The Effectiveness of a Non-Invasive Shot Blocking Device for Reducing Pain of In-office Injections in Hand Surgery.

Background: The gate control theory asserts that non-painful stimuli can block pain perception. The ShotBlocker™ device is a plastic disk with blunt projections that rests on the skin, and we hypothesize that it will reduce pain during hand injections. Methods: This is a prospective randomized trial of 117 patients undergoing injections for common hand conditions. Patients were randomized into 3 groups: device, placebo (device with projections removed), and control. Patients recorded on an analog pain scale the pain severity of the injection, as well as their most recent tetanus shot. A normalized pain score was obtained from the difference between the injection and tetanus shot pain scores. The mean non-normalized and normalized scores for each treatment group were compared to the control group using the Wilcoxon signed rank test. Results: There were 91 women and 26 men. Common diagnoses included trigger finger (n = 53), DeQuervain's tendonitis (n = 33), and basal joint arthritis (n = 22). The groups did not differ significantly in age, gender, or diagnosis. Mean pain score in the device group was 5.2 out of 10, and it was 5.7 for the control group. The normalized pain score in the device group was significantly lower than the control group. Normalized and non-normalized pain scores for the placebo group were not significantly lower than the control group. Conclusions: The shot blocking device effectively reduced pain of injection versus controls when pain scores were normalized for pain tolerance. The modified device did not reduce the pain of injection, suggesting that gate control is the mechanism of action.

Dynamic Rheology for the Prediction of Surgical Outcomes in Autologous Fat Grafting.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Murine Dermal Fibroblast Isolation by FACS.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis.

BACKGROUND: Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. METHODS: Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. RESULTS: In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. CONCLUSIONS: BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.

Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes.

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.

What Makes a Plastic Surgery Residency Program Attractive? An Applicant's Perspective.

BACKGROUND: Plastic surgery is among the most competitive specialties in medicine, but little is known about the attributes of programs that are most attractive to successful applicants. This study aimed to understand and provide insights regarding program characteristics that are most influential to students when ranking plastic surgery programs. METHODS: An anonymous online survey was conducted with newly matched plastic surgery residents for the integrated and combined Match in 2012 and 2013. Subjects were queried regarding their demographics, qualifications, application experiences, and motivations for residency program selection. RESULTS: A total of 92 of 245 matched plastic surgery residents (38 percent) responded to the survey. The perception of resident happiness was the most positive factor influencing program ranking, followed by high operative volume, faculty mentorship, and strong research infrastructure. Perception of a program as "malignant" was the most negative attribute. Applicants with Step 1 scores greater than 245 received significantly more interviews (p =0.001) and considered resident benefits less important (p < 0.05), but geographic location more important (p =0.005). Applicants who published more than two articles also received more interviews (p =0.001) and ranked a strong research infrastructure and program reputation as significantly more important (p < 0.05). Forty-two percent of applicants completed an away rotation at the program with which they matched, and these applicants were more likely to match at their number one ranked program (p = 0.001). CONCLUSIONS: Plastic surgery applicants have differing preferences regarding the ideal training program, but some attributes resonate. These trends can guide programs for improvement in attracting the best applicants.

RNA Sequencing for Identification of Differentially Expressed Noncoding Transcripts during Adipogenic Differentiation of Adipose-Derived Stromal Cells.

BACKGROUND: Adipose-derived stromal cells represent a relatively abundant source of multipotent cells, with many potential applications in regenerative medicine. The present study sought to demonstrate the use of RNA sequencing in identifying differentially expressed transcripts, particularly long noncoding RNAs, associated with adipogenic differentiation to gain a clearer picture of the mechanisms responsible for directing adipose-derived stromal cell fate toward the adipogenic lineage. METHODS: Human adipose-derived stromal cells were cultured in adipogenic differentiation media, and RNA was harvested at days 0, 1, 3, 5, and 7. Directional RNA sequencing libraries were prepared and sequenced. Paired-end reads were mapped to the human genome reference sequence hg19. Transcriptome assembly was performed and significantly differentially expressed transcripts were identified. Gene ontology term analysis was then performed to identify coding and noncoding transcripts of interest. Differential expression was verified by quantitative real-time polymerase chain reaction. RESULTS: Of 2868 significantly differentially expressed transcripts identified, 207 were noncoding. Enriched gene ontology terms among up-regulated coding transcripts notably reflected differentiation toward the adipogenic lineage. Enriched gene ontology terms among down-regulated coding transcripts reflected growth arrest. Guilt-by-association analysis revealed noncoding RNA candidates with potential roles in the process of adipogenic differentiation. CONCLUSIONS: The precise mechanisms that guide lineage-specific differentiation in multipotent cells are not yet fully understood. Defining long noncoding RNAs associated with adipogenic differentiation allows for potential manipulation of regulatory pathways in novel ways. The authors present RNA sequencing as a powerful tool for expanding the understanding of adipose-derived stromal cells and developing novel applications within regenerative medicine.

High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells.

Adipose tissue contains an abundant source of multipotent mesenchymal cells termed "adipose-derived stromal cells" (ASCs) that hold potential for regenerative medicine. However, the heterogeneity inherent to ASCs harvested using standard methodologies remains largely undefined, particularly in regards to differences across donors. Identifying the subpopulations of ASCs predisposed toward differentiation along distinct lineages holds value for improving graft survival, predictability, and efficiency. Human ASCs (hASCs) from three different donors were independently isolated by density-based centrifugation from adipose tissue and maintained in culture or differentiated along either adipogenic or osteogenic lineages using differentiation media. Undifferentiated and differentiated hASCs were then analyzed for the presence of 242 human surface markers by flow cytometry analysis. By comprehensively characterizing the surface marker profile of undifferentiated hASCs using flow cytometry, we gained novel insights into the heterogeneity underlying protein expression on the surface of cultured undifferentiated hASCs across different donors. Comparison of the surface marker profile of undifferentiated hASCs with hASCs that have undergone osteogenic or adipogenic differentiation allowed for the identification of surface markers that were upregulated and downregulated by osteogenic or adipogenic differentiation. Osteogenic differentiation induced upregulation of CD164 and downregulation of CD49a, CD49b, CD49c, CD49d, CD55, CD58, CD105, and CD166 while adipogenic differentiation induced upregulation of CD36, CD40, CD146, CD164, and CD271 and downregulation of CD49b, CD49c, CD49d, CD71, CD105, and CD166. These results lend support to the notion that hASCs isolated using standard methodologies represent a heterogeneous population and serve as a foundation for future studies seeking to maximize their regenerative potential through fluorescence-activated cell sorting-based selection before therapy.

Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent.

BACKGROUND: Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. METHODS: Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 µl of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. RESULTS: Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 µl of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone CONCLUSIONS: : The authors' study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.

Emerging drugs for the treatment of wound healing.

INTRODUCTION: Wound healing can be characterized as underhealing, as in the setting of chronic wounds, or overhealing, occurring with hypertrophic scar formation after burn injury. Topical therapies targeting specific biochemical and molecular pathways represent a promising avenue for improving and, in some cases normalizing, the healing process. AREAS COVERED: A brief overview of both normal and pathological wound healing has been provided, along with a review of the current clinical guidelines and treatment modalities for chronic wounds, burn wounds and scar formation. Next, the major avenues for wound healing drugs, along with drugs currently in development, are discussed. Finally, potential challenges to further drug development, and future research directions are discussed. EXPERT OPINION: The large body of research concerning wound healing pathophysiology has provided multiple targets for topical therapies. Growth factor therapies with the ability to be targeted for localized release in the wound microenvironment are most promising, particularly when they modulate processes in the proliferative phase of wound healing.

Scarless wound healing: chasing the holy grail.

Over 100 million patients acquire scars in the industrialized world each year, primarily as a result of elective operations. Although undefined, the global incidence of scarring is even larger, extending to significant numbers of burn and other trauma-related wounds. Scars have the potential to exert a profound psychological and physical impact on the individual. Beyond aesthetic considerations and potential disfigurement, scarring can result in restriction of movement and reduced quality of life. The formation of a scar following skin injury is a consequence of wound healing occurring through reparative rather than regenerative mechanisms. In this article, the authors review the basic stages of wound healing; differences between adult and fetal wound healing; various mechanical, genetic, and pharmacologic strategies to reduce scarring; and the biology of skin stem/progenitor cells that may hold the key to scarless regeneration.

Assessment of viability of human fat injection into nude mice with micro-computed tomography.

Lipotransfer is a vital tool in the surgeon's armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.(1) However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. (1-3) To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes. Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Osteoclast derivation from mouse bone marrow.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Wound healing: an update.

Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

The role of stem cells in aesthetic surgery: fact or fiction?

BACKGROUND: Stem cells are attractive candidates for the development of novel therapies, targeting indications that involve functional restoration of defective tissue. Although most stem cell therapies are new and highly experimental, there are clinics around the world that exploit vulnerable patients with the hope of offering supposed stem cell therapies, many of which operate without credible scientific merit, oversight, or other patient protection. METHODS: The authors review the potential and the drawbacks of incorporation of stem cells in cosmetic procedures. A review of U.S. Food and Drug Administration-approved indications and ongoing clinical trials with adipose stem cells is provided. Furthermore, a "snapshot" analysis of Web sites using the search terms "stem cell therapy" or "stem cell treatment" or "stem cell facelift" was performed. RESULTS: Despite the protective net cast by regulatory agencies such as the U.S. Food and Drug Administration and professional societies such as the American Society of Plastic Surgeons, the authors are witnessing worrying advertisements for procedures such as stem cell face lifts, stem cell breast augmentations, and even stem cell vaginal rejuvenation. The marketing and promotion of stem cell procedures in aesthetic surgery is not adequately supported by clinical evidence in the majority of cases. CONCLUSIONS: Stem cells offer tremendous potential, but the marketplace is saturated with unsubstantiated and sometimes fraudulent claims that may place patients at risk. With plastic surgeons at the forefront of stem cell-based regenerative medicine, it is critically important that they provide an example of a rigorous approach to research, data collection, and advertising of stem cell therapies.

Gene expression in fetal murine keratinocytes and fibroblasts.

BACKGROUND: Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis--the keratinocyte and fibroblast--during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration. MATERIALS AND METHODS: Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate<2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways. RESULTS: By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts. CONCLUSIONS: Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention.

BACKGROUND: Fat grafting has become increasingly popular for the correction of soft-tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, the authors compare the biological properties of fat following injection using two methods. METHODS: Lipoaspiration samples were obtained from five female donors, and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low-shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over 12 weeks after injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically. RESULTS: Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at the 4-, 6-, 8-, and 12-week time points. This corresponded to significantly greater histologic scores for healthy fat and lower scores for injury following injection with the device. CONCLUSION: Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and the authors' in vitro and in vivo data both support the use of the automated, low-shear devices compared with the modified Coleman technique.