related histological changes in surgical wounds after AV-ePTFE implantation. In addition, the antibacterial activities of AV-ePTFE were assessed by an in vitro bacterial adhesion assay and scanning electron microscope observation. RESULTS: The optimal time point of AV-ePTFE was 12 weeks after implantation. AV-ePTFE relieved inflammation based on an inflammation grading evaluation and expedited wound healing. Furthermore, AV-ePTFE effectively reduced the number of bacterial adhesions, inhibited bacterial biofilm formation, and prevented the occurrence of infection. CONCLUSIONS: We conclude that autologous vascularization is an effective method to improve the antibacterial adhesion properties and biocompatibility of ePTFE after implantation and that it may have a significant effect on clinical application of future porous biomaterials.

FGF-2 combined with bilayer artificial dermis composed of collagen matrix prompts generation of fat pad in subcutis of mice.

Fibroblast growth factor (FGF)-2 induces mitogenesis, angiogenesis and adipogenesis. In this study, the adipogenesis-inducing effects of FGF-2 combined with bilayer artificial dermis in mice were evaluated. FGF-2-impregnated bilayer artificial dermis composed of collagen matrix, PELNAC (Gunze Corp., Osaka, Japan) was implanted subcutaneously into the thoracic region of mice. At 1, 2, 3, and 4 weeks, samples were collected for H&E staining, von Willebrand factor immunostaining, and perilipin immunostaining to examine adipose tissue localization and angiogenesis. The collagen matrix-implanted group without the addition of FGF-2 was prepared as a control. At 2 weeks after the implantation of FGF-2 combined with dermal substitutes, adipocytes appeared in the collagen fibers. At 3-4 weeks, a fat pad was generated with neovascularization. The thickness of the fat pad had significantly increased at 2, 3, and 4 weeks. The remaining collagen was decreased by absorption over time. In the control group, no fat pad was newly formed. This study has identified a promising method to enhance adipogenic effects in the murine subcutis, representing a potential technique for soft tissue reconstruction.

Long-term Functioning of Allogeneic Islets in Subcutaneous Tissue Pretreated With a Novel Cyclic Peptide Without Immunosuppressive Medication.

BACKGROUND: There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes. METHODS: In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets. RESULTS: Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts. CONCLUSIONS: These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

Management of skin and subcutaneous tissue in complex open abdominal wall reconstruction.

PURPOSE: Open abdominal wall reconstruction is often a complex endeavor, usually performed on patients with multiple risk factors and co-morbidities. METHODS: In this article, we review soft tissue management techniques that can optimize the skin and subcutaneous tissue, with the goal of reducing surgical-site occurrences. RESULTS: Regardless of the hernia repair technique used, outcomes can be highly dependent on the appropriate management of the skin and subcutaneous tissue. Indeed, dehiscence and surgical-site infection can jeopardize the entire reconstruction, especially in cases where synthetic mesh might become exposed and/or infected, setting up a "vicious cycle" (Holihan et al. in J Am Coll Surg 221:478-485, 2015). CONCLUSION: Multidisciplinary cooperation between the general and plastic surgeon is useful in cases of tenuous blood supply to the abdominal skin, in cases of redundant, marginal or excessive skin, and in cases of deficient skin.

A case report of cutaneous polyarteritis nodosa in siblings.

Cutaneous polyarteritis nodosa (CPAN) is characterized by a necrotizing vasculitis of small and medium-sized arteries in the skin, which can be associated with fever, arthralgia, myalgia, and neuropathy, but, unlike polyarteritis nodosa (PAN), there is no visceral involvement. CPAN is rare in childhood. We report two siblings who developed CPAN during childhood. Interestingly, both had Mediterranean fever gene (MEFV) mutation, i.e. heterozygous E148Q. They also shared HLA-A24, -DR15 alleles. Simultaneous occurrence of MEFV mutation and HLA alleles with CPAN has never been reported in Japan. These cases could provide some hereditary clue for the development of CPAN.

Multi-site lymphatic venous anastomosis using echography to detect suitable subcutaneous vein in severe lymphedema patients.

BACKGROUND: The method of lymphatic venous anastomosis (LVA), including its indications or preoperative examinations, has not been established. The purpose of this study is to reveal the possible application of preoperative echography in surgical LVA outcome. METHODS: We performed a retrospective case-control study on patients with lower limb lymphedema who underwent LVA between August 15, 2013 and August 15, 2014. As a preoperative examination, we used venous echography to identify subcutaneous veins in the echo group, while we only used Accuvein visualizing system in the control group. The operation time, number of anastomoses, and limb circumference were compared between the two groups. RESULTS: Seventeen patients (34 limbs) were included in the echo group, and 21 patients (42 limbs) were included in the control group. The average follow-up period was 11.9 (6-16) and 12.4 (6-27) months, respectively. The average operation time in the echo group was 258.6 min, and that in the control group was 216.5 min. The average number of anastomoses was 9.8 and 7.0 in the echo and control group, respectively. The average time per anastomosis was 27.4 and 32.6 min, respectively. The diameter of the vein had a tendency to be larger in the echo group than in the control group. In 5.8% of the echo group, we observed a circumference increase, compared with 23.8% in the control group. CONCLUSIONS: Preoperative venous echography allowed surgeons to increase the number of anastomoses performed within the operating time, resulting in improvement of surgical outcomes.

Angiotensin Receptor Expression and Vascular Endothelial Dysfunction in Obstructive Sleep Apnea.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with vascular endothelial dysfunction (VED) in otherwise healthy patients. The role of renin-angiotensin system (RAS) in the OSA induced VED is not well understood. METHODS: Recently diagnosed OSA patients with very low cardiovascular disease (CVD) risk (Framingham score <5%) were studied at diagnosis and after 12 weeks of verified continuous positive airway pressure (CPAP) therapy. Participants underwent biopsy of gluteal subcutaneous tissue at baseline and after CPAP. Microcirculatory endothelial expression of angiotensin receptors type-1 (AT-1) and type-2 (AT-2) was measured in the subcutaneous tissue using quantitative confocal microscopy techniques. The ex-vivo effect of AT-1 receptor blockade (ARB) on endothelial superoxide production was also measured before and after CPAP treatment. RESULTS: In OSA patients (n = 11), microcirculatory endothelial AT1 expression decreased from 873 (200) (fluorescence units) at baseline to 393 (59) units after 12 weeks of CPAP (P = 0.02). AT2 expression did not decrease significantly in these patients (479 (75) to 329 (58) post CPAP (P = 0.08)). The ex-vivo addition of the losartan to the microcirculatory endothelium resulted in decreased superoxide expression in the vascular walls from 14.2 (2.2) units to 4.2 (0.8) P < 0.001; while it had no effect on post-CPAP patient tissue (P = 0.64). CONCLUSIONS: In OSA patients with no to minimal CVD risk, VED is associated with upregulation of AT-1 expression that is reversible with CPAP. Endothelial oxidative stress was reversible with ARB. RAS activation may play an important role in the development of early CVD risk in OSA patients.

Controlled basic fibroblast growth factor release device made of poly(ethyleneglycol) dimethacrylates for creating a subcutaneous neovascular bed for cell transplantation.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.