Nitric Oxide and Prostaglandin E<sub>2</sub> Secretion in Osteocytes Induced by Intermittent Cyclic Compressive Force_Journal of Biomedical Engineering

Authors：

YINJian ^1,2 , HAOZhichao ^1,2 , LIAOShuang ^1,2 , LIUYing ^1,2 , SHENJiefei ^1,2 , LIAOYunmao ¹ ,  WANGHang ^1,2

1. State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China;
2. Department of Prosthodontics, West China College of Stomatology, Sichuan University, Chengdu 610041, China;

Corresponding author：

WANGHang, Email: Wanghang@scu.edu.cn

Keywords：

mechanical stimulation; rest period; osteocyte; mechanosensitivity

DOI：

10.7507/1001-5515.20140116

Video：

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This paper is aimed to investigate the effect of rest-inserted loading on the mechanosensitivity of osteocytes. In the investigation, cultured MLO-Y4 osteocyte-like cells were strained on cyclic compressive force (CCF) by the self-made compressive loading device. Then we observed the effect of different rest periods-inserted loading (5 s, 15 s, 30 s, respectively) on the mechanosensitivity of osteocytes. We then determined the levels of secreted nitric oxide (NO) and prostaglandin E₂ (PGE₂) by Griess method and enzyme linked immunosorbent assay (ELISA), respectively. We then stained the cytoskeleton F-actin using immunofluorescence. We found that the expressions of NO and PGE₂ in rest-inserted strained groups (>15 s) were significantly increased compared to those in the continuous strained group. And rest-inserted loading promoted the parallel alignment of stress fibers. It indicates that rest-inserted loading could promote the mechanosensitivity of osteocytes, and this might be related to the parallel alignment of stress fibers.

Citation： YINJian, HAOZhichao, LIAOShuang, LIUYing, SHENJiefei, LIAOYunmao, WANGHang. Nitric Oxide and Prostaglandin E₂ Secretion in Osteocytes Induced by Intermittent Cyclic Compressive Force. Journal of Biomedical Engineering, 2014, 31(3): 619-624. doi: 10.7507/1001-5515.20140116 Copy

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2.	FROST H M. A determinant of bone architecture.The minimum effective strain[J]. Clin Orthop Relat Res, 1983, 175:286-292.
3.	RUBIN C T, LANYON L E. Regulation of bone mass by mechanical strain magnitude[J]. Calcif Tissue Int, 1985, 37(4):411-417.
4.	BATRA N N, LI Y J, YELLOWLEY C E, et al. Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells[J]. J Biomech, 2005, 38(9):1909-1917.
5.	RUBIN C T, MCLEOD K J. Promotion of bony ingrowth by frequency-specific, low-amplitude mechanical strain[J]. Clin Orthop Relat Res, 1994, 298:165-174.
6.	ROBLING A G, HINANT F M, BURR D B, et al. Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts[J]. J Bone Miner Res, 2002, 17(8):1545-1554.
7.	TURNER C H. Three rules for bone adaptation to mechanical stimuli[J]. Bone, 1998, 23(5):399-407.
8.	UMEMURA Y, ISHIKO T, YAMAUCHI T, et al. Five jumps per day increase bone mass and breaking force in rats[J]. J Bone Miner Res, 1997, 12(9):1480-1485.
9.	ROBLING A G, BURR D B, TURNER C H. Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading[J]. J Bone Miner Res, 2000, 15(8):1596-1602.
10.	ROBLING A G, BURR D B, TURNER C H. Recovery periods restore mechanosensitivity to dynamically loaded bone[J]. J Exp Biol, 2001, 204(Pt 19):3389-3399.
11.	PARFITT A M. The cellular basis of bone turnover and bone loss:a rebuttal of the osteocytic resorption--bone flow theory[J]. Clin Orthop Relat Res, 1977, 127:236-247.
12.	CHEN J H, LIU C, YOU L, et al. Boning up on Wolff's law:mechanical regulation of the cells that make and maintain bone[J]. J Biomech, 2010, 43(1):108-118.
13.	BONEWALD L F. Mechanosensation and transduction in osteocytes[J]. Bonekey Osteovision, 2006, 3(10):7-15.
14.	KLEIN-NULEND J, VAN DER PLAS A, SEMEINS C M, et al. Sensitivity of osteocytes to biomechanical stress in vitro[J]. FASEB J, 1995, 9(5):441-445.
15.	BONEWALD L F. The amazing osteocyte[J]. J Bone Miner Res, 2011, 26(2):229-238.
16.	JACOBS C R, TEMIYASATHIT S, CASTILLO A B. Osteocyte mechanobiology and pericellular mechanics[J]. Annu Rev Biomed Eng, 2010, 12:369-400.
17.	YOUNG W C, BUDYNAS B. Formulas for stress and strain[M]. New York:Mc Graw Hill, 1954.
18.	RUBIN C T, LANYON L E. Dynamic strain similarity in vertebrates:an alternative to allometric limb bone scaling[J]. J Theor Biol, 1984, 107(2):321-327.
19.	FRITTON S P, MCLEOD K J, RUBIN C T. Quantifying the strain history of bone:spatial uniformity and self-similarity of low-magnitude strains[J]. J Biomech, 2000, 33(3):317-325.
20.	SRINIVASAN S, WEIMER D A, AGANS S C, et al. Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle[J]. J Bone Miner Res, 2002, 17(9):1613-1620.
21.	SRINIVASAN S, AUSK B J, POLIACHIK S L, et al. Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles[J]. J Appl Physiol, 2007, 102(5):1945-1952.
22.	PLUNKETT N A, PARTAP S, O'BRIEN F J. Osteoblast response to rest periods during bioreactor culture of collagen-glycosaminoglycan scaffolds[J]. Tissue Eng Part A, 2010, 16(3):943-951.
23.	KLEIN-NULEND J, VELDHUIJZEN J P, DE JONG M, et al. Increased bone formation and decreased bone resorption in fetal mouse calvaria as a result of intermittent compressive force in vitro[J]. Bone Miner, 1987, 2(6):441-448.
24.	HUISKES R, RUIMERMAN R, VAN LENTHE G H, et al. Effects of mechanical forces on maintenance and adaptation of form in trabecular bone[J]. Nature, 2000, 405(6787):704-706.
25.	BANG J K, HWANG S J, KO C Y, et al. Heat treatment and rest-inserted exercise enhances EMG activity of the lower limb[J].International Science Index, 2007, 1(11):902-905.
26.	KLEIN-NULEND J, BAKKER A D, BACABAC R G, et al. Mechanosensation and transduction in osteocytes[J]. Bone, 2013, 54(2):182-190.
27.	KLEIN-NULEND J, SEMEINS C M, AJUBI N E, et al. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts--correlation with prostaglandin upregulation[J]. Biochem Biophys Res Commun, 1995, 217(2):640-648.
28.	XIONG J, ONAL M, JILKA R L, et al. Matrix-embedded cells control osteoclast formation[J]. Nat Med, 2011, 17(10):1235-1241.
29.	TURNER C H, TAKANO Y, OWAN I, et al. Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats[J]. Am J Physiol, 1996, 270(4 Pt 1):E634-E639.
30.	NAKASHIMA T, HAYASHI M, FUKUNAGA T, et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression[J]. Nat Med, 2011, 17(10):1231-1234.
31.	SAMY A S, IGWE O J. Regulation of IL-1β-induced cyclooxygenase-2 expression by interactions of Aβ peptide, apolipoprotein E and nitric oxide in human neuroglioma[J]. J Mol Neurosci, 2012, 47(3):533-545.
32.	BANSAL K, NARAYANA Y, PATIL S A, et al. M. bovis BCG induced expression of COX-2 involves nitric oxide-dependent and-independent signaling pathways[J]. J Leukoc Biol, 2009, 85(5):804-816.
33.	SALVEMINI D, MISKO T P, MASFERRER J L, et al. Nitric oxide activates cyclooxygenase enzymes[J]. Proc Natl Acad Sci U S A, 1993, 90(15):7240-7244.
34.	TUNÇTAN B, ALTUG S, ULUDAG O, et al. Effects of cyclooxygenase inhibitors on nitric oxide production and survival in a mice model of sepsis[J]. Pharmacol Res, 2003, 48(1):37-48.
35.	COOK S, VOLLENWEIDER P, MÉNARD B, et al. Increased eNO and pulmonary iNOS expression in eNOS null mice[J]. Eur Respir J, 2003, 21(5):770-773.
36.	ZHU Y, ZHU M, LANCE P. iNOS signaling interacts with COX-2 pathway in colonic fibroblasts[J]. Exp Cell Res, 2012, 318(16):2116-2127.
37.	SMITH W L, GARAVITO R M, DEWITT D L. Prostaglandin endoperoxide H synthases(cyclooxygenases)-1 and-2[J]. J Biol Chem, 1996, 271(52):33157-33160.
38.	AJUBI N E, KLEIN-NULEND J, NIJWEIDE P J, et al. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes--a cytoskeleton-dependent process[J]. Biochem Biophys Res Commun, 1996, 225(1):62-68.
39.	李晓东,黄跃生.机械信号的细胞感受与转导[J].中国病理生理杂志,2004(3):186-190.
40.	MCGARRY J G, KLEIN-NULEND J, PRENDERGAST P J. The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts[J]. Biochem Biophys Res Commun, 2005, 330(1):341-348.
41.	ALLEN F D, HUNG C T, POLLACK S R, et al. Serum modulates the intracellular Calcium response of primary cultured bone cells to shear flow[J]. J Biomech, 2000, 33(12):1585-1591.

1. WOLFF J. Das Gesetz der Transformation der Knochen[M]. Berlin:Hirschwald, 1892.
2. FROST H M. A determinant of bone architecture.The minimum effective strain[J]. Clin Orthop Relat Res, 1983, 175:286-292.
3. RUBIN C T, LANYON L E. Regulation of bone mass by mechanical strain magnitude[J]. Calcif Tissue Int, 1985, 37(4):411-417.
4. BATRA N N, LI Y J, YELLOWLEY C E, et al. Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells[J]. J Biomech, 2005, 38(9):1909-1917.
5. RUBIN C T, MCLEOD K J. Promotion of bony ingrowth by frequency-specific, low-amplitude mechanical strain[J]. Clin Orthop Relat Res, 1994, 298:165-174.
6. ROBLING A G, HINANT F M, BURR D B, et al. Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts[J]. J Bone Miner Res, 2002, 17(8):1545-1554.
7. TURNER C H. Three rules for bone adaptation to mechanical stimuli[J]. Bone, 1998, 23(5):399-407.
8. UMEMURA Y, ISHIKO T, YAMAUCHI T, et al. Five jumps per day increase bone mass and breaking force in rats[J]. J Bone Miner Res, 1997, 12(9):1480-1485.
9. ROBLING A G, BURR D B, TURNER C H. Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading[J]. J Bone Miner Res, 2000, 15(8):1596-1602.
10. ROBLING A G, BURR D B, TURNER C H. Recovery periods restore mechanosensitivity to dynamically loaded bone[J]. J Exp Biol, 2001, 204(Pt 19):3389-3399.
11. PARFITT A M. The cellular basis of bone turnover and bone loss:a rebuttal of the osteocytic resorption--bone flow theory[J]. Clin Orthop Relat Res, 1977, 127:236-247.
12. CHEN J H, LIU C, YOU L, et al. Boning up on Wolff's law:mechanical regulation of the cells that make and maintain bone[J]. J Biomech, 2010, 43(1):108-118.
13. BONEWALD L F. Mechanosensation and transduction in osteocytes[J]. Bonekey Osteovision, 2006, 3(10):7-15.
14. KLEIN-NULEND J, VAN DER PLAS A, SEMEINS C M, et al. Sensitivity of osteocytes to biomechanical stress in vitro[J]. FASEB J, 1995, 9(5):441-445.
15. BONEWALD L F. The amazing osteocyte[J]. J Bone Miner Res, 2011, 26(2):229-238.
16. JACOBS C R, TEMIYASATHIT S, CASTILLO A B. Osteocyte mechanobiology and pericellular mechanics[J]. Annu Rev Biomed Eng, 2010, 12:369-400.
17. YOUNG W C, BUDYNAS B. Formulas for stress and strain[M]. New York:Mc Graw Hill, 1954.
18. RUBIN C T, LANYON L E. Dynamic strain similarity in vertebrates:an alternative to allometric limb bone scaling[J]. J Theor Biol, 1984, 107(2):321-327.
19. FRITTON S P, MCLEOD K J, RUBIN C T. Quantifying the strain history of bone:spatial uniformity and self-similarity of low-magnitude strains[J]. J Biomech, 2000, 33(3):317-325.
20. SRINIVASAN S, WEIMER D A, AGANS S C, et al. Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle[J]. J Bone Miner Res, 2002, 17(9):1613-1620.
21. SRINIVASAN S, AUSK B J, POLIACHIK S L, et al. Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles[J]. J Appl Physiol, 2007, 102(5):1945-1952.
22. PLUNKETT N A, PARTAP S, O'BRIEN F J. Osteoblast response to rest periods during bioreactor culture of collagen-glycosaminoglycan scaffolds[J]. Tissue Eng Part A, 2010, 16(3):943-951.
23. KLEIN-NULEND J, VELDHUIJZEN J P, DE JONG M, et al. Increased bone formation and decreased bone resorption in fetal mouse calvaria as a result of intermittent compressive force in vitro[J]. Bone Miner, 1987, 2(6):441-448.
24. HUISKES R, RUIMERMAN R, VAN LENTHE G H, et al. Effects of mechanical forces on maintenance and adaptation of form in trabecular bone[J]. Nature, 2000, 405(6787):704-706.
25. BANG J K, HWANG S J, KO C Y, et al. Heat treatment and rest-inserted exercise enhances EMG activity of the lower limb[J].International Science Index, 2007, 1(11):902-905.
26. KLEIN-NULEND J, BAKKER A D, BACABAC R G, et al. Mechanosensation and transduction in osteocytes[J]. Bone, 2013, 54(2):182-190.
27. KLEIN-NULEND J, SEMEINS C M, AJUBI N E, et al. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts--correlation with prostaglandin upregulation[J]. Biochem Biophys Res Commun, 1995, 217(2):640-648.
28. XIONG J, ONAL M, JILKA R L, et al. Matrix-embedded cells control osteoclast formation[J]. Nat Med, 2011, 17(10):1235-1241.
29. TURNER C H, TAKANO Y, OWAN I, et al. Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats[J]. Am J Physiol, 1996, 270(4 Pt 1):E634-E639.
30. NAKASHIMA T, HAYASHI M, FUKUNAGA T, et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression[J]. Nat Med, 2011, 17(10):1231-1234.
31. SAMY A S, IGWE O J. Regulation of IL-1β-induced cyclooxygenase-2 expression by interactions of Aβ peptide, apolipoprotein E and nitric oxide in human neuroglioma[J]. J Mol Neurosci, 2012, 47(3):533-545.
32. BANSAL K, NARAYANA Y, PATIL S A, et al. M. bovis BCG induced expression of COX-2 involves nitric oxide-dependent and-independent signaling pathways[J]. J Leukoc Biol, 2009, 85(5):804-816.
33. SALVEMINI D, MISKO T P, MASFERRER J L, et al. Nitric oxide activates cyclooxygenase enzymes[J]. Proc Natl Acad Sci U S A, 1993, 90(15):7240-7244.
34. TUNÇTAN B, ALTUG S, ULUDAG O, et al. Effects of cyclooxygenase inhibitors on nitric oxide production and survival in a mice model of sepsis[J]. Pharmacol Res, 2003, 48(1):37-48.
35. COOK S, VOLLENWEIDER P, MÉNARD B, et al. Increased eNO and pulmonary iNOS expression in eNOS null mice[J]. Eur Respir J, 2003, 21(5):770-773.
36. ZHU Y, ZHU M, LANCE P. iNOS signaling interacts with COX-2 pathway in colonic fibroblasts[J]. Exp Cell Res, 2012, 318(16):2116-2127.
37. SMITH W L, GARAVITO R M, DEWITT D L. Prostaglandin endoperoxide H synthases(cyclooxygenases)-1 and-2[J]. J Biol Chem, 1996, 271(52):33157-33160.
38. AJUBI N E, KLEIN-NULEND J, NIJWEIDE P J, et al. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes--a cytoskeleton-dependent process[J]. Biochem Biophys Res Commun, 1996, 225(1):62-68.
39. 李晓东,黄跃生.机械信号的细胞感受与转导[J].中国病理生理杂志,2004(3):186-190.
40. MCGARRY J G, KLEIN-NULEND J, PRENDERGAST P J. The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts[J]. Biochem Biophys Res Commun, 2005, 330(1):341-348.
41. ALLEN F D, HUNG C T, POLLACK S R, et al. Serum modulates the intracellular Calcium response of primary cultured bone cells to shear flow[J]. J Biomech, 2000, 33(12):1585-1591.

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Nitric Oxide and Prostaglandin E₂ Secretion in Osteocytes Induced by Intermittent Cyclic Compressive Force

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Nitric Oxide and Prostaglandin E2 Secretion in Osteocytes Induced by Intermittent Cyclic Compressive Force

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Nitric Oxide and Prostaglandin E₂ Secretion in Osteocytes Induced by Intermittent Cyclic Compressive Force