The therapeutic and commercial landscape of stem cell vesicles in regenerative dermatology.

Extracellular vesicles (EVs) are lipid enveloped nanoparticles that are naturally produced by cells and function in the intercellular transfer of biological material such as proteins, RNAs and metabolites. They have been shown to act in an autocrine and paracrine manner to alter the functions of local and distant recipient cells, with a growing body of evidence highlighting their wide-ranging functions in regenerative processes such as stem cell maintenance, tissue repair and immune modulation. EVs offer several potential advantages over stem cell therapies such as improved safety profiles, scalability, and enhanced storage and quality control of the final product. In fact, many of the pro-regenerative outcomes of stem cell therapies have been attributed to the release of mesenchymal stem cell-derived EVs (MSC-EVs) and their potent effects on extracellular matrix turnover, local cell recruitment, proliferation and angiogenesis is now well described. These positive outcomes have led to clinical trials assessing the safety of MSC-EVs for applications in wound healing and the treatment of cutaneous ulcers, as well as the emergence of multiple commercial MSC-EV sources marketed for topical application in cosmetic medicine. However, regenerative EV therapeutics remain in their infancy and pertinent questions regarding product standardisation, potency and the regulatory landscape surrounding the development of these promising nano-therapeutics must be addressed to ensure safe and effective clinical adoption. In this article we provide an overview of the emerging landscape of MSC-EVs in regenerative dermatology and cosmetic science, highlighting the underlying biological mechanisms pertinent to their application and providing a perspective on current safety considerations, regulation and future directions in the field.

Bioengineered human skeletal muscle capable of functional regeneration.

BACKGROUND: Skeletal muscle (SkM) regenerates following injury, replacing damaged tissue with high fidelity. However, in serious injuries, non-regenerative defects leave patients with loss of function, increased re-injury risk and often chronic pain. Progress in treating these non-regenerative defects has been slow, with advances only occurring where a comprehensive understanding of regeneration has been gained. Tissue engineering has allowed the development of bioengineered models of SkM which regenerate following injury to support research in regenerative physiology. To date, however, no studies have utilised human myogenic precursor cells (hMPCs) to closely mimic functional human regenerative physiology. RESULTS: Here we address some of the difficulties associated with cell number and hMPC mitogenicity using magnetic association cell sorting (MACS), for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis within engineered tissues and force generation at maturity. In addition, these engineered human SkM regenerated following barium chloride (BaCl2) injury. Following injury, reductions in function (87.5%) and myotube number (33.3%) were observed, followed by a proliferative phase with increased MyoD+ cells and a subsequent recovery of function and myotube number. An expansion of the Pax7+ cell population was observed across recovery suggesting an ability to generate Pax7+ cells within the tissue, similar to the self-renewal of satellite cells seen in vivo. CONCLUSIONS: This work outlines an engineered human SkM capable of functional regeneration following injury, built upon an open source system adding to the pre-clinical testing toolbox to improve the understanding of basic regenerative physiology.

Annexin-enriched osteoblast-derived vesicles act as an extracellular site of mineral nucleation within developing stem cell cultures.

The application of extracellular vesicles (EVs) as natural delivery vehicles capable of enhancing tissue regeneration could represent an exciting new phase in medicine. We sought to define the capacity of EVs derived from mineralising osteoblasts (MO-EVs) to induce mineralisation in mesenchymal stem cell (MSC) cultures and delineate the underlying biochemical mechanisms involved. Strikingly, we show that the addition of MO-EVs to MSC cultures significantly (P < 0.05) enhanced the expression of alkaline phosphatase, as well as the rate and volume of mineralisation beyond the current gold-standard, BMP-2. Intriguingly, these effects were only observed in the presence of an exogenous phosphate source. EVs derived from non-mineralising osteoblasts (NMO-EVs) were not found to enhance mineralisation beyond the control. Comparative label-free LC-MS/MS profiling of EVs indicated that enhanced mineralisation could be attributed to the delivery of bridging collagens, primarily associated with osteoblast communication, and other non-collagenous proteins to the developing extracellular matrix. In particular, EV-associated annexin calcium channelling proteins, which form a nucleational core with the phospholipid-rich membrane and support the formation of a pre-apatitic mineral phase, which was identified using infrared spectroscopy. These findings support the role of EVs as early sites of mineral nucleation and demonstrate their value for promoting hard tissue regeneration.

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating condition defined by the de novo development of bone within non-osseous soft tissues, and can be either hereditary or acquired. The hereditary condition, fibrodysplasia ossificans progressiva is rare but life threatening. Acquired HO is more common and results from a severe trauma that produces an environment conducive for the formation of ectopic endochondral bone. Despite continued efforts to identify the cellular and molecular events that lead to HO, the mechanisms of pathogenesis remain elusive. It has been proposed that the formation of ectopic bone requires an osteochondrogenic cell type, the presence of inductive agent(s) and a permissive local environment. To date several lineage-tracing studies have identified potential contributory populations. However, difficulties identifying cells in vivo based on the limitations of phenotypic markers, along with the absence of established in vitro HO models have made the results difficult to interpret. The purpose of this review is to critically evaluate current literature within the field in an attempt identify the cellular mechanisms required for ectopic bone formation. The major aim is to collate all current data on cell populations that have been shown to possess an osteochondrogenic potential and identify environmental conditions that may contribute to a permissive local environment. This review outlines the pathology of endochondral ossification, which is important for the development of potential HO therapies and to further our understanding of the mechanisms governing bone formation.

A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp.

Stem-cell-based therapies provide a biological basis for the regeneration of mineralised tissues. Stem cells isolated from adipose tissue (ADSCs), bone marrow (BMSCs) and dental pulp (DPSCs) have the capacity to form mineralised tissue. However, studies comparing the capacity of ADSCs with BMSCs and DPSCs for mineralised tissue engineering are lacking, and their ability to regenerate dental tissues has not been fully explored. Characterisation of the cells using fluorescence-activated cell sorting and semi-quantitative reverse transcription PCR for MSC markers indicated that they were immunophenotypically similar. Alizarin red (AR) staining and micro-computed tomography (µCT) analyses demonstrated that the osteogenic potential of DPSCs was significantly greater than that of BMSCs and ADSCs. Scanning electron microscopy and AR staining showed that the pattern of mineralisation in DPSC cultures differed from ADSCs and BMSCs, with DPSC cultures lacking defined mineralised nodules and instead forming a diffuse layer of low-density mineral. Dentine matrix components (DMCs) were used to promote dentinogenic differentiation. Their addition to cultures resulted in increased amounts of mineral deposited in all three cultures and significantly increased the density of mineral deposited in BMSC cultures, as determined by µCT analysis. Addition of DMCs also increased the relative gene expression levels of the dentinogenic markers dentine sialophosphoprotein and dentine matrix protein 1 in ADSC and BMSC cultures. In conclusion, DPSCs show the greatest potential to produce a comparatively high volume of mineralised matrix; however, both dentinogenesis and mineral volume was enhanced in ADSC and BMSC cultures by DMCs, suggesting that these cells show promise for regenerative dental therapies.

The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues.

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73-CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

Laparoscopic Roux-en-Y gastric bypass for morbid obesity: technique and preliminary results of our first 400 patients.

HYPOTHESIS: A technique of the laparoscopic Roux-en-Y gastric bypass can be developed that is safe, effective, and practical in the community setting. DESIGN: A case series of 400 morbidly obese and superobese individuals who underwent the laparoscopic Roux-en-Y gastric bypass over a 22-month period. SETTING: Community private practice in Fresno, Calif. PATIENTS: A consecutive sample of 400 patients (70 males and 330 females) who met National Institutes of Health criteria for recommendation of a bariatric procedure. Only patients who had a previous gastric or bariatric procedure were excluded from this sample. INTERVENTION: Laparoscopic Roux-en-Y gastric bypass with a hand-sewn gastrojejunal anastomosis. MAIN OUTCOME MEASURES: Weight loss, complications, length of hospital stay, successful completion of the operation, and operative times were measured. RESULTS: Open conversion was required in 12 patients (6 males and 6 females) and a secondary operation for incomplete division of the stomach was required in 2 patients early in the case series. Alternative exposure and fixation techniques greatly reduced these occurrences. There were 6 staple-line failures owing to a change in the manufacture of the instrument. There were no leaks at the gastrojejunal anastomosis, but 21 patients required endoscopic balloon dilation for significant stenosis. The average hospital stay was 1.6 days for the patients who underwent laparoscopy and 2.7 days for patients requiring open conversion. Average excessive weight loss was 69% at 12 months. Operative times are between 60 and 90 minutes. Other complications are described. CONCLUSION: The Roux-en-Y gastric bypass can be safely and effectively performed in the community setting using advanced laparoscopic techniques.

Cryptic mycotic abdominal aortic aneurysms: diagnosis and management.

The incidence of cryptic mycotic abdominal aortic aneurysms has relatively increased since antibiotic therapy has become available. The causative organism is the salmonella group in about 50 per cent of cases. This diagnosis should be strongly entertained in patients with fever of unknown origin, vague abdominal pain, and progressive appearance of a pulsatile abdominal mass. Aortography may be helpful in establishing the diagnosis. Some postoperative graft infections may be due to unrecognized cryptic mycotic infection of the aorta and not from external contamination, as previously supposed. Construction of an axillofemoral bypass graft through clean tissue is advised for the successful treatment of the grossly infected infrarenal aortic aneurysm. Three surviving patients with cryptic mycotic abdominal aortic aneurysms are added to the sixteen surviving patients already reported in the literature.

Use of the nephrocholedochoscope in vascular surgery.

The nephrocholedochoscope designed for inspection of the ductal system of the urinary and bladder tracts has proved to be a useful method for examining the denuded vessel wall after limited blind endarterectomy. This technic is especially helpful in endarterectomy of the common iliac arteries and also has application in surgery of the profunda femoris, renal artery, and internal carotid artery.

Endarterectomy of the internal carotid artery.

Results of 219 operations in 171 patients for arteriosclerotic stenosis of the internal carotid artery were consistently good in patients with lateralizing, transient ischemic attacks. Although less consistent, relief of symptoms may be expected in a high proportion of patients with significant stenosis and more nonspecific symptoms. A small number of patients (10 percent) may have significant stenosis without a bruit. Asymptomatic stenosis, which has an unpredictable prognosis, may be operated upon with low mortality and morbidity. The use of local anesthesia and shunting when necessary proved to be the safest technique for the authors.