EXPERIMENTAL In Vitro Osteoinductive Effects of Hydroxycholesterol on Human AdiposeDerived Stem Cells Are Mediated through the Hedgehog Signaling Pathway Anisa Yalom, M.D. Akishige Hokugo, D.D.S., Ph.D. Sarah Sorice, M.D. Andrew Li, M.D. Luis A. Segovia Aguilar, M.D. Patricia Zuk, Ph.D. Reza Jarrahy, M.D. Los Angeles, Calif.
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 hydroxycholesterolmediated 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. (Plast. Reconstr. Surg. 134: 960, 2014.)
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raniofacial defects often present significant technical challenges to the plastic surgeon; furthermore, strategies for their correction can put a considerable financial burden on an already strained domestic health care system. The rate of spontaneous healing of cranial defects decreases substantially after 2 years From the Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA. Received for publication November 22, 2013; accepted April 2, 2014. The first two authors contributed equally to this work. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000601
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of age, necessitating surgical intervention in the setting of complex craniofacial defects. Currently accepted techniques include autologous bone grafting, the use of alloplastic materials, allografting, and distraction osteogenesis.1 Although effective, each of these modalities is associated with the Disclosure: The authors have no commercial associations or financial disclosures that might pose a conflict of interest with any information presented in this article. his work was supported by T THE PLASTIC SURGERY FOUNDATION.
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Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol potential for adverse effects or suboptimal longterm outcomes, including donor-site morbidity, graft failure, resorption, infection, limited sources of autologous donor bone, contour irregularities, and disease transmission.2–5 A large effort in the field of tissue engineering has been made to address current shortcomings associated with autologous bone grafts. Specifically, a significant body of research is dedicated to the study of stem cells that have the capacity to support bone regeneration. Bone marrow–derived mesenchymal stem cells, for example, have been successfully directed not only along osteogenic lines of differentiation, but also along chondrogenic, myogenic, and adipogenic pathways.6–9 Adipose-derived stem cells possess similar multipotent capabilities but are candidates for autologous tissue engineering and possess several distinct advantages compared with other mesenchymal stem cells. Adipose-derived stem cells have demonstrated the same multipotent differentiation capacity without the donor-site morbidity associated with the harvest of bone marrow–derived stem cells.8–12 The role of adipose-derived stem cells in healing in vivo skeletal defects has been well established in animal models.13–15 Successful examples of translational application also exist: Lendeckel et al.16 used adipose-derived stem cells to augment bone growth in a 7-year old patient who underwent reconstruction of a critical-size skull defect. Adiposederived stem cells can be harvested in large quantities through easily accessible subcutaneous deposits, enabling rapid and cost-effective expansion of multipotent stem cell populations. These features have made adipose-derived stem cells among the most popular multipotent stem cells in applied translational stem cell research. Hydroxycholesterols, also known as oxysterols, consist of a large family of 27-carbon oxygenated products of cholesterol. These molecules are present in the circulation and in human tissues.17 Endogenous hydroxycholesterols are formed in various ways, including auto-oxidation of cholesterol, lipid peroxidation, and enzymatically.18 Circulating hydroxycholesterols are also obtained from exogenous sources through dietary intake.19 Hydroxycholesterols are biologically active in diverse metabolic processes, including regulation of cholesterol homeostasis, atherosclerosis, sphingolipid metabolism, platelet aggregation, and apoptosis.20 Most relevant to this study, however, is the fact that hydroxycholesterols have been shown to have pro-osteogenic activity.20 Activation of the hedgehog signaling pathway has been implicated in the mechanism of action of oxysterols in modulating osteogenesis
in murine cells.21,22 Various naturally occurring hydroxycholesterol analogues, namely, 22(R)-, 20(S)-, and 22(S)-hydroxycholesterol, have been shown to induce osteogenic differentiation in the M2-10B4 murine multipotent bone mesenchymal stem cell line, and in primary murine mesenchymal stem cells.20 Despite the fact that hydroxycholesterols represent a promising new growth factor with the potential capacity for clinical application to bone regenerative medicine, and the widespread acceptance of adipose-derived stem cells as a promising tool in bone tissue engineering, no study to date has investigated the effects of hydroxycholesterols on human adipose-derived stem cells in the setting osteogenesis. In this study, we aim to investigate the potential translational significance of hydroxycholesterol-treated human adipose-derived stem cells by studying the effects of hydroxycholesterols on the osteogenic capacity of human adipose-derived stem cells and the mechanisms behind any such observed effects.
MATERIALS AND METHODS Dulbecco’s Modified Eagle Medium, l-glutamine, and penicillin-streptomycin were purchased from Invitrogen Corp. (Carlsbad, Calif.), and fetal bovine serum was obtained from Omega Scientific, Inc. (Tarzana, Calif.). A total RNA isolation kit (RNeasy Plus Mini Kit) was obtained from Qiagen (Valencia, Calif.) and reverse transcriptase was purchased from Bio-Rad (Hercules, Calif.). Ascorbic acid-2 phosphate, β-glycerol phosphate, dimethyl sulfoxide, 20(S)-hydroxycholesterol, and all other reagents were obtained from SigmaAldrich (St. Louis, Mo.). Isolation and Culture of Human Adipose-Derived Stem Cells Human adipose-derived stem cells were harvested from lipoaspirates obtained from female patients presenting for cosmetic liposuction procedures, as reported previously.9,10 Patients ranged in age between 35 and 50 years. Adipose-derived stem cell populations were isolated and expanded in culture using growth medium consisting of Dulbecco’s Modified Eagle Medium (4.5 g/liter, with l-glutamine and sodium pyruvate) supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 mg/ ml streptomycin, and 2.5 μg/ml amphotericin B. Cells were allowed to reach 90% confluence before they were passaged using 2.5% trypsin/0.23 mM ethylenediaminetetraacetic acid.
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Plastic and Reconstructive Surgery • November 2014 Second- or third-passage cell populations that consistently showed homogenous morphology and phenotype were used in this study.23 For various treatment conditions, cells were seeded into multiwell plates and cultured with either (1) control medium containing Dulbecco’s Modified Eagle Medium, 5% fetal bovine serum, 2 mM l-glutamine, and 1% penicillin-streptomycin; (2) osteogenic medium containing control medium with 3 mM β-glycerol phosphate and 50 μg/ml ascorbic acid-2 phosphate; or (3) osteogenic medium with various concentrations of 20(S)-hydroxycholesterol (1, 5, and 10 μM) dissolved in dimethyl sulfoxide. Medium was changed every 72 hours. All cell cultures were maintained at 37°C and 5% carbon dioxide. Alkaline Phosphatase Activity Assay After 4 days of cell culture under the various experimental conditions described above, colorimetric alkaline phosphatase activity assay on whole cell extracts was performed as reported previously.20 Cells were rinsed twice with phosphate-buffered saline and scraped into 200 μl of lysis buffer (0.2% NP-40 in 1 mM of magnesium chloride) with a rubber policeman and sonicated for 5 seconds. Reaction solution containing alkaline buffer stock solution and phosphatase substrate was added to each well. This mixture was then incubated for 10 to 15 minutes at 37°C. Yellow color was indicative of alkaline phosphatase activity. The reaction was stopped with the addition of 1N sodium hydroxide to each well, and absorbance was determined at 405 nm. Alkaline phosphatase activity was calculated using p-nitrophenol as a standard. Results were normalized to total protein determined using the Bradford protein assay.24 von Kossa Staining After 28 days of culture under the various described conditions, cells were washed twice with phosphate-buffered saline, fixed with 10% formalin in neutral buffer for 30 minutes, rinsed extensively with distilled water, and covered with 0.5 ml of 5% silver nitrate. The cells were incubated for 30 minutes in the dark and then rinsed with distilled water. The cells were exposed to ultraviolet light for 30 minutes and treated with 2.5% sodium thiosulfate solution for 5 minutes. Plates were finally rinsed extensively with tap water and airdried. Staining results were captured with a Nikon Coolpix 990 digital camera (Nikon Corp., Tokyo, Japan). The mineralized nodule area was measured using ImageJ software (National Institutes
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of Health, Bethesda, Md.), a digital image analysis system. Four independent cultures were evaluated in each of the experimental groups. Quantitative Real-Time Polymerase Chain Reaction After 5 days of culture under the various experimental conditions, cells were collected and total RNA was extracted using RNA isolation kits, according to the manufacturer’s instructions. RNA was reverse-transcribed using reverse transcriptase to make single-stranded cDNA templates. Real-time polymerase chain reaction for the osteogenic markers runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) was performed using Taqman Gene Expression Assay (Hs00231692_m1, Hs01587814_g1, and Hs00959010_m1 respectively; Applied Biosystems, Foster City, Calif.). Count values were normalized to glyceraldehyde 3-phosphate dehydrogenase (Hs02758991_g1) as an internal control, and relative expression levels versus untreated controls (i.e., control medium, harvested at the same time points) were determined using the 2−ΔΔCT method. Each gene was quantitated a minimum of three times. Inhibition of Hydroxycholesterol-Mediated Osteogenesis with the Hedgehog Signaling Inhibitor Cyclopamine To determine the mechanism by which hydroxycholesterol mediates its effects on osteogenic differentiation in human adipose-derived stem cells, we examined the hedgehog signaling pathway, which has been shown to play a role in the process of osteogenesis in murine cells.21,22 Human adipose-derived stem cells were pretreated for 2 hours with cyclopamine— a known inhibitor of hedgehog signaling—at a concentration of 4 μM. To control for the cyclopamine delivery vehicle, cells were also treated for 2 hours with osteogenic medium containing dimethyl sulfoxide only. Following pretreatment, the medium was replenished with 5 μM of hydroxycholesterol or control vehicle and the cells were cultured for 4 days. Alkaline phosphatase activity assays were performed as an indicator of osteogenic activity. Statistical Analysis Data are presented as means ± SD. Statistical differences between groups were evaluated by one-way analysis of variance, and post hoc multiple-comparison tests (Tukey-Kramer multiple
Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol comparison test) were performed to assess significance. A value of p < 0.05 was considered statistically significant.
RESULTS Effect of Hydroxycholesterol on Alkaline Phosphatase Activity in Human Adipose-Derived Stem Cells To determine the effect of hydroxycholesterol on the activity of proteins that are relevant to the osteogenic pathway, human adipose-derived stem cells were treated with varying doses of 20(S)hydroxycholesterol for 4 days, and alkaline phosphatase activity was measured (Fig. 1). Alkaline phosphatase activity by human adipose-derived stem cells treated with osteogenic medium increased approximately 1.5-fold compared with cells maintained in noninductive control medium. Treatment of human adipose-derived stem cells with 5 μM hydroxycholesterol significantly increased alkaline phosphatase activity relative to cells treated with osteogenic medium alone. However, the treatment of cells with lower (1 μM) and higher (10 μM) concentrations of hydroxycholesterol yielded no significant increases in alkaline phosphatase activity relative to controls.
Fig. 1. Hydroxycholesterol (OHC) induces alkaline phosphatase (ALP) activity in human adipose-derived stem cells. Human adipose-derived stem cells at confluence were treated with osteogenic medium (OM) and 1, 5, or 10 μM hydroxycholesterol. Control cultures were treated with control medium (CM) or osteogenic medium plus vehicle. After 4 days, alkaline phosphatase activity was measured in whole cell extracts. Data are normalized to alkaline phosphatase activity in the cells treated with control medium. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium.
Effect of Hydroxycholesterol on Osteogenic Differentiation and Mineralization in Human Adipose-Derived Stem Cells To determine the effects of hydroxycholesterol on osteogenic differentiation in human adipose-derived stem cells, as indicated by the ability of treated cells to form a mineralized matrix, cell cultures were maintained for 28 days and then assessed with von Kossa staining (Fig. 2). Cells cultured with control medium demonstrated a negligible mineralization response, whereas significant areas of positive von Kossa staining were observed in cultures of cells treated with osteogenic medium. Mineralization levels were increased slightly when human adipose-derived stem cells were treated with osteogenic medium containing either 1 μM or 10 μM hydroxycholesterol. However, a significant increase in mineralization relative to osteogenic medium–treated control human adipose-derived stem cells was noted in human adipose-derived stem cells treated with osteogenic medium and 5 μM of hydroxycholesterol. Effect of Hydroxycholesterol on Osteogenic Gene Expression in Human Adipose-Derived Stem Cells To determine the osteoinductive potential of hydroxycholesterol on human adipose-derived stem cells, human adipose-derived stem cells were treated with osteogenic medium supplemented with 5 μM hydroxycholesterol for 5 days, and the expression of three key osteogenic genetic markers was examined using real-time polymerase chain reaction (Fig. 3). Osteogenic medium supplemented with 5 μM hydroxycholesterol was chosen in light of its optimal effect on alkaline phosphatase activity and matrix mineralization. As shown in Figure 3, treatment of human adipose-derived stem cells with 5 μM hydroxycholesterol resulted in significant increases in RUNX2, OCN, and OPN expression relative to controls. Mechanism of Action of Hydroxycholesterol in Osteogenic Differentiation of Human AdiposeDerived Stem Cells The induction of alkaline phosphatase activity by 5 μM hydroxycholesterol was significantly inhibited by cyclopamine (Fig. 4). There was no adverse effect of vehicle on human adiposederived stem cell osteogenesis, as elevated alkaline phosphatase activity was measured in this control group.
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Fig. 2. Hydroxycholesterol (OHC) treatment increases mineralization capacity of human adipose-derived stem cells. Mineralization of culture plates by human adipose-derived stem cells treated with 1, 5, or 10 μM of hydroxycholesterol at day 14 was evaluated by von Kossa staining. (Above) Representative images of von Kossa stains are shown (original magnification, × 4). (Below) The percentage of von Kossa–positive area per plate is shown. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium. CM, control medium; OM, osteogenic medium.
Fig. 3. Hydroxycholesterol (OHC) induces expression of markers of osteogenic differentiation in human adipose-derived stem cells. Human adipose-derived stem cells at confluence were treated with 5 μM hydroxycholesterol. Control cells were cultured with osteogenic medium (OM) plus vehicle. After 5 days, mRNA expression was measured by quantitative realtime polymerase chain reaction. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium.
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Fig. 4. Alkaline phosphatase (ALP) activity is diminished by addition of cyclopamine. Human adipose-derived stem cells at confluence were pretreated with control vehicle or 4 μM cyclopamine (Cyc) for 2 hours. Next, 5 μM hydroxycholesterol (OHC) or control vehicle was added to cell cultures. Alkaline phosphatase activity after 4 days of treatment was measured. Data are normalized to alkaline phosphatase activity in the cells treated with control medium. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05). CM, control medium; OM, osteogenic medium.
DISCUSSION Advances in the field of cell-based regenerative medicine over the past decade have demonstrated that adipose-derived stem cells are an exciting alternative to bone marrow mesenchymal stem cells in bone regeneration models. In fact, their in vitro osteogenic capacity is well established and strongly supported by numerous in vivo studies.13,25–27 Such findings, paired with the advantages of adipose-derived stem cells compared with bone marrow mesenchymal stem cells in terms of availability, ease of harvest and isolation, higher stem cell yields per gram of tissue,28 and plasticity, are largely responsible for their popularity in tissue engineering. Although pro-osteogenic effects of bone morphogenetic proteins in human adiposederived stem cells have been reported,29–32 recent evidence calls these effects into question.33,34 Moreover, the drawbacks of bone morphogenetic protein use, including costs and adverse effects associated with therapy, have contributed to a growing interest in exploring alternative osteoinductive growth factors.35–38 Hydroxycholesterol has emerged as one such promising molecule. Its pro-osteogenic effects in murine cells have been
established.20,22,38,39 We have also demonstrated that a novel semisynthetic hydroxycholesterol, named Oxy49, induces the osteogenic differentiation of rabbit bone marrow stromal cells as effectively as bone morphogenetic protein-2 both in vitro40 and in vivo,41 inducing bone formation in critical-size rabbit calvarial defects. Because no prior studies have examined the effect of hydroxycholesterol on human adiposederived stem cells, and because adipose-derived stem cells are a target of intense focus in bone engineering research, this study was conducted to form an initial assessment of the pro-osteogenic effectiveness of hydroxycholesterol therapy on human adipose-derived stem cells. In previously performed studies, a concentration of 1 to 5 μM oxysterol significantly increased osteogenic activity in a murine multipotent bone mesenchymal stem cell line20,38 and primary rabbit bone marrow stromal cells.40,41 Based on these studies, we tested the effects of three different concentrations (1, 5, and 10 μM) of hydroxycholesterol on human adipose-derived stem cells. The results of this work demonstrate that hydroxycholesterol is in fact an effective inducer of osteogenic differentiation in human adipose-derived stem cells. At an optimal dose of 5 μM, osteogenic differentiation was observed both at the level of gene expression and protein function. Exposure of human adipose-derived stem cells to hydroxycholesterol significantly increased alkaline phosphatase activity and osteogenic gene expression in comparison with control cultures. Most importantly, matrix mineralization, a terminal functional measure of osteoblast function, was significantly induced on hydroxycholesterol treatment compared with controls. Our results show that hydroxycholesterol dosing must be optimized. Specifically, we observed a decrease in osteogenesis when human adiposederived stem cells were exposed to higher doses of hydroxycholesterol (i.e., 10 μM). This may indicate a decreased responsiveness to higher levels of hydroxycholesterol because of receptor saturation/down-regulation or an induction of apoptosis. Lemaire-Ewing et al. reported cytotoxicity caused by oxysterols, including 7β-hydroxycholesterol and 7- ketocholesterol, leading to cell apoptosis on human promonocytic leukemia cells.42 However, oxysterol-associated toxicity varies between cell types. Vicente López and colleagues43 found a similar phenomenon when incubating human adipose-derived stem cells with various concentrations of bone morphogenetic protein and measuring cell viability, proliferation,
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Plastic and Reconstructive Surgery • November 2014 and multipotency. Although low doses of bone morphogenetic protein resulted in a significant increase in overall cell viability parameters, high doses significantly inhibited cell proliferation and increased apoptosis. A similar effect may be occurring on hydroxycholesterol stimulation. As such, additional studies elucidating the binding kinetics of hydroxycholesterol to its receptors in adiposederived stem cells will be necessary to ascertain whether a dose-dependent response occurs at the receptor level. As experimental evidence describing the efficacy and potential clinical utility of hydroxycholesterol continues to expand, a growing interest has been dedicated to discovering the molecular mechanisms by which osteogenic differentiation of progenitor cells occurs. The critical role that the hedgehog signaling pathway plays in hydroxycholesterol-induced osteogenic differentiation in murine cells has been demonstrated by Kim et al.22 In embryogenesis, the hedgehog signaling pathway is crucial in modulating embryonic skeletal development. Hedgehog signaling is reactivated during adult repair processes, controlling the expression of a variety of growth factors and triggering the differentiation of mesenchymal stem cells toward osteogenic lineage.44 Hedgehog signaling is initiated by the binding of hedgehog ligand to Patched (Ptch), a 12-transmembrane protein receptor.45 In the absence of hedgehog interaction, Ptch suppresses the signal transducer activities of the seven-transmembrane protein smoothened (Smo). In contrast, binding of hedgehog to Ptch prevents the suppressive effects of Ptch, thus allowing activation of Smo, which in turn activates the intracytoplasmic Gli proteins (GliA, Gli2, and GliR). After its translocation into the nucleus, GliA can activate various gene targets through interactions with specific DNA-binding elements.46 Downstream gene targets include those involved in osteogenesis, which has been demonstrated in previous reports that link hedgehog signaling to the differentiation of pluripotent stem cells into osteoblasts.47,48 In addition, studies have investigated Indian hedgehog knockout mice and found that these mice subsequently lack normal osteoblast function and endochondral bone formation, leading to stunted appendages.49 These strongly suggest that the hedgehog signaling pathway is necessary for the normal progression of osteogenesis. Until recently, it was unclear how oxysterols activate hedgehog signaling. Unlike most synthetic hedgehog effectors, oxysterols do not compete with cyclopamine, a
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hedgehog signaling inhibitor for binding to Smo, a property that has been used to argue that Smo is not a direct target for oxysterols.21,50 Recently, Nachtergaele et al.50 demonstrated the presence of at least two binding sites on Smo, one that binds natural 20(S)-hydroxycholesterol and a second that binds cyclopamine and the small molecule Smoothened agonist (SAG), which activates Smo by competing for the cyclopamine binding site. They found various binding interactions between these three molecules: whereas SAG and natural 20(S)-hydroxycholesterol showed a positive allosteric interaction, SAG and cyclopamine showed a competitive interaction, while cyclopamine and natural 20(S)-hydroxycholesterol showed a noncompetitive interaction.50 James et al. reported that N-terminal Sonic hedgehog significantly increased in vitro osteogenic differentiation in mouse adipose-derived stem cells and grafting of adipose-derived stem cell–treated N-terminal Sonic hedgehog resulted in significantly increased bone regeneration within the mouse tibia bone defect site.51 Kim et al. have made further observations on hydroxycholesterol-mediated osteogenesis. In M2 cells pretreated with a known hedgehog pathway inhibitor cyclopamine, the expression of Notch signaling-associated genes was induced and the pro-osteogenic effects of oxysterol were mitigated.39 Like hedgehog, the Notch signaling pathway plays an important role in the embryonic development, and its target genes are involved in regulating a variety of biological processes, including osteogenesis.52 Consistent with these previous studies, we observed that the pro-osteogenic effect of hydroxycholesterol in human adipose-derived stem cells was neutralized on the inhibition of hedgehog signaling. Although the complex nature of events occurring on a molecular level during hydroxycholesterol-mediated osteogenic differentiation of adipose-derived stem cells has yet to be fully elucidated, the identification of hedgehog as an important contributor to this process provides us with a focus for continuing study as we work toward translational applications. The results of this study provide additional support for the application of oxysterols and mesenchymal stem cells to the field of tissue engineering. Expanded understanding of the molecular events involved in hydroxycholesterol-induced osteogenesis in human adipose-derived stem cells is required, and demonstration of the in vivo significance of the in vitro results obtained in this study is a prerequisite. This work is ongoing in our laboratory and will serve as the foundation for
Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol future clinical studies using this promising novel osteogenic agent.
CONCLUSIONS This is the first study to describe the osteoinductive activity of hydroxycholesterol on human adipose-derived stem cells. We have confirmed that hydroxycholesterol can effect osteogenic differentiation in human adipose-derived stem cells, and that the process is mediated at least in part by the hedgehog signaling pathway. These findings further support our earlier work demonstrating that hydroxycholesterols have potent osteogenic activity, now observed in a human stem cell line. Hydroxycholesterols potentially offer new strategies for eventual translational application to clinical skeletal tissue engineering. However, before translation becomes clinical practice, several important parameters must be investigated. Patient characteristics such as sex, age, and harvest site could influence the osteogenic responsiveness of adipose-derived stem cells to hydroxycholesterol. Reza Jarrahy, M.D. Division of Plastic and Reconstructive Surgery David Geffen School of Medicine at UCLA 200 UCLA Medical Plaza, Suite 465 Los Angeles, Calif. 90095-6960 [email protected]
acknowledgments
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Plastic and Reconstructive Surgery • November 2014 25. Hattori H, Sato M, Masuoka K, et al. Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source. Cells Tissues Organs 2004;178:2–12. 26. Hicok KC, Du Laney TV, Zhou YS, et al. Human adiposederived adult stem cells produce osteoid in vivo. Tissue Eng. 2004;10:371–380. 27. Conejero JA, Lee JA, Parrett BM, et al. Repair of palatal bone defects using osteogenically differentiated fat-derived stem cells. Plast Reconstr Surg. 2006;117:857–863. 28. Zhu X, Shi W, Tai W, Liu F. The comparition of biological characteristics and multilineage differentiation of bone marrow and adipose derived mesenchymal stem cells. Cell Tissue Res. 2012;350:277–287. 29. Cowan CM, Shi YY, Aalami OO, et al. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol. 2004;22:560–567. 30. Cui L, Liu B, Liu G, et al. Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials 2007;28:5477–5486. 31. Guenther C, Pantalena-Filho L, Kingsley DM. Shaping skeletal growth by modular regulatory elements in the Bmp5 gene. PLoS Genet. 2008;4:e1000308. 32. Tsuji K, Bandyopadhyay A, Harfe BD, et al. BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet. 2006;38:1424–1429. 33. Chou YF, Zuk PA, Chang TL, Benhaim P, Wu BM. Adiposederived stem cells and BMP2: Part 1. BMP2-treated adiposederived stem cells do not improve repair of segmental femoral defects. Connect Tissue Res. 2011;52:109–118. 34. Zuk P, Chou YF, Mussano F, Benhaim P, Wu BM. Adiposederived stem cells and BMP2: Part 2. BMP2 may not influence the osteogenic fate of human adipose-derived stem cells. Connect Tissue Res. 2011;52:119–132. 35. Chen K, Aenlle KK, Curtis KM, et al. Hepatocyte growth factor (HGF) and 1,25- dihydroxyvitamin D together stimulate human bone marrow-derived stem cells toward the osteogenic phenotype by HGF-induced up-regulation of VDR. Bone 2012;51:69–77. 36. Levi B, Nelson ER, Brown K, et al. Differences in osteogenic differentiation of adipose-derived stromal cells from murine, canine, and human sources in vitro and in vivo. Plast Reconstr Surg. 2011;128:373–386. 37. Duan F, Yang C, Yang H, et al. Effect of retinoic acid on alkaline phosphatase expression in rat adipose-derived stem cells. Nan Fang Yi Ke Da Xue Xue Bao 2012;32:32–36. 38. Johnson JS, Meliton V, Kim WK, et al. Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem. 2011;112:1673–1684.
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39. Kim WK, Meliton V, Tetradis S, et al. Osteogenic oxysterol, 20(S)- hydroxycholesterol, induces notch target gene expression in bone marrow stromal cells. J Bone Miner Res. 2010;25:782–795. 40. Hokugo A, Sorice S, Yalom A, et al. In vitro study of a novel oxysterol for osteogenic differentiation on rabbit bone marrow stromal cells. Plast Reconstr Surg. 2013;132:70e–80e. 41. Hokugo A, Sorice S, Parhami F, et al. A novel oxysterol promotes bone regeneration in rabbit cranial bone defects. J Tissue Eng Regen Med. (in press). 42. Lemaire-Ewing S, Prunet C, Montange T, et al. Comparison of the cytotoxic, pro- oxidant and pro-inflammatory characteristics of different oxysterols. Cell Biol Toxicol. 2005;21:97–114. 43. Vicente López MA, Vázquez García MN, Entrena A, et al. Low doses of bone morphogenetic protein 4 increase the survival of human adipose-derived stem cells maintaining their stemness and multipotency. Stem Cells Dev. 2011;20:1011–1019. 44. Fuchs S, Dohle E, Kirkpatrick CJ. Sonic Hedgehog-mediated synergistic effects guiding angiogenesis and osteogenesis. Vitam Horm. 2012;88:491–506. 45. Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev Mol Cell Biol. 2005;6:306–317. 46. Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med. 2009;15:417–429. 47. Nakamura T, Aikawa T, Iwamoto-Enomoto M, et al. Induction of osteogenic differentiation by hedgehog proteins. Biochem Biophys Res Commun. 1997;237:465–469. 48. Spinella-Jaegle S, Rawadi G, Kawai S, et al. Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. J Cell Sci. 2001;114:2085–2094. 49. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 1999;13:2072–2086. 50. Nachtergaele S, Mydock LK, Krishnan K, et al. Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol. 2012;8:211–220. 51. James AW, Leucht P, Levi B, et al. Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells. Tissue Eng Part A 2010;16:2605–2616. 52. Nobta M, Tsukazaki T, Shibata Y, et al. Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling. J Biol Chem. 2005;280:15842–15848.
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 hydroxycholesterolmediated 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. (Plast. Reconstr. Surg. 134: 960, 2014.)
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raniofacial defects often present significant technical challenges to the plastic surgeon; furthermore, strategies for their correction can put a considerable financial burden on an already strained domestic health care system. The rate of spontaneous healing of cranial defects decreases substantially after 2 years From the Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA. Received for publication November 22, 2013; accepted April 2, 2014. The first two authors contributed equally to this work. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000601
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of age, necessitating surgical intervention in the setting of complex craniofacial defects. Currently accepted techniques include autologous bone grafting, the use of alloplastic materials, allografting, and distraction osteogenesis.1 Although effective, each of these modalities is associated with the Disclosure: The authors have no commercial associations or financial disclosures that might pose a conflict of interest with any information presented in this article. his work was supported by T THE PLASTIC SURGERY FOUNDATION.
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Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol potential for adverse effects or suboptimal longterm outcomes, including donor-site morbidity, graft failure, resorption, infection, limited sources of autologous donor bone, contour irregularities, and disease transmission.2–5 A large effort in the field of tissue engineering has been made to address current shortcomings associated with autologous bone grafts. Specifically, a significant body of research is dedicated to the study of stem cells that have the capacity to support bone regeneration. Bone marrow–derived mesenchymal stem cells, for example, have been successfully directed not only along osteogenic lines of differentiation, but also along chondrogenic, myogenic, and adipogenic pathways.6–9 Adipose-derived stem cells possess similar multipotent capabilities but are candidates for autologous tissue engineering and possess several distinct advantages compared with other mesenchymal stem cells. Adipose-derived stem cells have demonstrated the same multipotent differentiation capacity without the donor-site morbidity associated with the harvest of bone marrow–derived stem cells.8–12 The role of adipose-derived stem cells in healing in vivo skeletal defects has been well established in animal models.13–15 Successful examples of translational application also exist: Lendeckel et al.16 used adipose-derived stem cells to augment bone growth in a 7-year old patient who underwent reconstruction of a critical-size skull defect. Adiposederived stem cells can be harvested in large quantities through easily accessible subcutaneous deposits, enabling rapid and cost-effective expansion of multipotent stem cell populations. These features have made adipose-derived stem cells among the most popular multipotent stem cells in applied translational stem cell research. Hydroxycholesterols, also known as oxysterols, consist of a large family of 27-carbon oxygenated products of cholesterol. These molecules are present in the circulation and in human tissues.17 Endogenous hydroxycholesterols are formed in various ways, including auto-oxidation of cholesterol, lipid peroxidation, and enzymatically.18 Circulating hydroxycholesterols are also obtained from exogenous sources through dietary intake.19 Hydroxycholesterols are biologically active in diverse metabolic processes, including regulation of cholesterol homeostasis, atherosclerosis, sphingolipid metabolism, platelet aggregation, and apoptosis.20 Most relevant to this study, however, is the fact that hydroxycholesterols have been shown to have pro-osteogenic activity.20 Activation of the hedgehog signaling pathway has been implicated in the mechanism of action of oxysterols in modulating osteogenesis
in murine cells.21,22 Various naturally occurring hydroxycholesterol analogues, namely, 22(R)-, 20(S)-, and 22(S)-hydroxycholesterol, have been shown to induce osteogenic differentiation in the M2-10B4 murine multipotent bone mesenchymal stem cell line, and in primary murine mesenchymal stem cells.20 Despite the fact that hydroxycholesterols represent a promising new growth factor with the potential capacity for clinical application to bone regenerative medicine, and the widespread acceptance of adipose-derived stem cells as a promising tool in bone tissue engineering, no study to date has investigated the effects of hydroxycholesterols on human adipose-derived stem cells in the setting osteogenesis. In this study, we aim to investigate the potential translational significance of hydroxycholesterol-treated human adipose-derived stem cells by studying the effects of hydroxycholesterols on the osteogenic capacity of human adipose-derived stem cells and the mechanisms behind any such observed effects.
MATERIALS AND METHODS Dulbecco’s Modified Eagle Medium, l-glutamine, and penicillin-streptomycin were purchased from Invitrogen Corp. (Carlsbad, Calif.), and fetal bovine serum was obtained from Omega Scientific, Inc. (Tarzana, Calif.). A total RNA isolation kit (RNeasy Plus Mini Kit) was obtained from Qiagen (Valencia, Calif.) and reverse transcriptase was purchased from Bio-Rad (Hercules, Calif.). Ascorbic acid-2 phosphate, β-glycerol phosphate, dimethyl sulfoxide, 20(S)-hydroxycholesterol, and all other reagents were obtained from SigmaAldrich (St. Louis, Mo.). Isolation and Culture of Human Adipose-Derived Stem Cells Human adipose-derived stem cells were harvested from lipoaspirates obtained from female patients presenting for cosmetic liposuction procedures, as reported previously.9,10 Patients ranged in age between 35 and 50 years. Adipose-derived stem cell populations were isolated and expanded in culture using growth medium consisting of Dulbecco’s Modified Eagle Medium (4.5 g/liter, with l-glutamine and sodium pyruvate) supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 mg/ ml streptomycin, and 2.5 μg/ml amphotericin B. Cells were allowed to reach 90% confluence before they were passaged using 2.5% trypsin/0.23 mM ethylenediaminetetraacetic acid.
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Plastic and Reconstructive Surgery • November 2014 Second- or third-passage cell populations that consistently showed homogenous morphology and phenotype were used in this study.23 For various treatment conditions, cells were seeded into multiwell plates and cultured with either (1) control medium containing Dulbecco’s Modified Eagle Medium, 5% fetal bovine serum, 2 mM l-glutamine, and 1% penicillin-streptomycin; (2) osteogenic medium containing control medium with 3 mM β-glycerol phosphate and 50 μg/ml ascorbic acid-2 phosphate; or (3) osteogenic medium with various concentrations of 20(S)-hydroxycholesterol (1, 5, and 10 μM) dissolved in dimethyl sulfoxide. Medium was changed every 72 hours. All cell cultures were maintained at 37°C and 5% carbon dioxide. Alkaline Phosphatase Activity Assay After 4 days of cell culture under the various experimental conditions described above, colorimetric alkaline phosphatase activity assay on whole cell extracts was performed as reported previously.20 Cells were rinsed twice with phosphate-buffered saline and scraped into 200 μl of lysis buffer (0.2% NP-40 in 1 mM of magnesium chloride) with a rubber policeman and sonicated for 5 seconds. Reaction solution containing alkaline buffer stock solution and phosphatase substrate was added to each well. This mixture was then incubated for 10 to 15 minutes at 37°C. Yellow color was indicative of alkaline phosphatase activity. The reaction was stopped with the addition of 1N sodium hydroxide to each well, and absorbance was determined at 405 nm. Alkaline phosphatase activity was calculated using p-nitrophenol as a standard. Results were normalized to total protein determined using the Bradford protein assay.24 von Kossa Staining After 28 days of culture under the various described conditions, cells were washed twice with phosphate-buffered saline, fixed with 10% formalin in neutral buffer for 30 minutes, rinsed extensively with distilled water, and covered with 0.5 ml of 5% silver nitrate. The cells were incubated for 30 minutes in the dark and then rinsed with distilled water. The cells were exposed to ultraviolet light for 30 minutes and treated with 2.5% sodium thiosulfate solution for 5 minutes. Plates were finally rinsed extensively with tap water and airdried. Staining results were captured with a Nikon Coolpix 990 digital camera (Nikon Corp., Tokyo, Japan). The mineralized nodule area was measured using ImageJ software (National Institutes
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of Health, Bethesda, Md.), a digital image analysis system. Four independent cultures were evaluated in each of the experimental groups. Quantitative Real-Time Polymerase Chain Reaction After 5 days of culture under the various experimental conditions, cells were collected and total RNA was extracted using RNA isolation kits, according to the manufacturer’s instructions. RNA was reverse-transcribed using reverse transcriptase to make single-stranded cDNA templates. Real-time polymerase chain reaction for the osteogenic markers runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) was performed using Taqman Gene Expression Assay (Hs00231692_m1, Hs01587814_g1, and Hs00959010_m1 respectively; Applied Biosystems, Foster City, Calif.). Count values were normalized to glyceraldehyde 3-phosphate dehydrogenase (Hs02758991_g1) as an internal control, and relative expression levels versus untreated controls (i.e., control medium, harvested at the same time points) were determined using the 2−ΔΔCT method. Each gene was quantitated a minimum of three times. Inhibition of Hydroxycholesterol-Mediated Osteogenesis with the Hedgehog Signaling Inhibitor Cyclopamine To determine the mechanism by which hydroxycholesterol mediates its effects on osteogenic differentiation in human adipose-derived stem cells, we examined the hedgehog signaling pathway, which has been shown to play a role in the process of osteogenesis in murine cells.21,22 Human adipose-derived stem cells were pretreated for 2 hours with cyclopamine— a known inhibitor of hedgehog signaling—at a concentration of 4 μM. To control for the cyclopamine delivery vehicle, cells were also treated for 2 hours with osteogenic medium containing dimethyl sulfoxide only. Following pretreatment, the medium was replenished with 5 μM of hydroxycholesterol or control vehicle and the cells were cultured for 4 days. Alkaline phosphatase activity assays were performed as an indicator of osteogenic activity. Statistical Analysis Data are presented as means ± SD. Statistical differences between groups were evaluated by one-way analysis of variance, and post hoc multiple-comparison tests (Tukey-Kramer multiple
Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol comparison test) were performed to assess significance. A value of p < 0.05 was considered statistically significant.
RESULTS Effect of Hydroxycholesterol on Alkaline Phosphatase Activity in Human Adipose-Derived Stem Cells To determine the effect of hydroxycholesterol on the activity of proteins that are relevant to the osteogenic pathway, human adipose-derived stem cells were treated with varying doses of 20(S)hydroxycholesterol for 4 days, and alkaline phosphatase activity was measured (Fig. 1). Alkaline phosphatase activity by human adipose-derived stem cells treated with osteogenic medium increased approximately 1.5-fold compared with cells maintained in noninductive control medium. Treatment of human adipose-derived stem cells with 5 μM hydroxycholesterol significantly increased alkaline phosphatase activity relative to cells treated with osteogenic medium alone. However, the treatment of cells with lower (1 μM) and higher (10 μM) concentrations of hydroxycholesterol yielded no significant increases in alkaline phosphatase activity relative to controls.
Fig. 1. Hydroxycholesterol (OHC) induces alkaline phosphatase (ALP) activity in human adipose-derived stem cells. Human adipose-derived stem cells at confluence were treated with osteogenic medium (OM) and 1, 5, or 10 μM hydroxycholesterol. Control cultures were treated with control medium (CM) or osteogenic medium plus vehicle. After 4 days, alkaline phosphatase activity was measured in whole cell extracts. Data are normalized to alkaline phosphatase activity in the cells treated with control medium. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium.
Effect of Hydroxycholesterol on Osteogenic Differentiation and Mineralization in Human Adipose-Derived Stem Cells To determine the effects of hydroxycholesterol on osteogenic differentiation in human adipose-derived stem cells, as indicated by the ability of treated cells to form a mineralized matrix, cell cultures were maintained for 28 days and then assessed with von Kossa staining (Fig. 2). Cells cultured with control medium demonstrated a negligible mineralization response, whereas significant areas of positive von Kossa staining were observed in cultures of cells treated with osteogenic medium. Mineralization levels were increased slightly when human adipose-derived stem cells were treated with osteogenic medium containing either 1 μM or 10 μM hydroxycholesterol. However, a significant increase in mineralization relative to osteogenic medium–treated control human adipose-derived stem cells was noted in human adipose-derived stem cells treated with osteogenic medium and 5 μM of hydroxycholesterol. Effect of Hydroxycholesterol on Osteogenic Gene Expression in Human Adipose-Derived Stem Cells To determine the osteoinductive potential of hydroxycholesterol on human adipose-derived stem cells, human adipose-derived stem cells were treated with osteogenic medium supplemented with 5 μM hydroxycholesterol for 5 days, and the expression of three key osteogenic genetic markers was examined using real-time polymerase chain reaction (Fig. 3). Osteogenic medium supplemented with 5 μM hydroxycholesterol was chosen in light of its optimal effect on alkaline phosphatase activity and matrix mineralization. As shown in Figure 3, treatment of human adipose-derived stem cells with 5 μM hydroxycholesterol resulted in significant increases in RUNX2, OCN, and OPN expression relative to controls. Mechanism of Action of Hydroxycholesterol in Osteogenic Differentiation of Human AdiposeDerived Stem Cells The induction of alkaline phosphatase activity by 5 μM hydroxycholesterol was significantly inhibited by cyclopamine (Fig. 4). There was no adverse effect of vehicle on human adiposederived stem cell osteogenesis, as elevated alkaline phosphatase activity was measured in this control group.
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Fig. 2. Hydroxycholesterol (OHC) treatment increases mineralization capacity of human adipose-derived stem cells. Mineralization of culture plates by human adipose-derived stem cells treated with 1, 5, or 10 μM of hydroxycholesterol at day 14 was evaluated by von Kossa staining. (Above) Representative images of von Kossa stains are shown (original magnification, × 4). (Below) The percentage of von Kossa–positive area per plate is shown. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium. CM, control medium; OM, osteogenic medium.
Fig. 3. Hydroxycholesterol (OHC) induces expression of markers of osteogenic differentiation in human adipose-derived stem cells. Human adipose-derived stem cells at confluence were treated with 5 μM hydroxycholesterol. Control cells were cultured with osteogenic medium (OM) plus vehicle. After 5 days, mRNA expression was measured by quantitative realtime polymerase chain reaction. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05) compared with osteogenic medium.
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Fig. 4. Alkaline phosphatase (ALP) activity is diminished by addition of cyclopamine. Human adipose-derived stem cells at confluence were pretreated with control vehicle or 4 μM cyclopamine (Cyc) for 2 hours. Next, 5 μM hydroxycholesterol (OHC) or control vehicle was added to cell cultures. Alkaline phosphatase activity after 4 days of treatment was measured. Data are normalized to alkaline phosphatase activity in the cells treated with control medium. Tukey-Kramer multiple comparison test; *significant differences (p < 0.05). CM, control medium; OM, osteogenic medium.
DISCUSSION Advances in the field of cell-based regenerative medicine over the past decade have demonstrated that adipose-derived stem cells are an exciting alternative to bone marrow mesenchymal stem cells in bone regeneration models. In fact, their in vitro osteogenic capacity is well established and strongly supported by numerous in vivo studies.13,25–27 Such findings, paired with the advantages of adipose-derived stem cells compared with bone marrow mesenchymal stem cells in terms of availability, ease of harvest and isolation, higher stem cell yields per gram of tissue,28 and plasticity, are largely responsible for their popularity in tissue engineering. Although pro-osteogenic effects of bone morphogenetic proteins in human adiposederived stem cells have been reported,29–32 recent evidence calls these effects into question.33,34 Moreover, the drawbacks of bone morphogenetic protein use, including costs and adverse effects associated with therapy, have contributed to a growing interest in exploring alternative osteoinductive growth factors.35–38 Hydroxycholesterol has emerged as one such promising molecule. Its pro-osteogenic effects in murine cells have been
established.20,22,38,39 We have also demonstrated that a novel semisynthetic hydroxycholesterol, named Oxy49, induces the osteogenic differentiation of rabbit bone marrow stromal cells as effectively as bone morphogenetic protein-2 both in vitro40 and in vivo,41 inducing bone formation in critical-size rabbit calvarial defects. Because no prior studies have examined the effect of hydroxycholesterol on human adiposederived stem cells, and because adipose-derived stem cells are a target of intense focus in bone engineering research, this study was conducted to form an initial assessment of the pro-osteogenic effectiveness of hydroxycholesterol therapy on human adipose-derived stem cells. In previously performed studies, a concentration of 1 to 5 μM oxysterol significantly increased osteogenic activity in a murine multipotent bone mesenchymal stem cell line20,38 and primary rabbit bone marrow stromal cells.40,41 Based on these studies, we tested the effects of three different concentrations (1, 5, and 10 μM) of hydroxycholesterol on human adipose-derived stem cells. The results of this work demonstrate that hydroxycholesterol is in fact an effective inducer of osteogenic differentiation in human adipose-derived stem cells. At an optimal dose of 5 μM, osteogenic differentiation was observed both at the level of gene expression and protein function. Exposure of human adipose-derived stem cells to hydroxycholesterol significantly increased alkaline phosphatase activity and osteogenic gene expression in comparison with control cultures. Most importantly, matrix mineralization, a terminal functional measure of osteoblast function, was significantly induced on hydroxycholesterol treatment compared with controls. Our results show that hydroxycholesterol dosing must be optimized. Specifically, we observed a decrease in osteogenesis when human adiposederived stem cells were exposed to higher doses of hydroxycholesterol (i.e., 10 μM). This may indicate a decreased responsiveness to higher levels of hydroxycholesterol because of receptor saturation/down-regulation or an induction of apoptosis. Lemaire-Ewing et al. reported cytotoxicity caused by oxysterols, including 7β-hydroxycholesterol and 7- ketocholesterol, leading to cell apoptosis on human promonocytic leukemia cells.42 However, oxysterol-associated toxicity varies between cell types. Vicente López and colleagues43 found a similar phenomenon when incubating human adipose-derived stem cells with various concentrations of bone morphogenetic protein and measuring cell viability, proliferation,
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Plastic and Reconstructive Surgery • November 2014 and multipotency. Although low doses of bone morphogenetic protein resulted in a significant increase in overall cell viability parameters, high doses significantly inhibited cell proliferation and increased apoptosis. A similar effect may be occurring on hydroxycholesterol stimulation. As such, additional studies elucidating the binding kinetics of hydroxycholesterol to its receptors in adiposederived stem cells will be necessary to ascertain whether a dose-dependent response occurs at the receptor level. As experimental evidence describing the efficacy and potential clinical utility of hydroxycholesterol continues to expand, a growing interest has been dedicated to discovering the molecular mechanisms by which osteogenic differentiation of progenitor cells occurs. The critical role that the hedgehog signaling pathway plays in hydroxycholesterol-induced osteogenic differentiation in murine cells has been demonstrated by Kim et al.22 In embryogenesis, the hedgehog signaling pathway is crucial in modulating embryonic skeletal development. Hedgehog signaling is reactivated during adult repair processes, controlling the expression of a variety of growth factors and triggering the differentiation of mesenchymal stem cells toward osteogenic lineage.44 Hedgehog signaling is initiated by the binding of hedgehog ligand to Patched (Ptch), a 12-transmembrane protein receptor.45 In the absence of hedgehog interaction, Ptch suppresses the signal transducer activities of the seven-transmembrane protein smoothened (Smo). In contrast, binding of hedgehog to Ptch prevents the suppressive effects of Ptch, thus allowing activation of Smo, which in turn activates the intracytoplasmic Gli proteins (GliA, Gli2, and GliR). After its translocation into the nucleus, GliA can activate various gene targets through interactions with specific DNA-binding elements.46 Downstream gene targets include those involved in osteogenesis, which has been demonstrated in previous reports that link hedgehog signaling to the differentiation of pluripotent stem cells into osteoblasts.47,48 In addition, studies have investigated Indian hedgehog knockout mice and found that these mice subsequently lack normal osteoblast function and endochondral bone formation, leading to stunted appendages.49 These strongly suggest that the hedgehog signaling pathway is necessary for the normal progression of osteogenesis. Until recently, it was unclear how oxysterols activate hedgehog signaling. Unlike most synthetic hedgehog effectors, oxysterols do not compete with cyclopamine, a
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hedgehog signaling inhibitor for binding to Smo, a property that has been used to argue that Smo is not a direct target for oxysterols.21,50 Recently, Nachtergaele et al.50 demonstrated the presence of at least two binding sites on Smo, one that binds natural 20(S)-hydroxycholesterol and a second that binds cyclopamine and the small molecule Smoothened agonist (SAG), which activates Smo by competing for the cyclopamine binding site. They found various binding interactions between these three molecules: whereas SAG and natural 20(S)-hydroxycholesterol showed a positive allosteric interaction, SAG and cyclopamine showed a competitive interaction, while cyclopamine and natural 20(S)-hydroxycholesterol showed a noncompetitive interaction.50 James et al. reported that N-terminal Sonic hedgehog significantly increased in vitro osteogenic differentiation in mouse adipose-derived stem cells and grafting of adipose-derived stem cell–treated N-terminal Sonic hedgehog resulted in significantly increased bone regeneration within the mouse tibia bone defect site.51 Kim et al. have made further observations on hydroxycholesterol-mediated osteogenesis. In M2 cells pretreated with a known hedgehog pathway inhibitor cyclopamine, the expression of Notch signaling-associated genes was induced and the pro-osteogenic effects of oxysterol were mitigated.39 Like hedgehog, the Notch signaling pathway plays an important role in the embryonic development, and its target genes are involved in regulating a variety of biological processes, including osteogenesis.52 Consistent with these previous studies, we observed that the pro-osteogenic effect of hydroxycholesterol in human adipose-derived stem cells was neutralized on the inhibition of hedgehog signaling. Although the complex nature of events occurring on a molecular level during hydroxycholesterol-mediated osteogenic differentiation of adipose-derived stem cells has yet to be fully elucidated, the identification of hedgehog as an important contributor to this process provides us with a focus for continuing study as we work toward translational applications. The results of this study provide additional support for the application of oxysterols and mesenchymal stem cells to the field of tissue engineering. Expanded understanding of the molecular events involved in hydroxycholesterol-induced osteogenesis in human adipose-derived stem cells is required, and demonstration of the in vivo significance of the in vitro results obtained in this study is a prerequisite. This work is ongoing in our laboratory and will serve as the foundation for
Volume 134, Number 5 • Osteogenic Effects of Hydroxycholesterol future clinical studies using this promising novel osteogenic agent.
CONCLUSIONS This is the first study to describe the osteoinductive activity of hydroxycholesterol on human adipose-derived stem cells. We have confirmed that hydroxycholesterol can effect osteogenic differentiation in human adipose-derived stem cells, and that the process is mediated at least in part by the hedgehog signaling pathway. These findings further support our earlier work demonstrating that hydroxycholesterols have potent osteogenic activity, now observed in a human stem cell line. Hydroxycholesterols potentially offer new strategies for eventual translational application to clinical skeletal tissue engineering. However, before translation becomes clinical practice, several important parameters must be investigated. Patient characteristics such as sex, age, and harvest site could influence the osteogenic responsiveness of adipose-derived stem cells to hydroxycholesterol. Reza Jarrahy, M.D. Division of Plastic and Reconstructive Surgery David Geffen School of Medicine at UCLA 200 UCLA Medical Plaza, Suite 465 Los Angeles, Calif. 90095-6960 [email protected]
acknowledgments
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