The Impact of Obesity on Success of Immediate Lymphatic Reconstruction for Prevention of Breast Cancer-Related Lymphedema.

BACKGROUND: Breast cancer-related lymphedema (BRCL) is a potential sequela of high-risk breast cancer treatment. Preventive treatment with immediate lymphatic reconstruction (ILR) at the time of axillary lymph node dissection (ALND) has emerged as the standard of care; however, there is relatively little known about factors that may contribute to procedural failure. METHODS: A retrospectively maintained, institutional review board-approved study followed patients who underwent ILR at the time of ALND at our tertiary care center between May 2018 and May 2023. Patients who presented for at least one follow-up visit in our multidisciplinary lymphedema clinic met the criteria for inclusion. Patients who developed lymphedema despite ILR and potential contributing factors were further explored. RESULTS: 349 patients underwent ILR at our institution between May 2018 and May 2023. 341 of these patients have presented for follow-up in our multidisciplinary lymphedema clinic. 32 (9.4%) patients developed lymphedema despite ILR. This cohort was significantly more likely to be obese (56% vs 35%, P = 0.04). Multivariate logistic regression demonstrates increased odds of procedural failure in patients with a body mass index (BMI) ≥30 kg/m2 (odds ratio 2.6 [1.2-5.5], P = 0.01). CONCLUSION: These data comment upon our institutions outcomes following ILR. Patients who develop lymphedema despite ILR tend to have a higher BMI, with a significantly increased risk in patients with a BMI of 30 or greater. Consideration of these data is critical for preprocedural counseling and may support a BMI cutoff when considering candidacy for ILR going forward, as well as when optimizing failures for secondary lymphedema procedures.

Immediate Lymphatic Reconstruction in 77 Consecutive Breast Cancer Patients: 2-year Follow-up.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a cyclical, progressive disease that begins at the time of axillary dissection and worsens in the setting of adjuvant oncologic therapies. The paradigm of lymphedema management in these patients is shifting from therapeutic surgeries and decongestive therapy to preventative surgery with immediate lymphatic reconstruction (ILR). METHODS: After institutional review board approval, a prospective database was maintained of all patients undergoing ILR. Patients were excluded if they had preoperative lymphedema or expired during the study period. All ILR were performed by the senior author. A control group was established with standardized physician delivered phone surveys of patients who had axillary dissection for breast cancer (same oncologic surgeon cohort) prior to the implementation of ILR at the same institution. The study and control groups were matched based on history of adjuvant radiation and body mass index. RESULTS: A cohort of patients between 2016 and 2019 with 2 years of follow-up after undergoing ILR (77 patients) were matched with those who did not undergo lymphatic reconstruction (94 patients). The incidence of lymphedema in the study group undergoing ILR was 10% (N = 8). In comparison, the incidence in the cohort who did not undergo lymphatic reconstruction was 38% (N = 36; p < 0.01). Patients with ILR had 92% lower odds of developing lymphedema (p < 0.01). CONCLUSION: ILR can significantly reduce the risk of developing BRCL in high-risk patients at 2 years of follow-up. Patients receiving adjuvant radiation therapy are more likely to develop BCRL after ILR compared with those who do not. Ongoing studies include investigation aimed at identifying patients most at risk for the development of BRCL to help target intervention as well as elucidate factors that contribute to the success of ILR.

Reliable Location of Upper Extremity Lymphatic Channels for Use in Immediate Lymphatic Reconstruction.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a limiting sequelae of breast cancer treatment that may negatively impact 30% to 50% of high-risk breast cancer survivors. Risk factors for development of BCRL include axillary lymph node dissection (ALND), and recently, axillary reverse lymphatic mapping and immediate lymphovenous reconstruction (ILR) at time of ALND have been implemented to prevent BCRL. Reliable anatomy of neighboring venules has been commented on in the literature; however, little information exists about anatomical location of local lymphatic channels amenable for bypass. METHODS: After institutional review board approval, patients who underwent ALND with axillary reverse lymphatic mapping and ILR at a tertiary cancer center from November 2021 to August 2022 were applicable for this study. The location and number of lymphatic channels used for ILR were identified and measured intraoperatively with the arm abducted to 90 degrees and soft tissue under no tension. Four measurements were taken to localize each lymphatic and were based on relationship with reliable anatomic landmarks including 4th rib, anterior axillary line, and lower border of the pectoralis major muscle. Demographics, oncologic treatments, intraoperative factors, and outcomes were prospectively maintained. RESULTS: Twenty-seven patients met inclusion for this study by August 2022 with a total of 86 lymphatic channels identified. Patients were on average 50 ± 12 years old with a body mass index of 30 ± 6 and had an average of 1 vein and 3 identifiable lymphatic channels amenable to bypass. Seventy percent of lymphatic channels were found in a cluster of 2 or more channels. The average horizontal location was 4.5 ± 1.4 cm lateral to the 4th rib. The average vertical location was 1.3 ± 0.9 cm from the superior border of the 4th rib. CONCLUSIONS: These data comment upon intraoperatively identified and consistent location of upper extremity lymphatic channels used for ILR. These lymphatic channels are often found in clusters with 2 or more lymphatic channels at the same location. Such insight may aid in easier intraoperative identification of amenable vessels for the unexperienced surgeon, decrease in intraoperative time, and higher success of ILR.

Efficacy of Immediate Lymphatic Reconstruction in Prevention of Breast Cancer-Related Lymphedema.

INTRODUCTION: Breast cancer-related lymphedema (BCRL) is a chronic condition that can negatively affect the quality of life of breast cancer survivors. Immediate lymphatic reconstruction (ILR) at the time of axillary lymph node dissection is emerging as a technique for the prevention of BCRL. This study compared the incidence of BRCL in patients who received ILR and those who were not amenable to ILR. METHODS: Patients were identified through a prospectively maintained database between 2016 and 2021. Some patients were deemed nonamenable to ILR due to a lack of visualized lymphatics or anatomic variability (eg, spatial relationships or size discrepancies). Descriptive statistics, independent t test, and Pearson χ 2 test were used. Multivariable logistic regression models were created to assess the association between lymphedema and ILR. A loose age-matched subsample was created for subanalysis. RESULTS: Two hundred eighty-one patients were included in this study (252 patients who underwent ILR and 29 patients who did not). The patients had a mean age of 53 ± 12 years and body mass index of 28.6 ± 6.8 kg/m 2 . The incidence of developing lymphedema in patients with ILR was 4.8% compared with 24.1% in patients who underwent attempted ILR without lymphatic reconstruction ( P = 0.001). Patients who did not undergo ILR had significantly higher odds of developing lymphedema compared with those who had ILR (odds ratio, 10.7 [3.2-36.3], P < 0.001; matched OR, 14.2 [2.6-77.9], P < 0.001). CONCLUSIONS: Our study showed that ILR was associated with lower rates of BCRL. Further studies are needed to determine which factors place patients at highest risk of developing BCRL.

Relationship of Adjuvant Radiation and the Lymphedema Index: A Valuable Trend to Follow Subclinical Lymphedema in Patients After Immediate Lymphatic Reconstruction.

BACKGROUND: Immediate lymphaticovenular bypass (immediate lymphatic reconstruction [ILR]) at the time of axillary lymph node dissection has emerged as a preventative paradigm to decrease the incidence of breast cancer-related lymphedema in high-risk patients. These patients are often treated with adjuvant therapies, including radiation. Bioimpedance spectroscopy is a validated tool for trending breast cancer-related lymphedema and identifying subclinical disease. Lymphedema Index (LDEX) values are commonly obtained in ILR patients; however, postoperative trends and relationships with adjuvant treatments are yet to be reported in the literature. METHODS: After International Review Board approval, 100 consecutive patients underwent axillary lymph node dissection with axillary reverse lymphatic mapping and ILR at a tertiary cancer center. These patients were then followed prospectively in a multidisciplinary lymphedema clinic at 3-month intervals with clinical examination, circumferential limb girth measurements and bioimpedance spectroscopy (LDEX). RESULTS: Seventy-two patients met inclusion for analysis at 3 months, 60 at 6 months, 51 at 9 months, 45 at 12 months, 41 at 15 months, and 22 at 18 months. A majority of the patients included underwent adjuvant radiation. Average LDEX score for patients who developed lymphedema was 3.02 at 3 months, at 29.1 months, 17.8 at 9 months, 15.05 at 12 months, 18.75 at 15 months, and 7.7 at 18 months. Patients who went on to develop lymphedema had a higher LDEX score at 6 months (29.1 vs 3.20, P = 0.1329), which reached a significant difference beginning at 9 months (17.8 vs 3.19, P = 0.0004). All patients who went on to develop lymphedema received adjuvant radiation. CONCLUSIONS: These data provide valuable insight guiding follow-up after ILR. Six-month LDEX is much higher in patients who developed lymphedema, all of which underwent adjuvant radiation therapy, which correlates with the time of completion of their treatment. Average LDEX value after this remains significantly higher in this population. Patients who demonstrate this increase in LDEX and received adjuvant radiation are at highest risk to develop lymphedema despite ILR. All patients who developed lymphedema despite ILR had adjuvant radiation, and this is likely a contributing factor. Injury from adjuvant radiation and its impact after ILR is not insignificant and warrants further studies.

Reverse Lymphatic Mapping and Immediate Microsurgical Lymphatic Reconstruction Reduces Early Risk of Breast Cancer-Related Lymphedema.

BACKGROUND: Breast cancer-related lymphedema is a progressive disease that poses tremendous physical, psychosocial, and financial burden on patients. Immediate lymphaticovenular anastomosis at the time of axillary lymph node dissection is emerging as a potential therapeutic paradigm to decrease the incidence of breast cancer-related lymphedema in high-risk patients. METHODS: Eighty-one consecutive patients underwent reverse lymphatic mapping and, when feasible, supermicrosurgical immediate lymphaticovenular anastomosis at the time of axillary lymph node dissection at a tertiary care cancer center. Patients were followed prospectively in a multidisciplinary lymphedema clinic (plastic surgery, certified lymphatic therapy, dietary, case management) at 3-month intervals with clinical examination, circumferential limb girth measurements, and bioimpedance spectroscopy. An institutional control cohort was assessed for the presence of objectively diagnosed and treated breast cancer-related lymphedema. Data were analyzed by a university statistician. RESULTS: Seventy-eight patients met inclusion, and 66 underwent immediate lymphaticovenular anastomosis. Mean follow-up was 250 days. When compared to a retrospective control group, the rate of lymphedema in patients who underwent immediate lymphaticovenular anastomosis was significantly lower (6 percent versus 44 percent; p < 0.0001). Patients with 6-month follow-up treated with combined adjuvant radiation therapy and chemotherapy had significantly greater risk of developing breast cancer-related lymphedema (p = 0.04) compared to those without combined adjuvant therapy. Arborized anastomotic technique had a statistically shorter operative time than end-to-end anastomosis (p = 0.005). CONCLUSIONS: This series of consecutive patients demonstrate a 6 percent incidence of early-onset breast cancer-related lymphedema with immediate lymphaticovenular anastomosis and an increased risk in those undergoing combined adjuvant treatment. These early data represent an encouraging and substantial decrease of breast cancer-related lymphedema in high-risk patients. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Hyaluronic acid synthesis is required for zebrafish tail fin regeneration.

Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-ß signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impairs tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduces the expression of genes associated with wound epithelium and blastema function. Treatment with glycogen synthase kinase 3 inhibitors rescues 4-MU-induced defects in cell proliferation and tail regeneration, while restoring a subset of wound epithelium and blastema markers. Our findings demonstrate a role for HA biosynthesis in zebrafish tail regeneration and delineate its epistatic relationships with other regenerative processes.

Nipple-areola complex reconstruction improves psychosocial and sexual well-being in women treated for breast cancer.

INTRODUCTION: Women choose to undergo nipple-areola complex (NAC) reconstruction as part of breast reconstruction following breast cancer treatment. However, the effect of this procedure on psychosocial and sexual well-being is not well studied. The present study aimed to evaluate how NAC reconstruction affects patient satisfaction with regard to psychosocial and sexual well-being. METHODS: A retrospective chart review was performed for all patients who underwent NAC reconstruction at Magee-Women's Hospital from January 1, 2004 to July 31, 2011. A letter and questionnaire based on the BREAST-Q were mailed to patients to request their participation in the study. Patient satisfaction and health-related quality of life were measured before and after NAC reconstruction. RESULTS: In total, 107 of 328 patients (32.6%) completed the survey. The BREAST-Q scale score for satisfaction with outcome following NAC reconstruction was 85.1 ± 15.8, with higher satisfaction scores for patients with a follow-up of <1.5 years than those with a follow-up of >2.5 years (82.5 ± 21.7 vs. 69.5 ± 19.5; p < 0.01). No significant differences were found in satisfaction with the breast mound before and after NAC reconstruction. Women scored significantly higher on the psychosocial and sexual well-being scales after NAC reconstruction (p < 0.002 and 0.00004, respectively). CONCLUSIONS: This study indicates that patients are highly satisfied after undergoing NAC reconstruction. Satisfaction with the procedure, however, may decrease over time. NAC reconstruction significantly contributes to patient psychosocial and sexual well-being, and this effect did not change over time. NAC reconstruction improves patient outcomes in those who choose to undergo the procedure.

Osteogenic differentiation of human multipotent mesenchymal stromal cells.

A comprehensive knowledge of the molecular biology underlying osteogenic differentiation in a controlled, laboratory setting may promise optimization of future cell-based tissue engineering strategies for clinical problems. The scope of this review encompasses a discussion of the methodology utilized to perform such studies. Our laboratory routinely performs both in vitro and in vivo assays underlying osteogenic differentiation, and the widespread use of singular methodology across multiple investigators and institutions promises great advancements for the skeletal tissue engineering community.

Elucidating mechanisms of osteogenesis in human adipose-derived stromal cells via microarray analysis.

INTRODUCTION: The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation. METHODS: We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining. RESULTS: IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium. CONCLUSIONS: Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.

Different endogenous threshold levels of Fibroblast Growth Factor-ligands determine the healing potential of frontal and parietal bones.

In the skull vault, neural crest derived frontal bones have an increased healing capacity and higher expression levels of Fibroblast Growth Factor-ligands as compared to mesoderm-derived parietal bones. Thus, we asked whether Fibroblast Growth Factor-ligands are responsible for the superior healing potential of frontal bones. Parietal defects in juvenile and adult mice treated with Fibroblast Growth Factor-2, -9 and -18 showed increased bone regeneration, comparable to frontal defects. Immunohistochemistry revealed increased recruitment of osteoprogenitors and activation of FGF-signaling pathways in FGF-treated parietal defects. Conversely, calvarial defects in Fgf-9(+/-) and Fgf-18(+/-) mice showed impaired calvarial healing which could be rescued by exogenous Fibroblast Growth Factor-ligands. Moreover, by utilizing Wnt1Cre/R26R mice, the migration and contribution of dura mater and pericranium cells to calvarial healing could be demonstrated. Taken together our results demonstrated that different endogenous threshold levels of Fibroblast Growth Factor-ligands in frontal and parietal bones have a profound impact on calvarial regeneration. The present study thereby opens new avenues for translational medicine.

A nonviral minicircle vector for deriving human iPS cells.

Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

Human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein-2 during in vitro osteogenic differentiation.

BACKGROUND: Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells. METHODS: Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody. RESULTS: Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody. CONCLUSIONS: These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.

Origin matters: differences in embryonic tissue origin and Wnt signaling determine the osteogenic potential and healing capacity of frontal and parietal calvarial bones.

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest-derived frontal and paraxial mesodermal-derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest-derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal-derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest-derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3beta and increases the nuclear levels of beta-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest-derived frontal bone.

Bone regeneration and repair.

In the face of mounting clinical demand, and armed with reconstructive techniques that are technically challenging and frequently result in suboptimal patient outcomes, increasing focus is being placed on tissue engineering and regenerative medicine as a potential source of novel skeletal reconstructive approaches. Specifically, evidence is accumulating that highlights the promise of osteoprogenitor cell-based reconstructive strategies to meet the needs of an expanding patient population. Historically, the study of cell and molecular biology guiding physiologic and pathologic skeletal development, as well as endogenous bone regeneration following injury, has provided a wealth of information that lends insight toward potential parallel processes that may regulate the osteogenic differentiation of progenitor cells. Multiple progenitor cell populations are now known to possess a capacity to undergo robust osteogenic differentiation in the presence of appropriate environmental cues (hESC, BMSC, ASC, etc.) Recent investigations have put forth multiple advantages of ASC relative to BMSC. Of note, ASC exist in relative abundance, lack the need for in vitro expansion prior to utilization, and can be harvested with relative ease and reduced donor morbidity. Collectively, these factors, paired with promising in vitro and in vivo observations that speak toward the substantial osteogenic potential of ASC, have spurred enthusiasm to pursue the application of ASC in the maturation of skeletal tissue engineering applications. Yet, elucidating what structural and functional properties of scaffolds designed for ASC-mediated skeletal tissue engineering applications (porosity, pore size, composition, mechanical stability, degradation kinetics, etc.), as well as evolving our understanding and capacity to deliver spatiotemporally specific pro-osteogenic targeted molecular manipulation to progenitor cells, remain important hurdles to clear. The scope of this review encompasses the current state of ongoing investigations along these fronts, as well as what future direction will be critical to the transition of cell-based skeletal tissue engineering strategies to the bedside.

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells.

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

Tissue harvest by means of suction-assisted or third-generation ultrasound-assisted lipoaspiration has no effect on osteogenic potential of human adipose-derived stromal cells.

BACKGROUND: Human adipose-derived stromal cells readily undergo osteogenic differentiation in vitro and in vivo. Thus, interest in their potential role in skeletal tissue engineering continues to escalate. Very little is known regarding the effects that energy delivered by means of third-generation ultrasound-assisted lipoaspiration may have on the osteogenic potential of these cells. The authors investigated whether differences in adipose-derived stromal cell yield, and the in vitro proliferation and osteogenic potential of these cells obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration, exist. METHODS: Adipose-derived stromal cells were harvested from lipoaspiration specimens of patients undergoing elective suction-assisted lipoaspiration and third-generation ultrasound-assisted lipoaspiration. Harvested cells were seeded to evaluate proliferative capacity and in vitro osteogenic potential. Alkaline phosphatase and alizarin red staining were performed to evaluate early and terminal osteogenic differentiation, respectively. Quantitative real-time polymerase chain reaction analysis was used to examine osteogenic gene expression patterns of RUNX2/CFBA1 (early differentiation) and osteocalcin (late differentiation). RESULTS: No significant differences in the proliferative capacity (n = 3), alkaline phosphatase staining (n = 3), or extracellular matrix mineralization (n = 3) of suction-assisted lipoaspiration- or third-generation ultrasound-assisted lipoaspiration-derived cells were appreciated. Transcript levels of markers of early and terminal osteogenic differentiation were not significantly different (n = 3). CONCLUSIONS: These findings suggest that exposure of adipose-derived stromal cells to ultrasound energy during tissue harvest by means of third-generation ultrasound-assisted lipoaspiration does not impart a negative consequence toward their proliferative capacity or osteogenic potential. Thus, the cells harvested using third-generation ultrasound-assisted lipoaspiration are comparable to those obtained by means of suction-assisted lipoaspiration for use in the study of osteogenic differentiation and skeletal tissue engineering.