Active Vitamin D3 (Calcitriol) Increases Adipose Graft Retention in a Xenograft Model.

BACKGROUND: Autologous fat grafting, although broadly indicated, is limited by unsatisfactory retention and often requires multiple procedures to achieve durable outcomes. Graft survival is strongly influenced by the magnitude and duration of post-engraftment ischemia. Calcitriol is a pleiotropic, safe nutrient with cell-specific influence on viability and metabolic flux. OBJECTIVES: Evaluate the efficacy of activated vitamin D3 (calcitriol) in improving grafting outcomes and examine its mechanisms. METHODS: Lipoaspirate was collected for ex vivo culture (7 unique donors), in vitro bioenergetic analysis (6 unique donors), and in vivo transplantation (5 unique donors). Ex vivo samples were incubated for up to 2 weeks before extraction of the stromal vascular fraction (SVF) for viability or flow cytometry. SVF was collected for Seahorse (Agilent; Santa Clara, CA) analysis of metabolic activity. Human endothelial cell lines were utilized for analyses of endothelial function. In vivo, samples were implanted into athymic mice with calcitriol treatment either (1) once locally or (2) 3 times weekly via intraperitoneal injection. Grafts were assessed photographically, volumetrically, and histologically at 1, 4, and 12 weeks. Hematoxylin and eosin (H&E), Sirius red, perilipin, HIF1α, and CD31 tests were performed. RESULTS: Calcitriol-treated lipoaspirate demonstrated dose-dependent increases in SVF viability and metabolic reserve during hypoxic stress. Calcitriol treatment enhanced endothelial mobility ex vivo and endothelial function in vitro. In vivo, calcitriol enhanced adipocyte viability, reduced fibrosis, and improved vascularity. Continuous calcitriol was sufficient to improve graft retention at 12 weeks (P < .05). CONCLUSIONS: Calcitriol increased fat graft retention in a xenograft model. Calcitriol has potential to be a simple, economical means of increasing fat graft retention and long-term outcomes.

A Step in the Right Direction: A Prospective Randomized, Controlled Crossover Trial of Autologous Fat Grafting for Rejuvenation of the Heel.

BACKGROUND: The shock-absorbing soft tissues of the heel are composed of dermis and specialized fat pads. Heel fat pad atrophy is common and can be painful and debilitating. In our previous work, autologous fat grafting was effective for treating pain from forefoot fat pad atrophy. OBJECTIVES: The authors hypothesized that autologous fat grafting to the heel would relieve pain and improve function in patients with heel fat pad atrophy. METHODS: Patients with heel fat pad atrophy and associated pain were recruited and randomized into 2 groups. Group 1 received autologous fat grafting on enrollment and was followed for 2 years. Group 2 received offloading and activity modification for 1 year, then crossed over, underwent autologous fat grafting, and was followed for 1 year afterward. Outcome measures included ultrasound-measured fat pad and dermal thickness; pedobarograph-measured foot pressures and forces; and patient-reported outcomes as measured by the Manchester Foot Pain and Disability Index. RESULTS: Thirteen patients met the inclusion criteria and completed the study. Seven (12 affected feet) were randomized into Group 1; and 6 (9 affected feet) were randomized into Group 2. The average age was 55 years and BMI was 30.5 kg/m2. Demographics did not significantly differ between groups. Heel fat pad thickness increased after autologous fat grafting but returned to baseline at 6 months. However, autologous fat grafting increased dermal thickness significantly and also increased fat pad thickness under a compressive load compared with controls at 6 and 12 months. Foot pain, function, and appearance were also significantly improved compared with controls at 6 and 12 months. CONCLUSIONS: Autologous fat grafting improved patient-reported foot pain, function, and appearance and may rejuvenate local soft tissues in patients with heel fat pad atrophy.

Adipose stem cells enhance excisional wound healing in a porcine model.

BACKGROUND: Adipose-derived stem cells (ASCs) are capable of secreting regenerative growth factors and replacing multiple tissue types. Although current literature suggests that ASCs accelerate wound healing and reduce scarring, the dose-response relationship has not been adequately investigated in large animals. We sought to establish a porcine model to optimize dose and delivery. METHODS: Four-centimeter circular, full thickness excisional wounds were created on the backs of Yorkshire pigs. Fluorescently labeled allogeneic porcine ASCs were injected into the superficial wound bed and around the wound perimeter at high (3.0 × 106 cells/cm2; n = 8), medium (1.0 × 106 cells/cm2; n = 8), and low (0.3 × 106 cells/cm2; n = 8) doses. Control wounds received saline injections (n = 8) or no treatment (n = 8). Dressings were changed twice per week, and wound closure was tracked by surface area tracing. Animals were sacrificed at 1 and 2 wk. Wounds were harvested for real-time quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and ASC tracking. RESULTS: Labeled ASCs integrated into treated wounds by 1 wk in a dose-dependent fashion. Epithelial coverage was achieved by 14 d in all wounds. Wounds receiving high-dose ASCs exhibited thicker granulating neodermis at 7 d and greater wound contraction at 14 d. real-time quantitative reverse transcriptase polymerase chain reaction revealed improved collagen 1:collagen 3 (Col1:Col3) ratio in the medium-dose group and enhanced α-smooth muscle actin in the high-dose group at 14 d. Western blot demonstrated increased cluster of differentiation 31 protein at 2 wk in wounds receiving >106 cells/cm2. CONCLUSIONS: Doses up to 3.0 × 106 cells/cm2 were well-tolerated. High-dose ASCs accelerate wound contraction, enhance neovascularization, and may improve scar quality in excisional wounds healing by secondary intention. Doses greater than those previously used may be necessary to achieve desired effects.

A Transition to Practice Curriculum for Anesthesiology Fellows.

Graduate medical education trainees must be well-versed in practice management principles and how the application of this knowledge affects their patient care. The lack of exposure of anesthesiology trainees to practice management topics remains an ongoing concern nationally. Given similar feedback regarding education on practice management and financial literacy topics across all of our department's fellowship specialties, a novel pilot curriculum comprising a virtual lecture and workshop series was delivered to anesthesiology fellows throughout the academic years 2020 to 2023. Lecture topics included (1) personal finance and contract negotiation, (2) interview preparation and well-being, (3) conflict management, and (4) diversity and inclusion lectures.

Comparison of Clinically Relevant Adipose Preparations on Articular Chondrocyte Phenotype in a Novel In Vitro Co-Culture Model.

Adipose therapeutics, including isolated cell fractions and tissue emulsifications, have been explored for osteoarthritis (OA) treatment; however, the optimal preparation method and bioactive tissue component for healing has yet to be determined. This in vitro study compared the effects of adipose preparations on cultured knee chondrocytes. De-identified human articular chondrocytes were co-cultured with adipose preparations for 36 or 72 h. Human adipose tissues were obtained from abdominal panniculectomy procedures and processed using three different techniques: enzymatic digestion to release stromal vascular fraction (SVF), emulsification with luer-to-luer transfer (nanofat), and processing in a bead-mill (Lipogems, Lipogems International SpA, Milan, Italy). Gene expression in both chondrocytes and adipose preparations was measured to assess cellular inflammation, catabolism, and anabolism. Results demonstrated that chondrocytes cultured with SVF consistently showed increased inflammatory and catabolic gene expression compared with control chondrocytes at both 36- and 72-h timepoints. Alternatively, chondrocytes co-cultured with either nanofat or bead-mill processed adipose derivatives yielded minimal pro-inflammatory effects and instead increased anabolism and regeneration of cartilage extracellular matrix. Interestingly, nanofat preparations induced transient matrix anabolism while Lipogems adipose consistently demonstrated increased matrix synthesis at both study timepoints after co-culture. This evaluation of the regenerative potential of adipose-derived preparations as a clinical tool for knee OA treatment suggests that mechanically processed preparations may be more efficacious than an isolated SVF cell preparation.

Do Lower Costs for Applicants Come at the Expense of Program Perception? A Cross-Sectional Survey Study of Virtual Residency Interviews.

Background: Travel costs and application fees make in-person residency interviews expensive, compounding existing financial burdens on medical students. We hypothesized virtual interviews (VI) would be associated with decreased costs for applicants compared to in-person interviews (IPI) but at the expense of gathering information with which to assess the program. Objective: To survey senior medical students and postgraduate year (PGY)-1 residents regarding their financial burden and program perception during virtual versus in-person interviews. Methods: The authors conducted a single center, multispecialty study comparing costs of IPI vs VI from 2020-2021. Fourth-year medical students and PGY-1 residents completed one-time surveys regarding interview costs and program perception. The authors compared responses between IPI and VI groups. Potential debt accrual was calculated for 3- and 7-year residencies. Results: Two hundred fifty-two (of 884, 29%) surveys were completed comprising 75 of 169 (44%) IPI and 177 of 715 (25%) VI respondents. The VI group had significantly lower interview costs compared to the IPI group (median $1,000 [$469-$2,050 IQR] $784-$1,216 99% CI vs $3,200 [$1,700-$5,500 IQR] $2,404-$3,996 99% CI, P<.001). The VI group scored lower for feeling the interview process was an accurate representation of the residency program (3.3 [0.5] vs 4.1 [0.7], P<.001). Assuming interview costs were completely loan-funded, the IPI group will have accumulated potential total loan amounts $2,334 higher than the VI group at 2% interest and $2,620 at 6% interest. These differences were magnified for a 7-year residency. Conclusions: Virtual interviews save applicants thousands of dollars at the expense of their perception of the residency program.

Molecular Mechanisms of Adipose Tissue Survival during Severe Hypoxia: Implications for Autologous Fat Graft Performance.

BACKGROUND: Variable retention outcomes remain a significant issue in autologous fat grafting procedures. Among seemingly similar patients, using identical harvesting procedures, variability in graft retention is noted. Recent data suggest that the inherent characteristics of donor adipose tissue dictate graft healing outcomes. The goal of this study was to elucidate intrinsic qualities of human adipose tissue that confer resistance to ischemic stress to therapeutically target such mechanisms and improve overall results of fat grafts. METHODS: Whole fat from 5 female patients was cultured in vitro under severe (1% O2) and mild (8% O2) hypoxic conditions. Microarray analysis of 44 hypoxia-related genes was performed. Perilipin was used to visualize viable adipocytes. Macrophage phenotypes were identified using PCR. RESULTS: Analysis of adipocyte survival with perilipin suggested improved viability for tissue obtained from high BMI donors. Microarray data revealed a significant positive correlation for induced expression of ANGPTL4, a survival gene, and subject BMI (P = 0.0313) during hypoxic conditions whereas HIF1α and HIF2α genes were negatively correlated with donor BMI (P = 0.0003 and 0.0303). Interestingly, induced differentiation of proinflammatory M1 macrophages was negatively correlated with BMI under hypoxia (P = 0.0177). CONCLUSIONS: The innate resilience of adipocytes to hypoxia and relative macrophage activation play a crucial role in fat graft retention. This study suggests that adipose tissue from high BMI donors demonstrates greater resistance to hypoxia-induced apoptosis associated with an increased expression of ANGPTL4. Therefore, therapeutic interventions that target this factor may improve clinical adipose graft survival.

The Architecture of Fat Grafting II: Impact of Cannula Diameter.

BACKGROUND: Fat grafting has emerged as the treatment of choice for soft-tissue augmentation and reconstruction. Variability of volume retention remains the greatest challenge for this technique, often requiring multiple operations to achieve the desired volume. Graft that is placed greater than 2 mm from the recipient bed will undergo necrosis. Improved understanding of the architecture of fat within the recipient bed is paramount to improving outcomes. The impact of cannula diameter on graft architecture is unknown. METHODS: Fat was harvested by liposuction and stained with methylene blue. Stained fat was grafted into 4 × 2 × 1-cm sections of excised abdominal tissue with 12-, 14-, 16-, and 19-gauge Coleman cannulas at three different volumes: 0.1, 0.5, and 1.0 cc. Each tissue block was sectioned for stained graft visualization. The diameter of each deposit and percentage with a radius greater than 2 mm were recorded. RESULTS: With an injection volume of 0.1 cc, no fat deposits had a radius greater than 2 mm, regardless of cannula size. A graft volume of 0.5 cc created globules greater than 2 mm with larger cannulas (0 percent with 19-gauge, 2.9 percent with 16-gauge, 6.1 percent with 14-gauge, and 4.3 percent with 12-gauge). Injecting 1.0 cc resulted in a significant increase in the percentage of fat parcels expected to undergo central necrosis (16 percent with 19-gauge, 21 percent with 16-gauge, 26 percent with 14-gauge, and 44 percent with 12-gauge). CONCLUSIONS: Injection cannulas of 14-gauge or larger are more likely to create deposits with dimensions that may be susceptible to central necrosis when injecting 1.0 cc per pass. Smaller cannula sizes or lower volumes per pass should be considered. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Lymphatic Endothelial Cells under Mechanical Stress: Altered Expression of Inflammatory Cytokines and Fibrosis.

BACKGROUND: Secondary lymphedema, resulting from damage to lymphatic vessels, is a common sequela following surgical removal of lymph nodes for cancer. Current therapeutics for treating lymphedema are limited and further research on underlying causes is warranted. Published studies on molecular mechanisms of lymphedema primarily focus on lymphatic endothelial cells (LECs), which comprise the innermost lining of lymphatic capillaries and collecting vessels. However, traditional static culture of LECs may not adequately recapitulate the lymphedemous cell phenotype as transcriptomal comparison of human dermal LECs has shown significant differences in ex vivo and in vitro LEC gene expression. In this study, we designed a dynamic culture system, in which LECs were exposed to physiologic and excess mechanical strain to determine if native and lymphedemous phenotypes could be reproduced in vitro. METHODS AND RESULTS: Purified human LECs were cultured in silicon dishes and subjected to 0% (control), 4%, and 8% mechanical strain for 72 hours. Our results indicate that control and stretched LECs maintained a mature phenotype. Extreme stretching at 8% strain significantly increased LEC proliferation and significantly increased Prox1 expression, suggesting a lymphedemous cell phenotype resulting with lymphangiogenesis. CONCLUSION: Mechanical strain reinforced a mature lymphatic phenotype and excess strain promoted lymphangiogenesis, while altering collagen deposition and cytokine secretion.

Controlled dexamethasone delivery via double-walled microspheres to enhance long-term adipose tissue retention.

Current materials used for adipose tissue reconstruction have critical shortcomings such as suboptimal volume retention, donor-site morbidity, and poor biocompatibility. The aim of this study was to examine a controlled delivery system of dexamethasone to generate stable adipose tissue when mixed with disaggregated human fat in an athymic mouse model for 6 months. The hypothesis that the continued release of dexamethasone from polymeric microspheres would enhance both adipogenesis and angiogenesis more significantly when compared to the single-walled microsphere model, resulting in long-term adipose volume retention, was tested. Dexamethasone was encapsulated within single-walled poly(lactic-co-glycolic acid) microspheres (Dex SW MS) and compared to dexamethasone encapsulated in a poly(lactic-co-glycolic acid) core surrounded by a shell of poly-l-lactide. The double-walled polymer microsphere system in the second model was developed to create a more sustainable drug delivery process. Dexamethasone-loaded poly(lactic-co-glycolic acid) microspheres (Dex SW MS) and dexamethasone-loaded poly(lactic-co-glycolic acid)/poly-l-lactide double-walled microspheres (Dex DW MS) were prepared using single and double emulsion/solvent techniques. In vitro release kinetics were determined. Two doses of each type of microsphere were examined; 50 and 27 mg of Dex MS and Dex DW MS were mixed with 0.3 mL of human lipoaspirate. Additionally, 50 mg of empty MS and lipoaspirate-only controls were examined. Samples were analyzed grossly and histologically after 6 months in vivo. Mass and volume were measured; dexamethasone microsphere-containing samples demonstrated greater adipose tissue retention compared to the control group. Histological analysis, including hematoxylin and eosin and CD31 staining, indicated increased vascularization (p < 0.05) within the Dex MS-containing samples. Controlled delivery of adipogenic factors, such as dexamethasone via polymer microspheres, significantly affects adipose tissue retention by maintaining healthy tissue formation and vascularization. Dex DW MS provide an improved model to former Dex SW MS, resulting in notably longer release time and, consequently, larger volumes of adipose retained in vivo. The use of microspheres, specifically double-walled, as vehicles for controlled drug delivery of adipogenic factors therefore present a clinically relevant model of adipose retention that has the potential to greatly improve soft tissue repair.

The Architecture of Fat Grafting: What Lies beneath the Surface.

BACKGROUND: Fat grafting is a powerful procedure limited by unpredictable volume loss. Grafted tissue survives via plasmatic imbibition until neovascularization occurs; therefore, fat that is deposited more than 0.2 cm from capillaries will undergo central necrosis. This study aims to determine the architecture of fat deposits within the recipient bed following fat grafting. METHODS: Fat was harvested by liposuction and stained with methylene blue. Stained fat was grafted into 4 × 4 × 2-cm sections of pannus tissue at graft-to-recipient volume ratios ranging from 1:10 to 1:1. Each tissue block was sectioned for stained graft visualization. The diameter of each deposit and the percentage with a radius greater than 0.2 cm were recorded. RESULTS: Average tunnel diameter was 0.20 ± 0.01 cm at a graft-to-recipient ratio of 1:10, 0.25 ± 0.01 cm at 1:8, 0.26 ± 0.01 cm at 1:6, 0.31 ± 0.01 cm at 1:4, 0.40 ± 0.01 cm at 1:2, and 0.57 ± 0.02 cm at 1:1. All comparisons reached statistical significance (p ≤ 0.05) except 1:8 versus 1:6 (p = 0.96). The percentage of fat parcels with a radius greater than 0.2 cm was 3.0 percent at 1:10, 5.3 percent at 1:8, 9.5 percent at 1:6, 20.9 percent at 1:4, 42.0 percent at 1:2, and 68.3 percent at 1:1. All percentage comparisons were significant except 1:10 versus 1:8 (p = 0.15). CONCLUSION: As the total volume transferred increases, grafted deposits coalesce to form larger globules, particularly at ratios beyond 1:4, likely contributing to central necrosis and subsequent volume loss. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Long-term Patency of Primary Arterial Repair and the Modified Cold Intolerance Symptom Severity Questionnaire.

BACKGROUND: The goal of this study was to assess the long-term arterial patency of repaired arteries in the upper extremity and any morbidity resulting from the subsequent occlusion of these vessels. Concurrently, a new questionnaire, the modified Cold Intolerance Symptom Severity (mod CISS) questionnaire, was developed to allow for better assessment of cold intolerance. METHODS: Thirteen patients who had undergone repair of the radial (4 patients), ulnar (6 patients), brachial (1 patient), digital (1), and an undefined lower arm artery (1) were examined using questionnaires, physical examination, and high-resolution ultrasound. RESULTS: Outcome measures that were statistically significantly worse in the group of patients who presented with nerve injuries included cold intolerance symptoms, Disabilities of the Arm, Shoulder, and Hand score, Michigan Hand Questionnaire, and grip strength (middle setting on dynamometer). The results from the mod CISS correlated with high statistical significance with the results of the CISS score for the injured hand. Of note, wrist extension was significantly better with patent arteries. CONCLUSIONS: Sixty-seven percent of arterial repairs remained patent at 6 years (mean) follow-up. The presence of nerve injury has a higher impact on the outcome metrics assessed in this study than arterial patency. Our modification of the CISS score enhances its utility as a survey of cold intolerance.