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.

MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro.

OVERVIEW: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. MATERIALS AND METHODS: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. RESULTS: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. CONCLUSIONS: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.

A novel oxysterol promotes bone regeneration in rabbit cranial bone defects.

Bone morphogenetic proteins (BMPs) have played a central role in the development of regenerative therapies for bone reconstruction. However, the high cost and side-effect profile of BMPs limits their broad application. Oxysterols, naturally occurring products of cholesterol oxidation, are promising osteogenic agents alternative to BMPs. The osteogenic capacity of these non-toxic and relatively inexpensive molecules has been documented in rodent models. We studied the impact of Oxy49, a novel oxysterol analogue, on the osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs). Moreover, we evaluated the capacity for in vivo bone regeneration with Oxy49 in rabbit cranial bone defects. We found that rabbit BMSCs treated with Oxy49 demonstrated differentiation along osteogenic pathways, and that complete bone regeneration occurred when cranial defects were treated with Oxy49. Collectively, these results demonstrate that Oxy49 has the ability to induce osteogenic differentiation in rabbit BMSCs with an efficacy comparable to that of BMP-2 and to promote significant bone regeneration in cranial defects. Oxysterols may be a viable novel agent in bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Natural Killer Cells Differentiate Human Adipose-Derived Stem Cells and Modulate Their Adipogenic Potential.

BACKGROUND: Natural killer cells are thought to represent more than 30 percent of all lymphocytes within the stromal vascular fraction of lipoaspirates. However, their physiologic interaction with adipocytes and their precursors has never been specifically examined. The authors hypothesized that natural killer cells, by means of cytokine secretion, are capable of promoting the differentiation of adipose-derived stem cells. METHODS: Human natural killer cells purified from healthy donors' peripheral blood mononuclear cells were activated with a combination of interleukin-2 and anti-CD16 monoclonal antibody; natural killer cell supernatant was collected. Adipose-derived stem cells isolated from raw human lipoaspirates from healthy patients were treated with growth media, growth media with natural killer cell supernatant, adipogenic media, and adipogenic media with natural killer cells supernatant. Flow cytometric analysis was performed on cells using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC-1. Adipogenic-related gene expression (PPAR-γ, LPL, GPD-1, and aP2) was assessed. Oil Red O staining was performed as a functional assay of adipocyte differentiation and adipogenesis. RESULTS: Adipose-derived stem cells maintained in growth media with natural killer cell supernatant lost markers of "stemness," including CD44, CD34, and CD29; and expressed markers of differentiation, including B7H1 and MHC-1. Adipose-derived stem cells treated with natural killer cell supernatant accumulated small amounts of lipid after 10 days of natural killer cell supernatant treatment. Adipose-derived stem cells treated with natural killer cell supernatant showed altered expression of adipogenesis-associated genes compared with cells maintained in growth media. Adipose-derived stem cells maintained in adipogenic media with natural killer cell supernatant accumulated less lipid than those cells in adipogenic media alone. CONCLUSIONS: The authors demonstrate that, through secreted factors, natural killer cells are capable of differentiating adipose-derived stem cells. In cells maintained in adipogenic media, treatment with natural killer cell supernatant modulated adipogenic potential.

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.

In vitro osteoinductive effects of hydroxycholesterol on human adipose-derived stem cells are mediated through the hedgehog signaling pathway.

BACKGROUND: Human adipose-derived stem cells have been identified as a potential source of cells for use in bone tissue engineering because of their ready availability, ease of harvest, and susceptibility to osteogenic induction. The authors have previously demonstrated the ability of an osteogenic molecule called hydroxycholesterol, an oxidative derivative of cholesterol, to induce osteogenic differentiation in pluripotent murine and rabbit bone marrow stromal cells. In this study, the authors examine the ability of hydroxycholesterol to induce osteogenesis in human adipose-derived stem cells. METHODS: Human adipose-derived stem cells were isolated from raw human lipoaspirates through standard isolation and expansion of the stromal vascular fraction. Cells were plated onto tissue culture plates in control medium and harvested between passages 2 and 3, incubated with conventional osteogenic media, and treated with various concentrations (1, 5, and 10 µM) of the 20(S) analogue of hydroxycholesterol. Evaluation of cellular osteogenic activity was performed. The role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was evaluated by hedgehog inhibition assays. RESULTS: Alkaline phosphatase activity, bone-related gene expression, and mineralization were all significantly increased in cultures of human adipose-derived stem cells treated with 5 µM of 20(S)-hydroxycholesterol relative to controls. In addition, induction of hydroxycholesterol-mediated osteogenesis was mitigated by the addition of the hedgehog pathway inhibitor to cell cultures, implicating the hedgehog signaling pathway in the osteogenic mechanism on human adipose-derived stem cells by hydroxycholesterol. CONCLUSION: These in vitro studies demonstrate that hydroxycholesterol exerts an osteoinductive influence on human adipose-derived stem cells and that these effects are mediated at least in part through the hedgehog signaling pathway.

Manual isolation of adipose-derived stem cells from human lipoaspirates.

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In vitro study of a novel oxysterol for osteogenic differentiation on rabbit bone marrow stromal cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. METHODS: Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. RESULTS: Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. CONCLUSIONS: Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion.

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.

Lamellar stack formation and degradative behaviors of hydrolytically degraded poly(ε-caprolactone) and poly(glycolide-ε-caprolactone) blended fibers.

Electrospun fibrous mats have gained popularity in bioengineering over the past decade, but few papers detail their degradative mechanisms. To address this, blends of hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic PGA-PCL-PGA triblock copolymer were electrospun into aligned fibrous mats to assess the copolymers' mechanical and degradative properties. Increased hydrophilic triblock content led to enhanced morphological uniformity of fiber, tightening of fiber diameters, increased storage and Young's modulus, and decreased elongation. The corresponding decrease in hydrophobic PCL content led to faster hydrolytic degradation rate, as reflected by enhanced decrease in mass, molecular weight, and modulus loss at 25, 37, and 45°C. The activation energy for hydrolytic degradation for 15:85 PCL: triblock copolymer was approximately half that of 85:15 PCL: triblock copolymer. Detailed examination of fiber morphology and crystallinity revealed initial surface erosion followed by the evolution of crystalline lamellar stacks and bulk degradation at 37°C. Because of the high surface to volume and short diffusion length scale of the small diameter fibers, surface and bulk degradation may both contribute to the hydrolytic degradative behavior of these electrospun fibrous mats. Electrospun mats' distinct architecture that embodies high specific surface to volume and interfiber porous ultrastructures that lead to their unique degradative behaviors hold much potential for significant impact in the field of tissue engineering and controlled drug delivery.

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.

Adipose-derived stem cells and BMP2: part 2. BMP2 may not influence the osteogenic fate of human adipose-derived stem cells.

Although recent studies have proposed that human adipose-derived stem cells (ASCs), together with BMP2, can heal critical-sized bony defects, a companion study in this issue suggests that ASCs may not respond to BMP2 in vivo. To examine why this may be occurring. ASCs were treated with BMP2 and the cells' in vitro osteogenic capacity assessed along with the canonical BMP2 signaling pathway. In vitro treatment of ASCs with BMP2 had no consistent and significant effect on matrix mineralization or their expression of several osteogenic markers. Consistent and significant changes to Smad1/5/8 phosphorylation levels were also not observed upon BMP2 induction. The removal of dexamethasone from the BMP2 induction conditions had no effect on the observed results nor did stimulating ASCs with BMP2 from an alternate source (INFUSE; Medtronic, Minneapolis, MN, USA). In addition, no BMP-induced nuclear translocation of Smad1/Smad4 complexes could be discerned, suggesting that the canonical BMP2 signaling pathway may not be functional in ASCs. Interestingly, three downstream BMP2 pathway genes, distal-less3 (dlx3), distal-less5 (dlx5), and osterix, were not expressed in BMP2-induced ASCs, calling the utility of BMP2 induced in ASCs into question. The results of this in vitro study were consistent with that of our companion in vivo study that suggests a lack of effect of BMP2 on the osteogenic capacity of ASCs. Taken together, the data from both studies suggest that ASC osteogenic differentiation may not be influenced by BMP2. Consequently, combining BMP2 treatment with adult stem cells, like ASCs, may not be a viable strategy for bony healing.

Differential effect of ionizing radiation exposure on multipotent and differentiation-restricted bone marrow mesenchymal stem cells.

Debilitating effects of bone marrow from ionizing radiation exposure has been well established for hematopoietic stem cells; however, radiation toxicity of mesenchymal stem cells (MSCs) has been controversial. The present study addressed if ionizing radiation exposure differently affected bone marrow MSCs with various differentiation commitments. Mouse bone-marrow-derived MSCs, D1 cells of early passages (≤ 5 passages; p5) maintained the complete characteristics of multipotent MSCs, whereas, after ≥ 45 passages (p45) the differentiation capability of D1 cells became partially restricted. Both p5 and p45 D1 cells were subjected to single dose irradiation by radioactive isotope (137)Cs. Radiation treatment impaired cell renewal and differentiation activities of p5 D1 cells; however, p45 D1 cells were less affected. Radiation treatment upregulated both pro- and anti-apoptotic genes of p5 D1 cells in a dose-dependent manner, potentially resulting in the various apoptosis thresholds. It was found that constitutive as well as radiation-induced phosphorylation levels of histone H2AX was significantly higher in p45 D1 cells than in p5 D1 cells. The increased repair activity of DNA double-strand breakage may play a role for p45 D1 cells to exhibit the relative radioresistance. In conclusion, the radiation toxicity predominantly affecting multipotent MSCs may occur at unexpectedly low doses, which may, in part, contribute to the catabolic pathology of bone tissue.

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.

Functional testing of a tissue-engineered vocal fold cover replacement.

OBJECTIVES: Tissue engineering may provide a treatment for severe vocal fold scars. This study quantifies mechanical properties and demonstrates vibration of a tissue-engineered vocal fold cover replacement. METHODS: Tissue-engineered constructs were produced from fibrin and adipose-derived stem cells. Optimized bilayered constructs contained epithelial and mesenchymal cell phenotypes in a stratified geometry. For comparison, homogeneous constructs did not have epithelial differentiation. Elastic modulus was determined using indentation. Immunohistochemical labeling for type I collagen was performed. A bilayered construct was also tested in phonation in an excised larynx model. RESULTS: Bilayered vocal fold cover replacements had indentation moduli similar to human vocal fold covers (mean construct modulus 6.8 kPa). Collagen deposition occurred in the middle of the construct. Homogeneous constructs had a mean modulus of 8.3 kPa, and collagen was concentrated at the surface. An excised larynx with unilateral vocal fold cover replacement phonated and exhibited mucosal waves at physiologic airflow. CONCLUSION: Bilayered tissue-engineered constructs were produced that exhibited indentation modulus, microstructure, and vibration similar to that exhibited by human vocal fold covers.

Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering.

OBJECTIVES/HYPOTHESIS: One potential treatment option for severe vocal fold scarring is to replace the vocal fold cover layer with a tissue-engineered structure containing autologous cells. As a first step toward that goal, we sought to develop a three-dimensional cell-populated matrix resembling the vocal fold layers of lamina propria and epithelium. STUDY DESIGN: Basic science investigation. METHODS: Adipose-derived stem cells were cultured in fibrin hydrogels with various growth factors. At the end of the culture period, matrices were sectioned and labeled with immunomarkers to identify cell phenotype. RESULTS: Adipose-derived stem cells survived, attached, and populated three-dimensional fibrin matrices. Under select conditions, a superficial layer of cells expressing epithelial marker proteins overlay a deeper mesenchymal cell layer. CONCLUSIONS: A three-dimensional structure of fibrin and adipose-derived stem cells was created as a prototype vocal fold replacement. Two segregated cell phenotypes occurred, producing a bilayered structure resembling epithelium over lamina propria. This preliminary work demonstrates the feasibility of tissue engineering to produce structures for vocal fold replacement.

The effect of age on osteogenic, adipogenic and proliferative potential of female adipose-derived stem cells.

Human adipose tissue is an ideal source of autologous cells that is both plentiful and easily obtainable in large quantities through the simple surgical procedure of liposuction. The stromal vascular fraction of adipose tissue contains a stem cell population, adipose-derived stem cells (ASCs), capable of adipogenic, osteogenic, myogenic and chondrogenic differentiation. These cells have already been recognized to possess great therapeutic potential in tissue engineering and regeneration. In this study, we sought to determine the effect of donor age on the growth kinetics and differentiation potential of ASCs. For this, ASCs were isolated from liposuctioned adipose tissue obtained from female patients in the age range 20-58 years. Population doubling time was calculated over 2 weeks and differentiation potential was determined by assaying for adipogenesis and osteogenesis. ASCs obtained from older donors appeared to have a slower rate of proliferation, but this relationship was not significant. While adipogenic potential was unrelated to donor age, a distinct relationship between donor age and osteogenic potential was observed. The aetiology of this age-dependent change in osteogenic potential was not due to any changes in the number of precursors with osteogenic capacity in the adipose sample. These findings have important implications for emerging cell-based therapeutic strategies, such as tissue engineering, in addition to treatment of various metabolic bone disorders including osteoporosis.