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Background Part one of this study identified that prescribing behaviours during 2020 differed significantly from 2016-2021. Part 2, presented here, covers a more detailed analysis of the impact of the early part of the COVID-19 pandemic is presented here. Aims To describe patterns in community prescribed and dispensed antibiotic volumes attributable to non-medical prescribers during 2020 compared to the previous 4 years and identify any changes of behaviour during this period and their impact on antimicrobial stewardship. Methods An analysis of retrospective non-medical prescribers' prescribing data was performed to determine the numbers of independent non-medical prescribers and the patterns of prescribed community dispensed antibiotic volumes between 2016-2021. The 2020 data were then compared to the data from previous years. Findings The total amount of dispensed non-medical prescribers antibiotic prescription items per 100000 population dropped by 12% in 2020 compared to 2019, driven predominantly by reductions in nurse prescribing. However, high-risk antibiotic prescribing rose from 5.3% of antibiotics prescribed in 2019 to 6.4% in 2020. Seasonal patterns of antibiotic prescribing differed in 2020, with significant reductions in prescribing of antibiotics normally associated with treating LRTI from the first lockdown in March 2020. Conclusions The COVID-19 pandemic forced rapid changes in practice, including increased remote working, which created a challenging prescribing environment that adversely affected the high-risk antibiotic prescribing behaviour of non-medical prescribers more than it affected their medical colleagues. There were unusually low levels of prescribing for respiratory tract infections over the last quarter of 2020 which, although likely multifactorial, may indicate over-prescribing for self-limiting viral infections in previous years. More research is needed to establish ongoing prescribing trends and patient outcomes, and how antimicrobial stewardship programmes and training can be better targeted to support prescribers to address quality and safety issues arising from changing modes of practice.

Background Non-medical prescriber numbers have increased rapidly over the last 10 years, with increasingly diverse roles and backgrounds. Previous evaluations of their antibiotic prescribing demonstrated it was generally of a high quality and guideline-driven, but recent evidence is lacking and the data are not easily accessible. Aims To describe changes in the non-medical prescriber population and patterns in dispensed antibiotic volumes between 2016–2021, highlighting evidence of good antimicrobial stewardship and where further interventions may be required. Methods An analysis of retrospective non-medical prescriber prescribing data was performed, to determine the numbers of independent non-medical prescribers and the patterns of prescribed community-dispensed antibiotics in England between 2016–2021. Findings Between 2016–2021, it was found that independent non-medical prescriber numbers in England rose by 54%. Whilst they remain predominantly nurses (76%), the numbers of pharmacists and allied health professionals have increased. Non-medical prescribers were responsible for 10.6% of all dispensed antibiotic items prescribed in primary care in England. However, the proportion of dispensed antibiotic items prescribed by these non-medical prescribers reduced by over 50%. Prescribing in 2020 differed from previous years, with increased high-risk antibiotic prescribing. Conclusion The quality of prescribing generally appears good and in accordance with national guidelines and principles of good antimicrobial stewardship. The 2020 COVID-19 pandemic prompted changes in antibiotic prescribing behaviour.

Research indicates that students’ participation in university-based extracurricular activities contributes to their graduate attributes such as leadership, teamwork, communication and resilience. However, it has yet to be determined which types of extracurricular activities are more impactful. This study inquired if participation in competitive sporting activities compared to social-cultural clubs have a greater impact on graduate attributes. Students attending a large metropolitan university in Sydney, Australia, who participated in extracurricular activities were surveyed (n = 844) with an instrument adapted to measure their degree of club engagement and questions on the skills, knowledge and experience they acquired. The findings indicate that engagement in competitive sport is more effective at contributing to graduate attributes when compared to social-cultural clubs. The study drew on the theories of serious leisure and leisure constraints to interpret this phenomenon. Participation in competitive sport was found to relate to more aspects of serious leisure such as study/work-life balance, stress reduction and skill development such as teamwork, time management and leadership skills. However, there are greater constraints to participating in competitive sport. The study concludes with implications for university administrators and recommendations for facilitating greater student opportunities to participate in all types of extracurricular activities.

Summary Background Carpometacarpal osteoarthritis of the thumb (CMC OA) is treated with various therapeutic approaches. However, the literature remains inconclusive regarding the ideal procedure for each disease stage. In this study, we assessed the international application of surgical treatment options including CMC I implants and non-surgical treatment options for CMC OA depending on the disease stage, with a strong focus on the detection of geographical disparities. Methods We conducted a large international online survey with members of hand surgical societies of the International Federation of Societies for Surgery of the Hand (IFSSH). The first part of the survey asked about general therapy options of CMC OA depending on the severity of the disease, whereas the second part specifically dealt with the use of prostheses. Results We could include 10 of 56 IFSSH member societies (6807 surgeons) and received answers from 1138 members (16.7%). Significant differences were detected in an increased use of corticosteroid injections in the USA, and a growing frequency of fat injections in Europe. Regarding use and frequency of the resection arthroplasty, we found similar results in all participating countries. Prosthetic implantation showed a significant difference between the USA and Europe, with far larger numbers stated by European hand surgeons. Conclusion CMC OA is treated differently in the participating countries depending on the stage of the disease. We give an insight into geographical differences in treatment paradigms, with corticosteroid injections being more prevalent in the USA, and prosthesis implantation being more frequently chosen in the selected European countries.

Introduction: In the surgical world of breast cancer reconstruction, fat grafting is commonly viewed as an oncogenic risk. Scientific studies add confusion, given the stark lack of clinical evidence suggesting pro-oncogenic links. Typically, classic migration assays (e.g., Boyden chamber) between adipose-derived stem cells and breast cancer cells define this cell relationship as pro-oncogenic. Objective: We sought to develop a new migration model which better explains existing clinical data. Methods: Silicon chambers were used to seed isolated populations of cells simultaneously in culture dish. Once cells had adhered, chambers were removed and cells were allowed to follow natural trophic cues. Multiple permutations of MDA-MB-231, MCF-7, HS-27, and ASCs were engineered. Cells were stained with MitoTracker for fluorescent visualization. A human cytokine array (RayBiotech) was performed on the media of migrating assays. Cellular tropism and blot intensity were quantitatively measured in Image J. Results: An in vitromodel was successfully constructed where ASCs reproducibly and freely migrated. Cytokine arrays reveal higher levels of IL-6 and CCL2 in the media of Boyden chambers containing ASCs and MDA-MB-231, compared to the novel assay, comprised of the same cell numbers, types, and incubation times. Conclusion: These data collectively show for the first time the attraction of ASCs to malignant breast cancer cells; a phenomenon which many ASC studies infer. The cytokine profile of the novel system described is less oncogenic than the commonly described Boyden chamber. These data integrate better into the clinical data, which fail to link cancer recurrence with fat grafting.

In 2006, a new model of invasive breast tumor emerged and, since 2011, is gaining recognition and research momentum. “Tumor-associated collagen signatures” describe 3 distinct layers of collagen which radiate outward in shells from the main body of the tumor. The outermost layer (TACS3) features branches of collagen radiating away from the tumor, 90° perpendicular to the tumor surface. TACS3 increases tumor span and correlates directly with metastasis, though presently difficult to detect in breast tissue. TACS is an emerging model but has been validated by multiple labs in vitro and in vivo, specifically for breast cancer prognostics. Newly recognized and accepted tumor borders will impact both R0 resections and downstream surgical reconstruction. This review aims to comprehensively introduce and connect the ranging literature on linearized collagen of invasive tumor borders. Using PubMed keyword searches containing “aligned,” “linear,” “oriented,” and “organized,” we have gathered the studies on TACS, integrated the concept into the clinic, and projected future platforms.

Background Hypoxia-inducible factor 1α (HIF-1α), a transcription factor responsible for tissue homeostasis and regeneration, presents reduced functionality in advanced age. In addition to absence of oxygen, sequestration of iron also stimulates HIF-1α. Therefore, we analyzed the efficacy of the iron-chelator deferiprone (DFP) at stimulating dermal fibroblasts. Objectives The main objective of this study was to quantify the DFP concentrations capable of stimulating dermal fibroblasts in vitro and to correlate the effective DFP concentrations with the ability of DFP to penetrate the epidermis, reach the dermis, and activate HIF-1α in vivo. Methods We measured cell proliferation, metabolic activity, HIF-1α expression, and lactate dehydrogenase levels of both young and aged fibroblasts after a 24-hour in vitro preconditioning with DFP. In addition, we evaluated cell survival rates and morphology with different cellular stainings. Finally, we performed a transdermal permeation study with a 1% DFP topical formulation to quantify the concentration required to reach the dermis. Results In vitro administration of iron-chelation therapy (156-312.5 µg/mL DFP ) on aged fibroblasts resulted in activation of various antiaging processes. The concentration required to reach the dermis within 24 hours was 1.5% (0.15 mg/mL), which corresponds well with the effective doses of our laboratory analyses. Conclusions The activation of HIF-1α by DFP enhances cell metabolism, proliferation, and survival of fibroblasts while reducing lactate dehydrogenase levels. Modulation of HIF-1α is linked to activation of key regeneration enzymes and proteins, and by proxy, antiaging. Therefore, the antiaging properties of DFP and its satisfactory dermal penetration make it a promising regenerative agent.

The triple-negative breast tumor boundary is made of aligned, linear collagen. The pro-oncogenic impact of linear collagen is well established; however, its mechanism of formation is unknown. An in vitro analogue of the tumor border is created by a co-culture of MDA-MB-231 cells, adipose derived stem cells, and dermal fibroblasts. Decellularization of this co-culture after seven days reveals an extracellular matrix that is linear in fashion, high in pro-oncogenic collagen type VI, and able to promote invasion of reseeded cells. Further investigation revealed linear collagen VI is produced by fibroblasts in response to a paracrine co-culture of adipose derived stem cells and MDA-MB-231, which together secrete high levels of the chemokine CCL5. The addition of monoclonal antibody against CCL5 to the co-culture results in an unorganized matrix with dramatically decreased collagen VI. Importantly, reseeded cells do not exhibit pro-oncogenic behavior. These data illustrate a cellular mechanism, which creates linear extracellular matrix (ECM) in vitro, and highlight a potential role of CCL5 for building striated tumor collagen in vivo.

Targeted genetic dissection of tissues to identify precise cell populations has vast biological and therapeutic applications. Here we develop an approach, through the packaging and delivery of 4-hydroxytamoxifen liposomes (LiTMX), that enables localized induction of CreERT2 recombinase in mice. Our method permits precise, in vivo, tissue-specific clonal analysis with both spatial and temporal control. This technology is effective using mice with both specific and ubiquitous Cre drivers in a variety of tissue types, under conditions of homeostasis and post-injury repair, and is highly efficient for lineage tracing and genetic analysis. This methodology is directly and immediately applicable to the developmental biology, stem cell biology and regenerative medicine, and cancer biology fields., Targeted genetic dissection of tissues can be used to identify cell populations and lineages. Here the authors develop 4-hydroxytamoxifen liposomes for the localized induction of CreERT2.

The constant intrinsic and extrinsic stress the skin is exposed to leads to significant impairments of the regenerative capacity of aging skin. Current skin rejuvenation approaches lack the ability to holistically support the biological processes that exhaust during aging skin degeneration, such as collagen production, cell migration and proliferation, and new vessel formation. Similar to chronic wounds, aged skin is characterized by dysfunction of key cellular regulatory pathways impairing regeneration. Recent evidence suggests that the same mechanisms hindering a physiologic healing response in chronic wounds are the basis of impaired tissue homeostasis in aged skin. Dysfunction of a main response-to-injury pathway, the hypoxia-inducible factor (HIF)-1α regulatory pathway, has been identified as pivotal both in chronic wounds and in aging skin degeneration. HIF-1α signaling is significantly involved in tissue homeostasis and neovascularization, resulting in the production of new collagen, elastin, and nourishing blood vessels. Modulating the functionality of this pathway has been demonstrated to significantly enhance tissue regeneration. In this review, we present an overview of the regenerative effects linked to the up-regulation of HIF-1α functionality, potentially resulting in skin rejuvenation on both the cellular level and the tissue level.

Studies evaluating fat grafting in mice have frequently used micro-computed tomography (micro-CT) as an accurate radiographic tool to measure longitudinal volume retention without killing the animal. Over the past decade, however, microultrasonography has emerged as an equally powerful preclinical imaging tool. Given their respective strengths in 3-dimensional reconstruction, there is no study to our knowledge that directly compares micro-CT with microultrasound in volumetric analysis. In this study, we compared the performance of micro-CT with microultrasound in the evaluation of adipose tissue graft volume in a murine model. Fifteen immunodeficient mice were given 200 μL of adipose tissue grafts. In vivo volumetric analysis of the grafts by micro-CT and microultrasound was conducted at discrete time points up to postoperative day 105. Three mice were killed at multiple time points, and explanted grafts were reimaged by CT and ultrasound, as mentioned previously. Analysis revealed that in vivo graft volumes measured by micro-CT do not differ significantly from those of microultrasound. Furthermore, both micro-CT and microultrasound were capable of accurately measuring fat grafts as in vivo volumes closely correlated with explanted volumes. Finally, ultrasound was found to yield improved soft tissue contrast compared with micro-CT. Therefore, either modality may be used, depending on experimental needs.

The current strategies for healing bone defects are numerous and varied. At the core of each bone healing therapy is a biomimetic mechanism, which works to enhance bone growth. These range from porous scaffolds, bone mineral usage, collagen, and glycosaminoglycan substitutes to transplanted cell populations. Bone defects face a range of difficulty in their healing, given the composite of dense outer compact bone and blood-rich inner trabecular bone. As such, the tissue possesses a number of inherent characteristics, which may be clinically harnessed as promoters of bone healing. These include mechanical characteristics, mineral composition, native collagen content, and cellular fraction of bone. This review charts multiple biomimetic strategies to help heal bony defects in large and small osseous injury sites, with a special focus on cell transplantation.

Because of the abundance and biocompatibility of fat, lipotransfer has become an attractive method for treating soft-tissue deficits. However, it is limited by unpredictable graft survival and retention. Currently, little is known about the viscoelastic properties of fat after various injection methods. Here, the authors assess the effects of cannula diameter, length, and shape on the viscoelastic properties, structure, and retention of fat.Human lipoaspirate was harvested using suction-assisted liposuction and prepared for grafting. A syringe pump was used to inject fat at a controlled flow rate through cannulas of varying gauges, lengths, and shapes. Processed samples were tested in triplicate on an oscillatory rheometer to measure their viscoelastic properties. Fat grafts from each group were placed into the scalps of immunocompromised mice. After 8 weeks, graft retention was measured using micro-computed tomography and grafts were explanted for histologic analysis.Lipoaspirate injected through narrower, longer, and bent cannulas exhibited more shear thinning with diminished quality. The storage modulus (G') of fat processed with 18-gauge cannulas was significantly lower than when processed with 14-gauge or larger cannulas, which also corresponded with inferior in vivo histologic structure. Similarly, the longer cannula group had a significantly lower storage modulus than the shorter cannula, and was associated with decreased graft retention.Discrete modifications in the methods used for fat placement can have a significant impact on immediate graft integrity, and ultimately on graft survival and quality. Respecting these biomechanical influences during the placement phase of lipotransfer may allow surgeons to optimize outcomes.Therapeutic, V.

Soft tissue reconstruction to restore volume to damaged or deficient tissue beneath the skin remains a challenging endeavor. Current techniques are centered around autologous fat transfer, or the use of synthetic substitutes, however, a great deal of scientific inquiry has been made into both the molecular mechanisms involved in, and limitations of, de novo adipogenesis, that is, the formation of new adipose tissue from precursor cells. To best comprehend these mechanisms, an understanding of defined markers for adipogenic differentiation, and knowledge of both commercially available and primary cell lines that enable in vitro and in vivo studies is necessary. We review the growth factors, proteins, cytokines, drugs, and molecular pathways that have shown promise in enhancing adipogenesis and vasculogenesis, in addition to the multitude of scaffolds that act as delivery vehicles to support these processes. While progress continues on these fronts, equally important is how researchers are optimizing clinically employed strategies such as autologous fat transfer through cell-based intervention, and the potential to augment this approach through isolation of preferentially adipogenic or angiogenic precursor subpopulations, which exists on the horizon. This review will highlight the novel molecular and synthetic modifications currently being studied for inducing adipose tissue regeneration on a cellular level, which will expand our arsenal of techniques for approaching soft tissue reconstruction.

Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft retention in both mouse and human studies. However, the literature varies as to whether fresh stromal vascular fraction or culture-expanded adipose-derived stromal cells are used to augment volume retention. The authors' study sought to determine whether there was a significant advantage to using adipose-derived stromal cells over unpurified stromal vascular fraction cells in a mouse model of cell-assisted lipotransfer.

Biomimetics and biomimicry are thought processes applied to biomaterial design, where biological implants have properties which mirror closely those of natural material. The authorsdiscuss the recipient site, mechanical stability of implant, grafts based on bone mineral components, grafts based on structural protein, and grafts based on cellular implants.

Skin damage heals with a scar that helps wound contraction and will bring wound boundaries closer together. The authors discuss wound creation and healing and treatment of scars.

Due to an unstoppable and ongoing shift in the distribution of western population towards old age, we recently experience a dramatic increase in comorbidities such as diabetes or cardiovascular insufficiency. Being accompanied with a raising number of chronic wounds, this aging shift has become not only an individual medical but also a significant economic burden, consuming more than 4% of health care budgets worldwide. Wound healing requires an exact interplay of several cell types, intra- and extracellular mechanisms, proteins, and signaling pathways. To either reverse age-related alterations or to significantly improve endogenous healing capacity, drugs need to interfere with these complex endogenous mechanisms involved in the response to injury. In this chapter, we summarize the most promising advances in wound healing therapeutics with its challenges and shed light on possible solutions for effective application.

BACKGROUND: The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol. METHODS: Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: die collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, die authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test die ability of the isolated cells to enhance adipogenesis, die cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning. RESULTS: The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with die conventional method. Cells harvested using die new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with die conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice. CONCLUSIONS: The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

An invaluable part of the plastic surgeon's technical arsenal for soft tissue contouring, fat grafting continues to be plagued by unpredictable outcomes, resulting in either reoperation and/or patient dissatisfaction. Thus, extensive research has been conducted into the effects of adipose tissue procurement, processing, and placement on fat graft quality at both the cellular level and in terms of overall volume retention. Herein, we present an overview of the vast body of literature in these areas, with additional discussion of cell-assisted lipotransfer as a therapy to improve volume retention, and on the controversial use of autologous fat in the setting of prior irradiation.

Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy.

Adipose-derived stromal cells (ASCs) are a promising population of cells that may be useful for the regeneration of human tissue defects. ASCs are capable of forming bone tissue in vitro and in vivo. Further work is required to determine the optimal conditions that will allow human ASCs to regenerate tissue in clinically significant tissue defects. Here we present three experimental protocols that are indispensable for the study of ASC osteogenic activity.

Targeted genetic dissection of tissues to identify precise cell populations has vast biological and therapeutic applications. Here we develop an approach, through the packaging and delivery of 4-hydroxytamoxifen liposomes (LiTMX), that enables localized induction of CreER

BACKGROUND Abdominoplasty is one of the most common procedures in plastic surgery, and energy-based tissue dissection techniques have become the gold standard. Despite its frequency, abdominoplasty is still associated with high complication rates. OBJECTIVES The authors compared clinical and economic data of 4 methods of energy-based tissue dissection in a randomized, open-label study. METHODS A total of 57 patients were preoperatively randomized into 4 groups: electrocautery, Ultracision Harmonic Scalpel, argon plasma coagulation, and PEAK-Plasmablade. Demographic and operational data as well as information on the postoperative course and complications were collected. For economic analysis, quotes were obtained from the device companies or official suppliers. RESULTS Duration of surgery, drainage quantity, and wound healing complications did not differ significantly between groups. The Ultracision method caused significantly greater blood loss compared with all other techniques (P < 0.01). PEAK and Ultracision devices entailed greater surgical costs compared with APC and electrocautery. CONCLUSIONS All methods evaluated can be applied safely and effectively in abdominoplasty procedures. However, these data demonstrate a significantly higher blood loss for the Ultracision Harmonic Scalpel. Considering the clinical data, the higher costs of PEAK and Ultracision methods appear unjustified. LEVEL OF EVIDENCE: 2

Chronic diabetic ulcers are a common complication in patients with diabetes, often leading to lower limb amputations and even mortality. Stem cells have shown promise in promoting cutaneous wound healing by modulating inflammation, angiogenesis, and re-epithelialization. However, more effective delivery and engraftment strategies are needed to prolong transplanted stem cell lifespan and their pro-healing functions in a chronic wound environment to improve skin regeneration. In this study, an injectable poly(ethylene glycol) (PEG)-gelatin-based hydrogel system is examined to create a functional stem cell niche for the delivery of adipose-derived stem cells (ASCs) into diabetic wounds. Human ASCs are encapsulated into the in situ crosslinked hydrogels and cultured in a 3D topography. The encapsulated cells are well attached and spread inside the hydrogels, retaining viability, proliferation, and metabolic activity up to three weeks in vitro. Allogeneic ASCs are delivered to diabetic wounds by this hydrogel vehicle. It is found that stem cell retention is significantly improved in vivo with vehicle-mediated delivery. The ASC-hydrogel-based treatment decreases inflammatory cell infiltration, enhances neovascularization, and remarkably accelerates wound closure in diabetic mice. Together, these findings suggest this conveniently-applicable ASC-hydrogel-based skin substitute provides a promising potential for the treatment of chronic diabetic wounds.

Breast reconstruction proceeding cancer treatment carries risk, regardless of the type of surgery. From fat grafting, to flap placement, to implants, there is no guarantee that reconstruction will not stimulate breast cancer recurrence. Research in this field is clearly divided into two parts: scientific interventional studies and clinical retrospective evidence. The reconstructive procedure offers hypoxia, a wound microenvironment, bacterial load, adipose derived stem cells; agents shown experimentally to cause increased cancer cell activity. This is compelling scientific evidence which serves to bring uncertainty and fear to the reconstructive procedure. In the absence of clinical evidence, this laboratory literature landscape is now informing surgical choices. Curiously, clinical studies have not shown a clear link between breast cancer recurrence and reconstructive surgery. Where does that leave us? This review aims to analyze the science and the surgery, thereby understanding the oncological fear which accompanies breast cancer reconstruction.

The regenerative abilities of adipose-derived mesenchymal stem cells (ASCs) harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration were evaluated. ASC yield and viability, and expression of most osteogenic, adipogenic, and key regenerative genes were equivalent between the two methods. Cells harvested via UAL showed comparable abilities to enhance cutaneous regeneration and appear suitable for cell therapy and tissue engineering applications., Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31−/CD45−), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.

Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose-derived stromal cells (ASCs) from human lipoaspirate, compared to the standard protocol for harvesting ASCs established in 2001. Human ASC isolation is performed using two methods, and resultant cells are compared through cell yield, cell viability, cell proliferation and regenerative potential. The osteogenic and adipogenic potential of ASCs isolated using both protocols are assessed in vitro and gene expression analysis is performed. The focus of this series of protocols is the regenerative potential of both cell populations in vivo. As such, the two in vivo animal models described are fat graft retention (soft tissue reconstruction) and calvarial defect healing (bone regeneration). The techniques described comprise fat grafting with cell assisted lipotransfer, and calvarial defect creation healed with cell-seeded scaffolds. © 2017 by John Wiley & Sons, Inc. Keywords: adipose derived stromal cell; calvarial defect; digest; fat graft; liposuction

Significance: Wound healing requires a highly orchestrated coordination of processes that are not yet fully understood. Therefore, available clinical therapies are thus far limited in their efficacy in preventing and treating both chronic wounds and scars. Current gene-based therapeutics is largely based on our understanding of the protein-coding genome and proteins involved in known wound healing pathways. Recent Advances: Noncoding RNAs such as microRNAs and long noncoding RNAs have recently been found to be significant modulators of gene expression in diverse cellular pathways. Research has now implicated noncoding RNAs in nearly every stage of the wound healing process, suggesting that they may serve as clinical therapeutic targets. Noncoding RNAs are critical regulators in processes such as angiogenesis and cutaneous cell migration and proliferation, including classically described biological pathways previously attributed to mostly protein constituents. Critical Issues: The complexity and diversity ...

Background aims Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. Methods After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34+CD31–CD45–). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. Results Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. Conclusions Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an “off the shelf” prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration.

Background Adipose-derived stem cells (ASCs) have been identified as a population of multipotent cells with promising applications in tissue engineering and regenerative medicine. ASCs are abundant in fat tissue, which can be safely harvested through the minimally invasive procedure of liposuction. However, there exist a variety of different harvesting methods, with unclear impact on ASC regenerative potential. The aim of this study was thus to compare the functionality of ASCs derived from the common technique of suction-assisted lipoaspiration (SAL) versus resection. Methods Human adipose tissue was obtained from paired abdominoplasty and SAL samples from three female donors, and was processed to isolate the stromal vascular fraction. Fluorescence-activated cell sorting was used to determine ASC yield, and cell viability was assayed. Adipogenic and osteogenic differentiation capacity were assessed in vitro using phenotypic staining and quantification of gene expression. Finally, ASCs were applied in an in vivo model of tissue repair to evaluate their regenerative potential. Results SAL specimens provided significantly fewer ASCs when compared to excised fat tissue, however, with equivalent viability. SAL-derived ASCs demonstrated greater expression of the adipogenic markers FABP-4 and LPL, although this did not result in a difference in adipogenic differentiation. There were no differences detected in osteogenic differentiation capacity as measured by alkaline phosphatase, mineralization or osteogenic gene expression. Both SAL- and resection-derived ASCs enhanced significantly cutaneous healing and vascularization in vivo, with no significant difference between the two groups. Conclusion SAL provides viable ASCs with full capacity for multi-lineage differentiation and tissue regeneration, and is an effective method of obtaining ASCs for cell-based therapies.