A Closed-system Technology for Mechanical Isolation of High Quantities of Stromal Vascular Fraction from Fat for Immediate Clinical Use.

Adipose tissue stromal vascular fraction (SVF) is increasingly used in the clinic. SVF separation from fat by enzymatic disruption is currently the gold standard for SVF isolation. However, enzymatic SVF isolation is time-consuming (~1.5 h), costly and significantly increases the regulatory burden of SVF isolation. Mechanical fat disruption is rapid, cheaper, and less regulatory challenging. However, its reported efficacy is insufficient for clinical use. The current study evaluated the efficacy of a novel rotating blades (RBs) mechanical SVF isolation system. Methods: SVF cells were isolated from the same lipoaspirate sample (n = 30) by enzymatic isolation, massive shaking (wash), or engine-induced RBs mechanical isolation. SVF cells were counted, characterized by flow cytometry and by their ability to form adipose-derived stromal cells (ASCs). Results: The RBs mechanical approach yielded 2 × 105 SVF nucleated cells/mL fat, inferior to enzymatic isolation (4.17 × 105) but superior to cells isolating from fat by the "wash" technique (0.67 × 105). Importantly, RBs SVF isolation yield was similar to reported yields achieved via clinical-grade enzymatic SVF isolation. RBs-isolated SVF cells were found to contain 22.7% CD45-CD31-CD34+ stem cell progenitor cells (n = 5) yielding quantities of multipotent ASCs similar to enzymatic controls. Conclusions: The RBs isolation technology provided for rapid (<15 min) isolation of high-quality SVF cells in quantities similar to those obtained by enzymatic digestion. Based on the RBs platform, a closed-system medical device for SVF extraction in a rapid, simple, safe, sterile, reproducible, and cost-effective manner was designed.

The Age Factor in Postbariatric Body Contouring Surgery Outcome.

BACKGROUND: Increasing life expectancy will likely lead greater numbers of older patients to seek postbariatric body contouring plastic surgery. The impact of age on body contouring plastic surgery outcome is undetermined. METHODS: A retrospective cohort study of 317 postbariatric body contouring plastic surgery cases was performed. Patient demographics and operative and postoperative data were collected. Patients were categorized into three age groups, and univariate analysis examined group differences. Multivariate logistic regression analysis assessed independent associations between age and surgical outcome measures. RESULTS: Patients 60 years and older had a higher mean preoperative body mass index (30.8 ± 3.6 kg/m2, p < 0.001) and higher rates of hypertension (48.9 percent, p < 0.001), dyslipidemia (38.3 percent, p < 0.001), and diabetes mellitus (17 percent, p = 0.012) compared to the younger age groups. They also sustained significantly higher complication rates (any minor complications, p = 0.004; minor surgical site infections, p = 0.005; minor hematomas, p = 0.007; any major complications, p < 0.001; major surgical-site infections, p < 0.001; and major dehiscence, p < 0.001). Increasing age was a significant risk factor for any major complications (p = 0.005), reoperation (p = 0.02), and readmission (p = 0.001). Age greater than or equal to 60 years was a significant risk factor solely for readmission (OR, 3.32; p = 0.03). CONCLUSIONS: Increasing age was a risk factor for adverse postoperative outcome in postbariatric body contouring plastic surgery patients; however, age greater than or equal to 60 years in and of itself was an independent risk factor for readmission only. These findings may aid plastic surgeons in patient consultation and in decision making regarding suitable candidates for these procedures. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Tellurium compound provides pro-apoptotic signaling in drug resistant multiple myeloma.

Multiple Myeloma, effectively treated by chemotherapeutic drugs, relapses due to drug resistance. We tested here the capacity of mesenchymal stromal cells, from the bone marrow of patients or from adipose tissue of healthy individuals, to induce drug resistance in Myeloma cell lines. We show that drug resistance can be achieved by factors secreted by the various MSC's. Mass spectrometry analysis of MSC's conditioned media revealed that fibronectin, was particularly instrumental in providing anti-apoptotic signals to MM cells. Moreover, we demonstrate that SAS ([octa-O-bis-(R,R)tartarate ditellurane]), an immunomodulator Tellurium compound, is not only able of blocking the physical interaction between MM cells and fibronectin but is also capable of re-sensitizing the cells to the chemotherapeutic drugs. Finally, we show that this re-sensitization is coupled with the blocking of pAKT induction, in MM cells, by the MSC's. These results indicate that SAS may be useful in the treatment of drug resistant MM.

[FACIAL NERVE PARALYSIS - THERAPEUTIC APPROACH, FACIAL REANIMATION AND ADJUNCTIVE TREATMENT].

INTRODUCTION: Paralysis of the facial mimetic muscles causes loss of voluntary and non-voluntary muscle function, as well as facial tone. This is a devastating condition with profound functional, aesthetic and psychological consequences. Etiologies include congenital paralysis and acquired paralysis following viral infection, trauma, head and neck tumors, iatrogenic damage and more. Clinical presentation includes ocular symptoms (dry eye, epiphora, corneal irritation), nasal symptoms (nasal obstruction) and oral symptoms (drooling and speech disturbances). Reconstruction of facial nerve function is based on renewing the neural input to the paralyzed face in parallel with transferring a functioning muscle. The gold standard in long term facial paralysis reanimation includes a two-stage procedure that involves cross-face nerve grafting and later on a free gracilis muscle transfer. This method allows reconstruction of a symmetric, spontaneous and voluntary smile. In cases when cross-face nerve grafting is impossible, a free-gracilis muscle transfer is performed with neural coaptation to another cranial nerve, most commonly the motor nerve to the masseter muscle (of the trigeminal nerve). Non-microsurgical methods for facial reanimation exist, however, nowadays they are rarely performed. In addition to the surgical reconstruction, other surgical and non-surgical procedures are performed for functional and aesthetic symmetrization purposes. These include fat injection to the face, botulinum toxin injection, oculoplastic procedures and more. In this article we describe our patient population with facial nerve paralysis, common facial reanimation procedures, considerations in choosing the appropriate reconstruction procedure and the general approach for treatment of facial paralysis in our multidisciplinary facial paralysis clinic.

Cryopreservation of Stromal Vascular Fraction Cells Reduces Their Counts but Not Their Stem Cell Potency.

Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation. METHODS: SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at -196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone. RESULTS: While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45-CD34+CD31- stem cell progenitor cells compared to fresh SVF cells. CONCLUSIONS: Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.

Fas-L promotes the stem cell potency of adipose-derived mesenchymal cells.

Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.

Incomplete Facial Paralysis: The Use of the Ipsilateral Residual Facial Nerve as a Donor Nerve for Facial Reanimation.

BACKGROUND: The reconstructive approach for incomplete facial paralysis is not yet determined. In this article, the authors present a new surgical approach for patients with incomplete facial paralysis in which residual, ineffective movement is detected preoperatively in the ipsilateral buccozygomatic territory of the paretic facial nerve. METHODS: Sixteen patients with incomplete facial paralysis were found eligible for the procedure and underwent one-stage facial reanimation performed by the senior author (E.G.). Reanimation was performed using free gracilis muscle transfer with neural coaptation to an active facial nerve branch(es) responsible for the predetected buccozygomatic residual movement. Patients were reviewed in a systematic fashion using a combined still photographic and video scoring scale for symmetry at rest and at dynamic states. RESULTS: Following surgery, improved symmetry was observed in the majority of observations of the mouth region at rest and while smiling and of the nasolabial fold region while smiling. There was no significant change in symmetry in the majority of observations of the eye region at rest and while smiling and the nasolabial fold region at rest. Video assessment of dynamic facial symmetry while smiling demonstrated improved symmetry in 91 percent of the observations (n = 191 observations). Comparison of mean scores for dynamic smile symmetry produced a statistically significant improvement of 1.68 points following surgery (p < 0.001). CONCLUSION: Based on this series, the authors recommend that use of the ipsilateral facial nerve buccozygomatic residual branch be considered as a donor nerve for facial reanimation using a free gracilis muscle transfer in patients with incomplete facial paralysis with residual preoperative movement in the midface. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Intraoperative factors associated with postoperative complications in body contouring surgery.

BACKGROUND: Several preoperative factors have been shown to influence outcome of body contouring surgeries. The effect of intraoperative features, including fluid volume administered, hemodynamic and respiratory parameters, and body temperature on postoperative complication, has not been reported to date. MATERIALS AND METHODS: All subsequent patients undergoing body contouring surgery in the Tel Aviv Medical Center between 2007 and 2012 were enrolled. Demographic and intraoperative data were collected and analyzed for possible associations with postoperative complications, including formation of seroma, hematoma/bleeding, other surgical site complications (infection, adhesiolysis, or need for debridement), formation of a hypertrophic scar, any documented, infection or a composite outcome of any of the previously mentioned. RESULTS: Data of 218 patients were assessed. Mean (standard deviation) age of patients was 41(14) y. Intraoperative administration of higher volumes of fluids was significantly associated with formation of seroma (P = 0.01), hematoma/bleeding (P = 0.03), hypertrophic scar (P = 0.01), surgical site complications (P = 0.01), and a composite outcome (P < 0.001). Development of hematoma/bleeding was associated with longer periods of low (<35.6°C) intraoperative core temperature (72% versus 50% of surgery duration in patients who did not develop this complication, P < 0.05). Surgical site complications were associated with longer periods of intraoperative oxygen desaturation (saturation ≤92%, 4.2% versus 0.9% of surgery duration in patients who did not develop surgical site complications, P < 0.01). CONCLUSIONS: Intraoperative moderate hypothermia, hypoxemia, and liberal fluid administration are associated with worse surgical outcome in patients undergoing body contouring surgery. Increased awareness of the potential adverse effects of these factors in body contouring surgery will enhance interventions aimed at avoiding and promptly treating such events.

Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress.

Oxidative stress leads to the degeneration of retinal pigment epithelial (RPE) and photoreceptor cells. We evaluated the potential of adipose-derived mesenchymal stem cells (ASCs) as a therapeutic tool by studying the migration capacity of ASCs in vitro and their protective effect against RPE cell death under oxidative stress in vitro and in vivo. ASCs exhibited enhanced migration when exposed to conditioned medium of oxidative stressed RPE cells obtained by hydrogen peroxide. Migration-related axis SDF-1/CXCR4 was studied, and upregulation of SDF-1 in stressed RPE and of CXCR4 in ASCs was detected. Moreover, ASCs' conditioned medium prevented H2O2-induced cell death of RPE cells. Early passage ASCs had high expression level of HGF, low VEGF levels, and unmodulated IL-1ß levels, compared to late passage ASCs. Thus, early passage ASCs show the potential to migrate towards damaged RPE cells and protect them in a paracrine manner from cell death induced by oxidative stress. In vivo, mice received systemic injection of NaIO3, and 72 h later, ASCs were transplanted in the subretinal space. Seven days after ASC transplantation, the eyes were enucleated fixed and frozen for immunohistochemical analysis. Under such conditions, ASC-treated mice showed preservation of nuclear layers in the outer nuclear layer and stronger staining of RPE and photoreceptor layer, compared to PBS-treated mice. Taken together, our results indicate that ASCs are able to home in on damaged RPE cells and protect against damage to the RPE and PR layers caused by oxidative stress. These data imply the potential that ASCs have in regenerating RPE under oxidative stress, providing the basis for a therapeutic approach to retinal degeneration diseases related to oxidative stress that could help save the eyesight of millions of people worldwide.

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation.

Power-Assisted Liposuction Versus Tissue Resection for the Isolation of Adipose Tissue-Derived Mesenchymal Stem Cells: Phenotype, Senescence, and Multipotency at Advanced Passages.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ASCs) can be isolated from subcutaneous fat harvested by tissue resection or liposuction. OBJECTIVES: The authors compared ASCs isolated by tissue resection or power-assisted liposuction (PAL) to determine whether either surgical procedure yielded ASCs with improved purity and competence that was preserved for several passages. METHODS: For this experimental study, ASCs were isolated from fat harvested by tissue resection or PAL from six patients who underwent abdominoplasty. ASCs were counted to determine cell yields, and viabilities were assessed with an amine-reactive dye and by fluorescence-activated cell sorting (FACS). Cell phenotypes were determined by immunostaining and FACS, and doubling times were calculated. Senescence ratios of the cells were detected by gene profiling and by assaying ß-galactosidase activity. Multipotency was evaluated by induced differentiation analyses. RESULTS: No significant differences were observed in cell numbers or viabilities of ASCs isolated following either surgical method of fat harvesting. Both populations of cultured ASCs expressed markers of mesenchymal stem cells and preserved this expression pattern through the third passage. PAL and tissue resection yielded ASCs with similar division rates, similar senescence ratios into the fourth passage, and similar capacities to differentiate into osteocytes or adipocytes. CONCLUSIONS: Fat harvested by PAL or tissue resection yielded uniform cultures of ASCs with high division rates, low senescence ratios, and multipotency preserved into passages 3 and 4. Because PAL is less invasive, it may be preferable for the isolation of ASCs.

RBM28, a protein deficient in ANE syndrome, regulates hair follicle growth via miR-203 and p63.

Alopecia-neurological defects-endocrinopathy (ANE) syndrome is a rare inherited hair disorder, which was shown to result from decreased expression of the RNA-binding motif protein 28 (RBM28). In this study, we attempted to delineate the role of RBM28 in hair biology. First, we sought to obtain evidence for the direct involvement of RBM28 in hair growth. When RBM28 was downregulated in human hair follicle (HF) organ cultures, we observed catagen induction and HF growth arrest, indicating that RBM28 is necessary for normal hair growth. We also aimed at identifying molecular targets of RBM28. Given that an RBM28 homologue was recently found to regulate miRNA biogenesis in C. elegans and given the known pivotal importance of miRNAs for proper hair follicle development, we studied global miRNA expression profile in cells knocked down for RBM28. This analysis revealed that RBM28 controls the expression of miR-203. miR-203 was found to regulate in turn TP63, encoding the transcription factor p63, which is critical for hair morphogenesis. In conclusion, RBM28 contributes to HF growth regulation through modulation of miR-203 and p63 activity.

Extraskeletal Ewing's sarcoma in a great toe of a young boy.

Extraskeletal Ewing's sarcoma (EES) is a rare, soft tissue, malignant neoplasm histologically similar to skeletal Ewing's sarcoma. It occurs mainly in adolescents and young adults, and affects extremities in 36% of cases and central locations (commonly paravertebral regions) in the remainder. The differential diagnosis includes other small, blue, round cell tumours. A clinical case of EES involving a great toe in a young boy is reported. EES diagnosis was confirmed by features of histological analysis and immunohistochemistry, and by the presence of the t(11;22) chromosomal translocation.

Treatment of congenital facial nevi.

The treatment of congenital facial nevi is often difficult and challenging. Previous authors have reported their techniques, results, and complications when treating these lesions. Our objectives are to simplify the treatment planning by subdividing the lesions with a new classification and using this to formulate a surgical algorithm. One hundred and two patients with congenital facial nevi were reviewed. All of these patients have had surgical excision for the lesions. We have subgrouped the lesions into three groups, according to size, number of aesthetic units involved, and number of reconstructive stages required. Group I included lesions 1 to 3 cm in maximal diameter, within one aesthetic unit, and requiring one or two reconstructive stages. This group included 29 patients. Group II included lesions 3 to 12 cm in maximal diameter, covering one or two aesthetic units, and requiring not more than two stages of reconstruction. This group had 41 patients. Group III consisted of extensive lesions, over 12 cm in maximal diameter, covering several aesthetic units, and requiring several stages of reconstruction. In this group, we had 32 patients. On the basis of our experience in treating congenital facial nevi in this series, we have developed a surgical algorithm for reconstruction. We are optimistic that this will assist the surgeon in surgical planning and treating this complex patient population. The algorithm is arranged according to the new classification of congenital facial nevi that is presented.