Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects.

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.

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.

Treatment of axillary hyperhidrosis/bromidrosis using VASER ultrasound.

BACKGROUND: Current methods of treatment for axillary hyperhidrosis and/or bromidrosis are palliative (use of topical aluminum chloride or injections of botulinum toxin type A) or surgically based for more permanence (excisional surgery, endoscopic transthoracic sympathectomy, liposuction/curettage). The surgical approaches have mixed effectiveness and incur the risk of significant side effects and complications. METHODS: Thirteen patients (3 males, 10 females) with significant axillary hyperhidrosis and/or bromidrosis were recruited, treated with the VASER ultrasound, and followed for 6 months. Preoperative assessment of the impact of hyperhidrosis and/or bromidrosis on lifestyle and the degree of sweat/odor were completed. Postoperative assessment of changes relative to lifestyle and degree of sweat/odor reduction and patient and surgeon satisfaction were completed. RESULTS: Eleven of 13 patients had significant reduction in sweat/odor and had no recurrence of significant symptoms at 6 months. Two patients had a reduction in sweat/odor but not to the degree desired by the patients. No significant complications were noted. A simple amplitude and time protocol was established that provides consistent and predictable therapy. The complete procedure takes less than 1 h to treat two axillae using local anesthetic. CONCLUSION: The VASER is safe and effective for treatment of axillary hyperhidrosis/bromidrosis. The method is minimally invasive with immediate return to basic activities and only temporary minor restriction of arm movement. At 6 months the treatment appears to be long-lasting, but further follow-up is required for verification of permanence. This method has become the standard of care for the treatment of axillary hyperhidrosis/bromidrosis in the authors' practice.

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.

Deleterious effects of freezing on osteogenic differentiation of human adipose-derived stromal cells in vitro and in vivo.

Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze-thaw process.

Human adipose-derived stromal cells stimulate autogenous skeletal repair via paracrine Hedgehog signaling with calvarial osteoblasts.

Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P<0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.

Elastic properties of induced pluripotent stem cells.

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

Acute skeletal injury is necessary for human adipose-derived stromal cell-mediated calvarial regeneration.

BACKGROUND: Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects. METHODS: Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction). RESULTS: Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression. CONCLUSIONS: Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.

Differences in osteogenic differentiation of adipose-derived stromal cells from murine, canine, and human sources in vitro and in vivo.

BACKGROUND: Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species. METHODS: Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology. RESULTS: Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells. CONCLUSIONS: Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal 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.

Divergent modulation of adipose-derived stromal cell differentiation by TGF-beta1 based on species of derivation.

BACKGROUND: Adipose-derived stromal cells hold promise for skeletal tissue engineering. However, various studies have observed that adipose-derived stromal cells differ significantly in their biology depending on species of derivation. In the following study, the authors sought to determine the species-specific response of adipose-derived stromal cells to recombinant TGF-beta1 (rTGF-beta1). METHODS: Adipose-derived stromal cells were derived from mouse and human sources. Recombinant TGF-beta1 was added to culture medium (2.5 to 10 ng/ml); proliferation and osteogenic and adipogenic differentiation were assessed by standardized parameters, including cell counting, alkaline phosphatase, alizarin red, oil red O staining, and quantitative real-time polymerase chain reaction. RESULTS: Recombinant TGF-beta1 was found to significantly repress cellular proliferation in both mouse and human adipose-derived stromal cells (p < 0.01). Recombinant TGF-beta1 was found to significantly repress osteogenic differentiation in mouse adipose-derived stromal cells. In contrast, osteogenic differentiation of human adipose-derived stromal cells proceeded unimpeded in either the presence or the absence of rTGF-beta1. Interestingly, rTGF-beta1 induced expression of a number of osteogenic genes in human adipose-derived stromal cells, including BMP2 and BMP4. CONCLUSIONS: The authors' results further detail an important facet in which mouse and human adipose-derived stromal cells differ. Mouse adipose-derived stromal cell osteogenesis is completely inhibited by rTGF-beta1, whereas human adipose-derived stromal cell osteogenesis progresses in the presence of rTGF-beta1. These data highlight the importance of species of derivation in basic adipose-derived stromal cell biology. Future studies will examine in more detail the species-specific differences among adipose-derived stromal cell populations.

Depot-specific variation in the osteogenic and adipogenic potential of human adipose-derived stromal cells.

BACKGROUND: Adipose-derived stromal cells hold promise for use in tissue regeneration. However, multiple facets of their biology remain unclear. The authors examined the variations in osteogenesis and adipogenesis in adipose-derived stromal cells between subcutaneous fat depots and potential molecular causes. METHODS: Adipose-derived stromal cells were isolated from human patients from subcutaneous fat depots, including arm, flank, thigh, and abdomen (n = 5 patients). Osteogenic and adipogenic differentiation was performed (alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction). Co-cultures were established to assess the paracrine effect of human adipose-derived stromal cells on mouse osteoblasts. Finally, HOX gene expression was analyzed by quantitative real-time polymerase chain reaction. RESULTS: Subcutaneous fat depots retain markedly different osteogenic and adipogenic potentials. Osteogenesis was most robust in adipose-derived stromal cells from the flank and thigh, as compared with those from the arm and abdomen (p < 0.05 by all markers examined). This was accompanied by elevations of BMP4 and BMPR1B (p < 0.05 by all markers examined). The osteogenic advantage of cells from the flank and thigh was again observed when analyzing the paracrine effects of these cells. Conversely, those cells isolated from the flank had a lesser ability to undergo adipogenic differentiation. Adipose-associated HOX genes were less expressed in flank-derived adipose-derived stromal cells. CONCLUSIONS: Variations exist between fat depots in terms of adipose-derived stromal cell osteogenic and adipogenic differentiation. Differences in HOX expression and bone morphogenetic protein signaling may underlie these observations. This study indicates that the choice of fat depot derivation of adipose-derived stromal cells may be an important one for future efforts in tissue engineering.

Paracrine interaction between adipose-derived stromal cells and cranial suture-derived mesenchymal cells.

BACKGROUND: Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings. METHODS: Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (10 to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate. RESULTS: Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p < 0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p < 0.05). This was accompanied by an increase in bone morphogenetic protein expression (p < 0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p < 0.05). CONCLUSIONS: Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect.

Regulation of human adipose-derived stromal cell osteogenic differentiation by insulin-like growth factor-1 and platelet-derived growth factor-alpha.

BACKGROUND: Human adipose-derived stromal cells possess a great potential for tissue engineering purposes. The authors' laboratory is interested in harnessing human adipose-derived stromal cells for skeletal tissue regeneration and identifying those factors that enhance human adipose-derived stromal cell osteogenic differentiation. The authors hypothesized that insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) would stimulate human adipose-derived stromal cell osteogenesis and that IGF would stimulate adipogenesis. METHODS: Adipose-derived stromal cells were harvested from human lipoaspirate. Previously, a microarray analysis examined gene expression throughout osteogenic differentiation. In a candidate fashion, the authors added recombinant IGF-1 and PDGF-alpha individually and in combination. Osteogenesis and adipogenesis were assessed by alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction (RUNX2, ALP, OCN, IGF1, PPARG, LPL, AP2, and GCP1). Finally, intersection between IGF and PDGF signaling pathways was evaluated. RESULTS: IGF-1 was observed to increase osteogenic differentiation by all markers (p < 0.01). However, PDGF-alpha when added alone primarily did not affect osteogenic markers. PDGF-alpha positively regulated transcription of IGF1. Addition of PDGF-alpha in combination with or before IGF-1 enhanced osteogenesis more than either alone. IGF-1 increased whereas PDGF-alpha diminished human adipose-derived stromal cell adipogenesis. CONCLUSIONS: IGF signaling significantly increased osteogenesis in human adipose-derived stromal cells and may be used for tissue-engineering purposes. The combination of PDGF and IGF may be more beneficial than either alone in driving adipose-derived stromal cell osteogenesis. Future in vivo applications will focus on the combination of adipose-derived stromal cells, biomimetic scaffolds, and recombinant IGF.

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.