Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations.

BACKGROUND: Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. METHODS: We obtained levator veli palatini muscle fragments (3-5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. RESULTS: Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. CONCLUSION: Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Development of chemotactic smart scaffold for use in tissue regeneration.

BACKGROUND: Regenerative medicine aims to obviate the need for autologous grafting through the use of bioengineered constructs that combine stem cells, growth factors, and biocompatible vehicles. Human mesenchymal stem cells and vascular endothelial growth factor (VEGF) have both shown promise for use in this context, the former because of their pluripotent capacity and the latter because of its chemotactic activity. The authors harnessed the regenerative potential of human mesenchymal stem cells and VEGF to develop a chemotactic scaffold for use in tissue engineering. METHODS: Human mesenchymal stem cells were transduced with human VEGF via lentivirus particles to secrete VEGF. The chemotactic activity of the VEGF-transduced stem cells was evaluated via a trans-well assay. Migration through semipermeable membranes was significantly greater in chambers filled with medium conditioned by VEGF-transduced cells. VEGF-transduced cells were then seeded on apatite-coated poly(lactic-co-glycolic acid) scaffolds, thereby creating the Smart Scaffold. To determine in vivo angiogenesis, the Smart Scaffolds were implanted into subcutaneous pockets in the backs of nude mice. RESULTS: Significantly larger numbers of capillaries were observed in the Smart Scaffold compared with control implants on immunohistologic studies. For the chemotactic in vivo study, human mesenchymal stem cells tagged with a fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) were injected intravenously via tail vein after the subcutaneous implantation of the Smart Scaffolds. In vivo fluorescent imaging revealed that fluorescent dye-tagged human mesenchymal stem cells successfully accumulated within the Smart Scaffolds. CONCLUSION: These observations suggest that VEGF may play a vital role in the design of clinically relevant tissue regeneration graft substitutes through its angiogenic effects and ability to chemoattract mesenchymal stem cells.

The suitability of human adipose-derived stem cells for the engineering of ligament tissue.

Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports-related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament-derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose-derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFß1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential.

Osteoblast interactions within a biomimetic apatite microenvironment.

Numerous reports have shown that accelerated apatites can mediate osteoblastic differentiation in vitro and bone formation in vivo. However, how cells interact within the apatite microenvironment remains largely unclear, despite the vast literature available today. In response, this study evaluates the in vitro interactions of a well-characterized osteoblast cell line (MC3T3-E1) with the apatite microenvironment. Specifically, cell attachment, spreading, and viability were evaluated in the presence and absence of serum proteins. Proteins were found to be critical in the mediation of cell-apatite interactions, as adherence of MC3T3-E1 cells to apatite surfaces without protein coatings resulted in significant levels of cell death within 24 h in serum-free media. In the absence of protein-apatite interaction, cell viability could be "rescued" upon treatment of MC3T3-E1 cells with inhibitors to phosphate (PO(4) (3-)) transport, suggesting that PO(4) (3-) uptake may play a role in viability. In contrast, rescue was not observed upon treatment with calcium (Ca(2+)) channel inhibitors. Interestingly, a rapid "pull-down" of extracellular Ca(2+) and PO(4) (3-) ions onto the apatite surface could be measured upon the incubation of apatites with α-MEM, suggesting that cells may be subject to changing levels of Ca(2+) and PO(4) (3-) within their microenvironment. Therefore, the biomimetic apatite surface may significantly alter the microenvironment of adherent osteoblasts and, as such, be capable of affecting both cell survival and differentiation.

Viral transduction of adipose-derived stem cells.

Increasing numbers of regenerative approaches now involve use of adult stem cells, like the bone marrow MSC or the adipose-derived ASC. With their ease of in vitro manipulation and successful tissue integration in vivo, the ASC makes an attractive candidate for gene delivery in vivo using viral-based gene therapy strategies. As such, this chapter describes methods for the transduction of human ASCs with two popular types of recombinant viruses: adenovirus and lentivirus.

Adipose-derived stem cells and BMP2: part 1. BMP2-treated adipose-derived stem cells do not improve repair of segmental femoral defects.

Recombinant human bone morphogenetic protein-2 (rhBMP2) has been shown to induce both in vitro osteogenic differentiation and in vivo bone formation, with the capacity of rhBMP2 to elicit the repair of numerous bony defects (calvaria, spinal fusion, femora, and so on) well documented. In addition, rhBMP2 has been approved by the Food and Drug Administration (FDA) for selected human indications. Despite the fact that healing is often achieved, the challenge still remains to optimize the therapeutic use of rhBMP2. One avenue may be through the combination of rhBMP2 with stem cells capable of osteogenic differentiation. This study investigates the ability of rhBMP2 at various doses in combination with human adipose-derived stem cells (ASCs) to heal critical-sized rat segmental femoral defects. For this, different doses of rhBMP2 were incorporated with apatite-coated porous poly(l-lactide-co-dl-lactide) (70 : 30) (PLDLA) scaffolds, seeded with ASCs, and implanted into athymic rats. After 8 weeks, all implants were harvested and processed for bone formation using micro computed tomography (microCT) analysis and histology. Despite the findings that indicate no adverse effect of the apatite surface on ASC osteogenesis, no significant difference in bone formation could be qualitatively or quantitatively determined upon the implantation of ASC-seeded scaffolds absorbed to increasing doses of rhBMP2. Such results would suggest that the presence of ASCs within rhBMP2-absorbed scaffolds does not improve the bone-forming ability of the construct and that the formation of bone may be driven by the rhBMP2 alone. Based on these results, the addition of ASCs to rhBMP2-treated scaffolds may provide no significant advantage in terms of the ability to heal bone.

The adipose-derived stem cell: looking back and looking ahead.

In 2002, researchers at UCLA published a manuscript in Molecular Biology of the Cell describing a novel adult stem cell population isolated from adipose tissue-the adipose-derived stem cell (ASC). Since that time, the ASC has gone on to be one of the most popular adult stem cell populations currently being used in the stem cell field. With multilineage mesodermal potential and possible ectodermal and endodermal potentials also, the ASC could conceivably be an alternate to pluripotent ES cells in both the lab and in the clinic. In this retrospective article, a historical perspective on the ASC is given together with exciting new applications for the stem cell being considered today.

The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used.

To characterize ES cells, researchers have at their disposal a list of pluripotent markers, such as OCT4. In their quest to determine if adult stem cell populations, such as MSCs and ASCs, are pluripotent, several groups have begun to report the expression of these markers in these cells. Consistent with this, human ASCs (hASCs) are shown in this study to express a plethora of ES pluripotent markers at the gene and protein level, including OCT4, Sox2, and Nanog. When intracellular distribution is examined in hASCs, both OCT4 and Sox2 are expressed within the nuclei of hASCs, consistent with their expression patterns in ES cells. However, a significant amount of expression can be noted within the hASC cytoplasm and a complete absence of nuclear expression is observed for Nanog. Recent descriptions of OCT4 transcript variants may explain the cytoplasmic expression of OCT4 in hASCs and consistent with this, hASCs do express both the OCT4A and 4B transcript variants at the gene level. However, discrepancies arise when these three pluripotent markers are studied at the protein level. Specifically, distinct differences in intracellular expression patterns were noted for OCT4, Sox2, and Nanog from commercial antibody to commercial antibody. These antibody discrepancies persisted when hMSCs and rat ASCs and MSCs were examined. Therefore, confirming the expression of OCT4, Sox2, and Nanog in adult stem cells with today's commercial antibodies must be carefully considered before the designation of pluripotent can be granted.

BMP-2 does not influence the osteogenic fate of human adipose-derived stem cells.

BACKGROUND: Recent studies have shown that bone morphogenetic protein (BMP)-2, a potent osteogenic growth factor, in combination with human adipose-derived stem cells can heal critical-sized bony defects. However, whether BMP-2 induces an osteogenic response in the adipose-derived stem cells remains unknown. METHODS: : In vitro calcium production, osteogenic gene expression, and BMP-2 receptor expression on the adipose-derived stem cell surface were analyzed in BMP-2-stimulated adipose-derived stem cells. The cells (2 x 10(7) cells) maintained in osteogenic medium were treated with an initial pulse of BMP-2 for 48 hours or 7 days or were given continuous BMP-2. To assess the response of these cells to BMP-2 in vivo, they (250,000 cells) were seeded into polylactic-co-glycolic acid (PLGA) collagraft scaffolds treated with 5 microg of BMP-2 and implanted into critical-sized femoral rat defects (n = 40). Healing was assessed histologically and quantitated by micro-computed tomography. RESULTS: In vitro treatment of adipose-derived stem cells with BMP-2 revealed decreased ability of the cells to undergo matrix calcification, demonstrated by decreased calcium production and decreased osteogenic gene expression of transcription factor Cbfa-1 and key extracellular proteins. Flow cytometry demonstrated decreased expression of BMP-2 receptors 1a and 1b in osteogenically differentiated adipose-derived stem cells stimulated with BMP-2. In vivo implantation of adipose-derived stem cell-seeded PLGA did not result in healing of critical-sized femoral defects in rodents, whereas implantation of BMP-2-absorbed PLGA, with or without adipose-derived stem cells, consistently healed these defects. CONCLUSIONS: The data suggests that osteogenic differentiation of adipose-derived stem cells is marginally affected by the addition of BMP-2. Consequently, stem cells in combination with BMP-2 may not be a viable strategy for the bony healing.

Freezing adipose tissue grafts may damage their ability to integrate into the host.

In this study, the effect of freezing on the morphology, viability, and VEGF synthesis of human adipose tissue grafts is examined. Currently, storage of adipose grafts involves freezing in simple saline solutions. However, the effect of freezing on the morphology and function of adipose tissue remains unclear. As a result, this study attempts to determine whether freezing adipose grafts should be considered prior to soft-tissue augmentation. In this study, the freezing of adipose grafts in saline for only 24 hr resulted in morphological changes in vivo and affected their ability to synthesize VEGF. The use of a simple cryopreservation medium containing sucrose appeared to maintain VEGF synthetic levels by the grafts and improved both their morphology and retention in vivo. However, the benefits of this cryopreservation medium were directly linked to storage time as long-term storage did not result in any noticeable benefit to graft retention. Finally, as an alternative to freezing, adipose grafts were combined with human adipose-derived stem cells (ASCs) to determine if their presence could enhance in vivo graft structure. The presence of ASCs did appear to improve graft structure in vivo over the short term and was also capable of improving tissue morphology when combined with grafts frozen in PBS. In conclusion, the successful use of adipose grafts may require a closer examination of the graft's storage conditions and time. Specifically, it now appears that the practice of freezing in saline may not be advisable if graft viability, activity, and structure are to be maintained in vivo.

Stem cells from human fat as cellular delivery vehicles in an athymic rat posterolateral spine fusion model.

BACKGROUND: Mesenchymal stem cells derived from human liposuction aspirates, termed processed lipoaspirate cells, have been utilized as cellular delivery vehicles for the induction of bone formation in tissue engineering and gene therapy strategies. In this study, we sought to evaluate the efficacy of bone morphogenetic protein (BMP)-2-producing adipose-derived stem cells in inducing a posterolateral spine fusion in an athymic rat model. METHODS: Single-level (L4-L5) intertransverse spinal arthrodesis was attempted with use of a type-I collagen matrix in five groups of athymic rats, with eight animals in each group. Group I was treated with 5 x 10(6) adipose-derived stem cells transduced with an adenoviral vector containing the BMP-2 gene; group II, with 5 x 10(6) adipose-derived stem cells treated with osteogenic media and 1 microg/mL of recombinant BMP-2 (rhBMP-2); group III, with 10 microg of rhBMP-2; group IV, with 1 microg of rhBMP-2; and group V, with 5 x 10(6) adipose-derived stem cells alone. The animals that showed radiographic evidence of healing were killed four weeks after cell implantation and were examined with plain radiographs, manual palpation, microcomputed tomography scanning, and histological analysis. RESULTS: All eight animals in group I demonstrated successful spinal fusion, with a large fusion mass, four weeks postoperatively. Furthermore, group-I specimens consistently revealed spinal fusion at the cephalad level (L3 and L4), where no fusion bed had been prepared surgically. In contrast, despite substantial BMP-2 production measured in vitro, group-II animals demonstrated minimal bone formation even eight weeks after implantation. Of the groups treated with the application of rhBMP-2 alone, the one that received a relatively high dose (group III) had a higher rate of fusion (seen in all eight specimens) than the one that received the low dose (group IV, in which fusion was seen in four of the eight specimens). None of the group-V animals (treated with adipose-derived stem cells alone) demonstrated successful spine fusion eight weeks after the surgery. CONCLUSIONS: Adipose-derived stem cells show promise as gene transduction targets for inducing bone formation to enhance spinal fusion in biologically stringent environments.

Comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model.

STUDY DESIGN: Rat spinal fusion model. OBJECTIVE: To compare the efficacy of human adipose tissue-derived mesenchymal stem cells (HATDMSCs) and human bone marrow-derived mesenchymal stem cells (HBMDMSCs) transduced with an adenovirus containing the cDNA for bone morphogenetic proteins (BMP)-2 for inducing spinal fusion in an athymic rat model. SUMMARY OF BACKGROUND DATA: Recombinant BMPs have successfully induced spinal fusion in clinical trials. However, large doses are required for adequate bone repair. Regional gene therapy may deliver proteins to specific anatomic sites more efficiently. Gene transfer techniques using HATDMSCs have recently been tested. METHODS: Spinal fusion was performed in rats with different treatments: Group I (n = 10) collagen sponge containing HATDMSCs transfected with adeno-BMP-2, Group II (n = 10) collagen sponge containing HBMDMSCs transfected with adeno-BMP-2, Group III (n = 10) collagen sponge containing recombinant BMP-2 (10 mug), Group IV (n = 6) collagen sponge containing HATDMSCs transfected with adeno-LacZ, Group V (n = 6) collagen sponge containing HBMDMSCs transfected with adeno-LacZ, and Group VI (n = 6) collagen sponge alone. Radiographs were obtained at 4, 6, and 8 weeks. After sacrifice, the rat spines were assessed by manual palpation, microcomputed tomography, and histologic analysis. RESULTS: At 8 weeks, spinal fusion was observed in all Groups I, II, and III rats. 75% (15 of 20) of the gene therapy treatment animals (Groups I and II rats) had spontaneous extension of the fusion to a second level. No Groups IV, V, and VI rats developed fusion. New bone volume was significantly greater in Groups I and II than in Group VI. CONCLUSION: HATDMSCs transfected with adeno-BMP-2 induce abundant bone formation and have a similar posterolateral spinal fusion in rats as similarly genetically modified HBMDMSCs. Both are potential strategies for spinal fusion and may be a more efficient method of obtaining spinal fusion over currently used grafting substances.

Tissue engineering craniofacial defects with adult stem cells? Are we ready yet?

Over three-quarters of all craniofacial defects observed in the US per year are cleft palates. Usually involving significant bony defects in both the hard palate and alveolar process of the maxilla, repair of these defects is typically performed surgically using autologous bone grafts taken from appropriate sites (i.e., iliac crest). However, surgical intervention is not without its complications. As such, the reconstructive surgeon has turned to the scientist and engineer for help. In this review, the application of the field of tissue engineering to craniofacial defects (e.g., cleft palates) is discussed. Specifically the use of adult stem cells, such as mesenchymal stem cells from bone marrow and Adipose-derived Stem Cells (ASCs) in combination with currently available biomaterials is presented in the context of healing craniofacial defects like the cleft palate. Finally, future directions with regards to the use of ASCs in craniofacial repair are discussed, including possible scaffold-driven and gene-driven approaches.

Human adipose stem cells: a potential cell source for cardiovascular tissue engineering.

BACKGROUND/AIMS: A crucial step in providing clinically relevant applications of cardiovascular tissue engineering involves the identification of a suitable cell source. The objective of this study was to identify the exogenous and endogenous parameters that are critical for the differentiation of human adipose stem cells (hASCs) into cardiovascular cells. METHODS: hASCs were isolated from human lipoaspirate samples, analyzed, and subjected to two differentiation protocols. RESULTS: As shown by fluorescence-activated cell sorter (FACS) analysis, a population of hASCs expressed stem cell markers including CXCR4, CD34, c-kit, and ABCG2. Further, FACS and immunofluorescence analysis of hASCs, cultured for 2 weeks in DMEM-20%-FBS, showed the expression of smooth muscle cell (SMC)-specific markers including SM alpha-actin, basic calponin, h-caldesmon and SM myosin. hASCs, cultured for 2 weeks in endothelial cell growth medium-2 (EGM-2), formed a network of branched tube-like structures positive for CD31, CD144, and von Willebrand factor. The frequency of endothelial cell (EC) marker-expressing cells was passage number-dependent. Moreover, hASCs attached and formed a confluent layer on top of electrospun collagen-elastin scaffolds. Scanning electron microscopy and DAPI staining confirmed the integration of hASCs with the fibers and formation of a cell-matrix network. CONCLUSION: Our results indicate that hASCs are a potential cell source for cardiovascular tissue engineering; however, the differentiation capacity of hASCs into SMCs and ECs is passage number- and culture condition-dependent.

The role of cytoprotective cytokines in cardiac ischemia/reperfusion injury.

The mechanism(s) underlying the beneficial effects of adult mesenchymal stem cells (MSCs) after myocardial infarction (MI) is poorly understood. One possible explanation is the ability of MSCs to secrete cytokines, which modulate cardiomyocyte survival and function. MSCs express at least two cytoprotective cytokines, hepatocyte growth factor (HGF) and stromal cell-derived factor-1 alpha (CXCL12). The aim of our study was to compare the effects of these two cytokines administered acutely post-MI. We subjected adult male Lewis rats to myocardial ischemia/reperfusion injury. Immediately upon reperfusion, polymers saturated with HGF or CXCL12 were placed onto the infarcted anterior wall and the rats were allowed to recover. Echocardiographic analysis at 4 wk post-MI to assess left ventricular (LV) function revealed that LV ejection fraction was increased in the HGF treated group compared with the phosphate-buffered saline (PBS) control group. Likewise, LV end diastolic dimension was reduced in the HGF treated group compared with the PBS control group. Similarly, invasive hemodynamics at 12 wk showed improved contractility and relaxation in the HGF treated group compared with the PBS control group. In contrast, no significant effect on LV function was seen in the CXCL12 treated group. To determine the potential mechanism for this effect, infarct size (IFS) at 72 h was determined. IFS was decreased 4.2-fold in the HGF treated group compared with the PBS control group. Thus, HGF acutely post-MI using polymer delivery reduces IFS, leading to beneficial effects on post-MI LV remodeling.

Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells.

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.

Healing full-thickness cartilage defects using adipose-derived stem cells.

The purpose of this study was to evaluate the use of adipose-derived stem cells (ADSCs) as a source for full-thickness cartilage repair in an animal model. Autologous ADSCs were isolated and induced with growth medium and placed in a fibrin glue scaffold and into 3-mm x 4-mm full-thickness chondral defects in rabbits with negative controls. Specimens were evaluated for early healing using immunostaining, Western blotting, reverse transcriptase polymerase chain reaction, transfection with the Lac Z gene, and quantitative assessment. Twelve of 12 (100%) articular surface defects containing tissue-engineered stem cell constructs healed with hyaline-like cartilage, versus 1 of 12 (8%) in the control group (p < .001). There was complete healing to subchondral bone in 12 of 12 experimental defects (100%), and 10 of 12 (83%) had seamless annealing to the native cartilage. Aggrecan, superficial zone protein, collagen type II messenger ribonucleic acid, and Lac-Z gene products were identified in 12 of 12 experimental specimens, which exhibited a collagen type II:I protein ratio similar to that of normal rabbit cartilage. Quantitative histologic analysis revealed an average score of 18.2 of 21 in the experimental group, compared with 10.0 in the controls (p = .001). Induced ADSCs supported in a fibrin glue matrix are a promising cell source for cartilage tissue engineering.

Osteogenic potentiation of human adipose-derived stem cells in a 3-dimensional matrix.

Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n = 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n = 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen I, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group 1 (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase [P < 0.05], 8.4-fold greater expression of osteonectin [P < 0.05], 6.4-fold greater expression of osteopontin [P < 0.05], 2.9-fold greater expression of collagen I [P < 0.05], and 2.5-fold greater expression of JNK2 [P < 0.05]). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer.

In vitro microdistraction of preosteoblasts: distraction promotes proliferation and oscillation promotes differentiation.

Osteoblast biology is influenced in vivo by a 3-dimensional (3D) extracellular matrix that mediates their adhesion and interaction and by a constant state of compressive and tensile forces. To study the role of mechanical stress on osteoblasts in vitro, these parameters must be addressed. Therefore, this study describes the use of a novel, in vitro system that subjects cells to distractive and compressive forces in a 3D environment. This system, termed a microdistractor system, was used to apply linear forces to 3D collagen type I gels containing preosteoblasts. Gels were induced for up to 16 days in osteogenic medium and subjected to either constant linear distraction (distraction gels) or to repeating cycles of distraction and compression (oscillation gels). The effect of these stresses was evaluated over time by measuring proliferation rates, protein synthesis (i.e., cellular activity), and osteogenic differentiation levels. While linear forces in general appeared to increase protein synthesis, force-specific effects on proliferation and differentiation were observed. Specifically, distraction forces appeared to enhance MC3T3 proliferation while distraction/compressive forces appeared to accelerate their osteogenic differentiation program. Therefore, these results suggest that the microdistraction system may be an appropriate in vitro system for the study of mechanobiology in osteoblast phenotype.

Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction.

PURPOSE: We performed a pilot study to investigate the ability of human adipose derived, multipotent stem cells to be delivered to and survive within bladder and urethral smooth muscle. MATERIALS AND METHODS: Lipoaspirate was acquired from female patients undergoing liposuction. The lipoaspirate was processed to yield a pluripotent population of processed lipoaspirate (PLA) cells. For tissue delivery PLA cells were fluorescent labeled and suspended in Hanks' balanced salt solution (Sigma Chemical Co., St. Louis, Missouri). To assess PLA viability in multiple animal models 8 Rnu athymic rats (Charles River, Wilmington, Massachusetts) and 6 SCID mice (Taconic Farms, Oxnard, California) underwent laparotomy and injection of PLA cells into the bladder and urethra. An additional 8 rats underwent sham injection of Hanks' balanced salt solution alone. Experimental and control animals were sacrificed 2, 4, 8 and 12 weeks after injection, and the bladders and urethras were analyzed. RESULTS: Self-regenerating, pluripotent PLA cells were easily isolated from human adipose tissue. Evaluation 2, 4, 8 and 12 weeks after injection demonstrated PLA cell viability and incorporation into the recipient smooth muscle. Eight weeks following injection PLA cells demonstrated in vivo expression of alpha-smooth muscle actin, an early marker of smooth muscle differentiation. CONCLUSIONS: PLA cells are an easily accessible source of pluripotent cells, making them ideal for tissue regeneration. PLA cells remain viable up to 12 weeks in the lower urinary tract. Human PLA cells injected into the urinary tract show morphological and phenotypic evidence of smooth muscle incorporation and differentiation with time. PLA cells may provide a feasible and cost-effective cell source for urinary tract reconstruction.