Dynamic Fluid Flow Mechanical Stimulation Modulates Bone Marrow Mesenchymal Stem Cells

Abstract

Osteoblasts are derived from mesenchymal stem cells (MSCs), which initiate and regulate bone formation. New strategies for osteoporosis treatments have aimed to control the fate of MSCs. While functional disuse decreases MSC growth and osteogenic potentials, mechanical signals enhance MSC quantity and bias their differentiation toward osteoblastogenesis. Through a non-invasive dynamic hydraulic stimulation (DHS), we have found that DHS can mitigate trabecular bone loss in a functional disuse model via rat hindlimb suspension (HLS). To further elucidate the downstream cellular effect of DHS and its potential mechanism underlying the bone quality enhancement, a longitudinal in vivo study was designed to evaluate the MSC populations in response to DHS over 3, 7, 14, and 21 days. Five-month old female Sprague Dawley rats were divided into three groups for each time point: age-matched control, HLS, and HLS+DHS. DHS was delivered to the right mid-tibiae with a daily “10 min on-5 min off-10 min on” loading regime for five days/week. At each sacrifice time point, bone marrow MSCs of the stimulated and control tibiae were isolated through specific cell surface markers and quantified by flow cytometry analysis. A strong time-dependent manner of bone marrow MSC induction was observed in response to DHS, which peaked on day 14. After 21 days, this effect of DHS was diminished. This study indicates that the MSC pool is positively influenced by the mechanical signals driven by DHS. Coinciding with our previous findings of mitigation of disuse bone loss, DHS induced changes in MSC number may bias the differentiation of the MSC population towards osteoblastogenesis, thereby promoting bone formation under disuse conditions. This study provides insights into the mechanism of time-sensitive MSC induction in response to mechanical loading, and for the optimal design of osteoporosis treatments.

References

[1] Lau RY, Guo X. A review on current osteoporosis research: with special focus on disuse bone loss. J Osteoporos, 2011, 2011: 293808

[2] Turner AS. Animal models of osteoporosis—necessity and limitations. Eur Cell Mater, 2001, 1: 66-81

[3] Garnero P, Delmas PD. Contribution of bone mineral density and bone turnover markers to the estimation of risk of osteoporotic fracture in postmenopausal women. J Musculoskelet Neuronal Interact, 2004, 4: 50-63

[4] Lu X, Li S, Cheng J. Bone marrow mesenchymal stem cells: progress in bone/cartilage defect repair. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002, 19: 135-139

[5] Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell, 2002, 2: 389-406

[6] Kelly DJ, Jacobs CR. The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. Birth Defects Res C Embryo Today, 2010, 90: 75-85

[7] Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem, 2003, 278: 45969-45977

[8] David V, Martin A, Lafage-Proust MH, Malaval L, Peyroche S, Jones DB, Vico L, Guignandon A. Mechanical loading down-regulates peroxisome proliferator-activated receptor gamma in bone marrow stromal cells and favors osteoblastogenesis at the expense of adipogenesis. Endocrinology, 2007, 148: 2553-2562

[9] Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest, 2006, 116: 1202-1209

[10] Menuki K, Mori T, Sakai A, Sakuma M, Okimoto N, Shimizu Y, Kunugita N, Nakamura T. Climbing exercise enhances osteoblast differentiation and inhibits adipogenic differentiation with high expression of PTH/PTHrP receptor in bone marrow cells. Bone, 2008, 43: 613-620

[11] Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung UI, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H. PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest, 2004, 113: 846-855

[12] Castillo AB, Jacobs CR. Mesenchymal stem cell mechanobiology. Curr Osteoporos Rep, 2010, 8: 98-104

[13] Holtorf HL, Jansen JA, Mikos AG. Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone. J Biomed Mater Res A, 2005, 72: 326-334

[14] Datta N, Pham QP, Sharma U, Sikavitsas VI, Jansen JA, Mikos AG. In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci U S A, 2006, 103: 2488-2493

[15] Bjerre L, Bunger CE, Kassem M, Mygind T. Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds. Biomaterials, 2008, 29: 2616-2627

[16] Duty AO, Oest ME, Guldberg RE. Cyclic mechanical compression increases mineralization of cell-seeded polymer scaffolds in vivo. J Biomech Eng, 2007, 129: 531-539

[17] Emans PJ, Pieper J, Hulsbosch MM, Koenders M, Kreijveld E, Surtel DA, van Blitterswijk CA, Bulstra SK, Kuijer R, Riesle J. Differential cell viability of chondrocytes and progenitor cells in tissue-engineered constructs following implantation into osteo-chondral defects. Tissue Eng, 2006, 12: 1699-1709

[18] LeBaron RG, Athanasiou KA. Ex vivo synthesis of articular cartilage. Biomaterials, 2000, 21: 2575-2587

[19] Bryant SJ, Chowdhury TT, Lee DA, Bader DL, Anseth KS. Crosslinking density influences chondrocyte metabolism in dynamically loaded photocrosslinked poly(ethylene glycol) hydrogels. Ann Biomed Eng, 2004, 32: 407-417

[20] Luu YK, Capilla E, Rosen CJ, Gilsanz V, Pessin JE, Judex S, Rubin CT. Mechanical stimulation of mesenchymal stem cell proliferation and differentiation promotes osteogenesis while preventing dietary-induced obesity. J Bone Miner Res, 2009, 24: 50-61

[21] Sen B, Xie Z, Case N, Styner M, Rubin CT, Rubin J. Mechanical signal influence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen. J Biomech, 2011, 44: 593-599

[22] Sen B, Xie Z, Case N, Ma M, Rubin C, Rubin J. Mechanical strain inhibits adipogenesis in mesenchymal stem cells by stimulating a durable beta-catenin signal. Endocrinology, 2008, 149: 6065-6075

[23] Patel MJ, Liu W, Sykes MC, Ward NE, Risin SA, Risin D, Jo H. Identification of mechanosensitive genes in osteoblasts by comparative microarray studies using the rotating wall vessel and the random positioning machine. J Cell Biochem, 2007, 101: 587-599

[24] Pardo SJ, Patel MJ, Sykes MC, Platt MO, Boyd NL, Sorescu GP, Xu M, van Loon JJ, Wang MD, Jo H. Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts. Am J Physiol Cell Physiol, 2005, 288: C1211-C1221

[25] Carmeliet G, Nys G, Stockmans I, Bouillon R. Gene expression related to the differentiation of osteoblastic cells is altered by microgravity. Bone, 1998, 22: 139S-143S

[26] Zayzafoon M, Gathings WE, McDonald JM. Modeled micro-gravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis. Endocrinology, 2004, 145: 2421-2432

[27] Visigalli D, Strangio A, Palmieri D, Manduca P. Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells. BMC Musculoskelet Disord, 2010, 11: 147

[28] Pan Z, Yang J, Guo C, Shi D, Shen D, Zheng Q, Chen R, Xu Y, Xi Y, Wang J. Effects of hindlimb unloading on ex vivo growth and osteogenic/adipogenic potentials of bone marrow-derived mesenchymal stem cells in rats. Stem Cells Dev, 2008, 17: 795-804

[29] Hu M, Cheng J, Qin YX. Dynamic hydraulic flow stimulation on mitigation of trabecular bone loss in a rat functional disuse model. Bone, 2012, 51: 819-825

[30] Lam H, Qin YX. The effects of frequency-dependent dynamic muscle stimulation on inhibition of trabecular bone loss in a disuse model. Bone, 2008, 43: 1093-1100

[31] Zangi L, Rivkin R, Kassis I, Levdansky L, Marx G, Gorodetsky R. High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin microbeads. Tissue Eng, 2006, 12: 2343-2354

[32] Harting M, Jimenez F, Pati S, Baumgartner J, Cox C Jr. Immunophenotype characterization of rat mesenchymal stromal cells. Cytotherapy, 2008, 10: 243-253

[33] Wang C, Xu Y, Song WG, Chang WS. Isolation and culturation, phenotype detection of rat bone marrow mesenchymal stem cells. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007, 23: 466-468

[34] Mafi P, Hindocha S, Mafi R, Griffin M, Khan WS. Adult mesenchymal stem cells and cell surface characterization — a systematic review of the literature. Open Orthop J, 2011, 5: 253-260

[35] Marcus AJ, Coyne TM, Rauch J, Woodbury D, Black IB. Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane. Differentiation, 2008, 76: 130-144

[36] Phinney DG, Kopen G, Righter W, Webster S, Tremain N, Prockop DJ. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem, 1999, 75: 424-436

[37] Barou O, Palle S, Vico L, Alexandre C, Lafage-Proust MH. Hind-limb unloading in rat decreases preosteoblast proliferation assessed in vivo with BrdU incorporation. Am J Physiol, 1998, 274: E108-E114

[38] Basso N, Bellows CG, Heersche JN. Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats. Bone, 2005, 36: 173-183

[39] Raitakari M, Nuutila P, Ruotsalainen U, Teras M, Eronen E, Laine H, Raitakari OT, Iida H, Knuuti MJ, Yki-Jarvinen H. Relationship between limb and muscle blood flow in man. J Physiol, 1996, 496: 543-549

[40] Kreke MR, Huckle WR, Goldstein AS. Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. Bone, 2005, 36: 1047-1055