低能体外冲击波和低剂量间歇甲状旁腺素干预成骨细胞的增殖和分化

doi:10.3969/j.issn.2095-4344.2014.11.006

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (11): 1672-1679.doi: 10.3969/j.issn.2095-4344.2014.11.006

• 骨组织构建 bone tissue construction • 上一篇下一篇

低能体外冲击波和低剂量间歇甲状旁腺素干预成骨细胞的增殖和分化

刘长剑¹，王李²，罗宗键³

¹大连医科大学附属第一医院骨科，辽宁省大连市 116001；²大连市友谊医院医学影像科，辽宁省大连市 116001；³长春市中医药大学附属第一医院骨科，吉林省长春市 130011

修回日期:2014-01-07 出版日期:2014-03-12 发布日期:2014-03-12
通讯作者: 罗宗键，博士，副教授。长春市中医药大学附属第一医院骨科，吉林省长春市 130011
作者简介:Liu Chang-jian, M.D., Associate professor, Department of Orthopedics, First Affiliated Hospital of Dalian Medical University, Dalian 116001, Liaoning Province, China

Low-density extracorporeal shock wave and low-dose intermittent recombinant human parathyroid hormone 1-34 influence proliferation and differentiation of osteoblasts

Liu Chang-jian¹, Wang Li², Luo Zong-jian³

¹ Department of Orthopedics, First Affiliated Hospital of Dalian Medical University, Dalian 116001, Liaoning Province, China; ²Department of Radiology, Dalian Friendship Hospital, Dalian 116001, Liaoning Province, China ; ³Department of Orthopedics, First Affiliated Hospital, Changchun University of Chinese Medicine, Changchun 130011, Jilin Province, China

Revised:2014-01-07 Online:2014-03-12 Published:2014-03-12
Contact: Luo Zong-jian, M.D., Associate professor, Department of Orthopedics, First Affiliated Hospital, Changchun University of Chinese Medicine, Changchun 130011, Jilin Province, China
About author:刘长剑，男，1973年生，辽宁省大连市人，汉族，2008年吉林大学白求恩医学部毕业，博士，副教授。

摘要/Abstract

摘要：

背景：体外冲击波等应力刺激可促进成骨，甲状旁腺激素激素也参与调控骨代谢。

目的：实验探讨低剂量间歇人重组甲状旁腺素1-34和低能体外冲击波对体外培养大鼠成骨细胞增殖及成骨分化的作用。

方法：采用改良胶原酶消化法培养大鼠乳鼠颅骨来源成骨细胞备用。分别用60-150次0.18 mJ/mm²低能体外冲击波刺激体外培养大鼠成骨细胞，不同浓度(10^-¹² mol/L-10^-¹⁰ mol/L)及作用方式的人重组甲状旁腺素1-34刺激，以及低能体外冲击波和间歇低剂量(10^-11 mol/L)间歇人重组甲状旁腺素1-34刺激共同作用后，用锥虫蓝法进行细胞计数、MTT和流式细胞术分析检测大鼠成骨细胞的增殖情况；用酶标仪检测碱性磷酸酶活性，用免疫组化检测Ⅰ型胶原表达来观察大鼠成骨细胞的成骨分化。

结果与结论：60-150次0.18 mJ/mm²低能体外冲击波刺激、间歇人重组甲状旁腺素1-34 (10^-11和10^-10 mol/L)刺激以及低能体外冲击波+间歇人重组甲状旁腺素1-34 (10^-11 mol/L)刺激均可显著促进体外培养大鼠成骨细胞增殖和成骨分化(P < 0.05)，其中60-150次低能体外冲击波刺激+间歇人重组甲状旁腺素1-34刺激各组作用最强(P < 0.05)。结果证实，适当的低能体外冲击波应力刺激和低剂量间歇人重组甲状旁腺素1-34刺激联合应用可显著促进体外培养大鼠成骨细胞的增殖和成骨分化。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 骨组织构建, 成骨细胞, 体外冲击波, 人重组甲状旁腺素1-34, 甲状旁腺素, 细胞周期, 碱性磷酸酶, 细胞增殖, 成骨

Abstract:

BACKGROUND: Suitable stress signals such as extracorporeal shock wave (ESW) and parathyroid hormone can be involved in regulation of bone formation and metabolism.

OBJECTIVE: To investigate the effects of low-density ESW and low-dose intermittent recombinant human parathyroid hormone 1-34 (rhPTH1-34) on the proliferation and differentiation of osteoblasts from rats cultured in vitro.

METHODS: Osteoblasts from the skull of neonatal rats were cultured using collagenase digestion method. After 60-150 times of stimulations of 0.18 mJ/mm² ESW, different patterns of rhPTH1-34 at 10^-¹²-10^-¹⁰ mol/L and ESW+intermittent rhPTH1-34 (10^-¹¹ mol/L), osteoblasts were collected to observe cell proliferation by cell counting, MTT and flow cytometry analysis and to observe cell differentiation by measuring alkaline phosphatase and type I collagen expression.

RESULTS AND CONCLUSION: 0.18 mJ/mm² ESW (60-150 times), intermittent rhPTH1-34 (10^-11 and 10^-10 mol/L) and ESW+intermittent rhPTH1-34 (10^-11 mol/L) stimulations could all improve proliferation and differentiation of rat osteoblasts cultured in vitro (P < 0.05). ESW (60-150 times)+intermittent rhPTH1-34 stimulation showed better effects than ESW and intermittent rhPTH1-34 alone (P < 0.05). These findings indicate that the combination of suitable ESW stimulation and low-dose intermittent rhPTH1-34 stimulation can improve the proliferation and differentiation of rat osteoblasts significantly.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: fractures, open, fractures, stress, bone diseases

中图分类号:

R318

刘长剑，王李，罗宗键. 低能体外冲击波和低剂量间歇甲状旁腺素干预成骨细胞的增殖和分化[J]. 中国组织工程研究, 2014, 18(11): 1672-1679.

Liu Chang-jian, Wang Li, Luo Zong-jian. Low-density extracorporeal shock wave and low-dose intermittent recombinant human parathyroid hormone 1-34 influence proliferation and differentiation of osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1672-1679.

图/表（结果） 4

Detection of cell proliferation by cell counting with typan blue staining

Different times of 0.18 mJ/mm²ESW stimulations’ effect on cell growth

The cell count was promoted in turn as stimulating times of ESW ranging from 30 to 120, and the differences were significant (P < 0.05). But the cell number of the control and 30-time ESW stimulation group showed no difference (Table 1).

Different patterns of rhPTH1-34 stimulations’ effect on cell growth

Constant stimulations of 10^-¹²，10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on cell number. Intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote cell number significantly compared with controls and constant stimulation groups (P < 0.05). Cell number of 10^-¹⁰ mol/L rhPTH1-34 group was higher than that of 10^-¹¹ mol/L rhPTH1-34 group (P < 0.05; Table 2).

Cooperation of 30-150 times of ESW and 10^-¹¹mol/L intermittent rhPTH1-34 stimulations’ effect on cell growth

Cell number of 30-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups was respectively higher than that of only ESW stimulation groups (P < 0.05; Table 1). Cell number of 60-150 times of ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were higher than that of only 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Tables 1, 2). Detection of cell proliferation by MTT test

Different times of 0.18 mJ/mm² ESW stimulations’ effect on cell proliferation

As stimulation time rose from 30 to 120 in turn, A_{490 nm} value rose also. Except the differences between 30 times and control group, 90 and 150 times group, the differences of A_{490 nm} among 30-150 times ESW stimulation groups were significant (P < 0.05; Table1).

Different patterns of rhPTH1-34 stimulations’ effect on cell proliferation

Constant stimulations of 10^-¹², 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on cell proliferation. Intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote A_{490 nm} value significantly as compared with controls and constant stimulation groups (P < 0.05). The A_{490 nm} value of 10^-¹⁰ mol/L intermittent rhPTH1-34 stimulation group was higher than that of 10^-¹¹ mol/L intermittent rhPTH1-34 stimulation group (P < 0.05; Table 2).

Cooperation of 30-150 times of ESW and 10^-¹¹mol/L intermittent rhPTH1-34 stimulations’ effect on cell proliferation

A_{490 nm} values of 30-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were respectively higher than those of only ESW stimulation groups (P < 0.05; Table 3). A_{490 nm} values of 60-150 times of ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were higher than those of only 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Tables 1, 2).

Detection of PI value by flow cytometry

Different times of 0.18 mJ/mm² ESW stimulations’ effect on PI

As stimulation time rose from 30 to 120 in turn, PI value rose also. Except the differences between 30 times and control groups, and between 120 and 150 times groups, the differences of PI among 30-150 times ESW stimulation groups were significant (P < 0.05; Table 1).

Different patterns of rhPTH1-34 stimulates’ effect on PI

Constant stimulations of 10^-¹², 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on PI. Intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote PI value significantly compared with controls and constant stimulation groups (P < 0.05). PI value of 10^-¹⁰ mol/L rhPTH1-34 intermittent stimulation group was higher than that of 10^-¹¹ mol/L intermittent rhPTH1-34 stimulation group (P < 0.05; Table 3).

Cooperation of 30-150 times of ESW and 10^-¹¹ mol/L pulse rhPTH1-34 stimulations’ effect on PI

PI values of 30-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were respectively higher than those of only ESW stimulation groups (P < 0.05; Table 1). PI values of 60-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were higher than those of only 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Tables 1, 2).

Detection of osteoblast differentiation through ALP activity measurement

Different times of 0.18 mJ/mm² ESW stimulations’ effect on ALP activity

As stimulation time rose from 30 to 120 in turn, ALP activity rose also. Except the differences between 30 times and control groups, and between 90 and 150 times groups, the differences of ALP activity among 30-150 times ESW stimulation groups were significant (P < 0.05; Table 3).

Different patterns of rhPTH1-34 stimulations’ effect on ALP activity

Constant stimulations of 10^-¹², 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on ALP activity. Intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote ALP activity significantly compared with controls and constant stimulation groups (P < 0.05). ALP activity of 10^-¹⁰ mol/L rhPTH1-34 intermittent stimulation group was higher than that of 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Table 4).

Cooperation of 30-150 times of ESW and 10^-¹¹ mol/L intermittent rhPTH1-34 stimulations’ effect on ALP activity

ALP activities of 30-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were respectively higher than those of only ESW stimulation groups (P < 0.05; Table 3). ALP activities of 60-150 times of ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were higher than that of only 10^-¹¹ mol/L rhPTH1-34 stimulation group (P < 0.05; Tables 3, 4).

Detection of type I collagen expression by immunocytochemistry

Different times of 0.18 mJ/mm² ESW stimulations’ effect on type I collagen expression

As the stimulation time rose from 30 to 150 in turn, the ratio of type I collagen positive cells rose also. Except the differences between 30 times and control groups, and between 120 and 150 times groups, the differences of positive cell ratio among 30-150 times ESW stimulation groups were significant (P < 0.05; Table 3).

Different patterns of rhPTH1-34 stimulates’ effect on type I collagen expression

Constant stimulations of 10^-¹², 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on type I collagen positive cell ratio. Intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote type I collagen positive cell ratio significantly compared with controls and constant stimulation groups (P < 0.05). Type I collagen positive cell ratio of 10^-¹⁰ mol/L rhPTH1-34 intermittent stimulation group was higher than that of 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Table 4).

Cooperation of 30-150 times of ESW and 10^-¹¹mol/L intermittent rhPTH1-34 stimulations’ effect on type I collagen expression

Type I collagen positive cell ratios of 30-150 times ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were respectively higher than those of only ESW stimulation groups (P < 0.05; Table3). Type I collagen positive cell ratios of 60-150 times of ESW+10^-¹¹ mol/L rhPTH1-34 intermittent stimulation groups were higher than that of only 10^-¹¹ mol/L rhPTH1-34 intermittent stimulation group (P < 0.05; Tables 3, 4).

参考文献

[1] Saussine C. Extracorporeal shock wave lithotripsy. Prog Urol. 2013;23(14):1168-1171.

[2] Kolk A, Auw Yang KG, Tamminga R, et al. Radial extracorporeal shock-wave therapy in patients with chronic rotator cuff tendinitis: a prospective randomised double-blind placebo-controlled multicentre trial. Bone Joint J. 2013;95-B(11):1521-1526.

[3] Hausner T, Nógrádi A.The use of shock waves in peripheral nerve regeneration: new perspectives? Int Rev Neurobiol. 2013;109:85-98.

[4] Tailly GG. Extracorporeal shock wave lithotripsy today. Indian J Urol. 2013;29(3):200-207.

[5] Liu J, Zang YJ. Comparative study between three analgesic agents for the pain management during extracorporeal shock wave lithotripsy. Urol J. 2013;26; 10(3):942-945.

[6] Mikami Y, Matsumoto T, Kano K, et al. Current status of drug therapies for osteoporosis and the search for stem cells adapted for bone regenerative medicine. Anat Sci Int. 2014;89(1):1-10.

[7] Teotia PK, Hussein KE, Park KM, et al. Mouse adipose tissue-derived adult stem cells expressed osteogenic specific transcripts of osteocalcin and parathyroid hormone receptor during osteogenesis. Transplant Proc. 2013;45(8): 3102-3107.

[8] Sheyn D, Cohn Yakubovich D, Kallai I, et al. PTH promotes allograft integration in a calvarial bone defect. Mol Pharm. 2013;10(12):4462-4471.

[9] Smith BJ, Bu SY, Wang Y, et al. Comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat. Bone. 2013;58C:151-159.

[10] Zhong G, Pei FX, Li SF, et al. Experimental study on the proliferation and function of osteoblast cell induced by pBLAST49-mVEGF gene transfection. Sichuan Da Xue Xue Bao Yi Xue Ban. 2006;37(1):44-47.

[11] Ejersted C, Andreassen TT, Nilsson MHL, et al. Human parathyroid hormone(1-34) increases bone formation and strength of cortical bone in aged rats. Europe J Endocrinol. 1994;130(2):201-207.

[12] Xu J, Rong H, Ji H, et al. Effects of different dosages of parathyroid hormone-related protein 1-34 on the bone metabolism of the ovariectomized rat model of osteoporosis. Calcif Tissue Int. 2013;93(3):276-287.

[13] Chen YJ, Kuo YR, Yang KD, et al. Activation of extracellular signal-regulated kinase (ERK) and p38 kinase in shock wave-promoted bone formation of segmental defect in rats. Bone. 2004;34(3):466-477.

[14] Wang FS, Yang KD, Kuo YR, et al. Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect. Bone. 2003;32(4):387-396.

[15] 15 Ishizuya T, Yokose S, Hori M, et al. Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest. 1997;99(12):2961-2970.

[16] UzawaT, HoriM, Ejiri S, et al. Comparison of the effects of intermittent and continuous administration of human Parathyroid hormone (1-34) on rat bone. Bone. 1995;16(4): 477-484.

[17] Wang YH, Qiu Y, Han XD, et al. Haploinsufficiency of endogenous parathyroid hormone-related peptide impairs bone fracture healing. Clin Exp Pharmacol Physiol. 2013; 40(11):715-723.

[18] Khan MP, Mishra JS, Sharan K, et al. A novel flavonoid C-glucoside from Ulmus wallichiana preserves bone mineral density, microarchitecture and biomechanical properties in the presence of glucocorticoid by promoting osteoblast survival: a comparative study with human parathyroid hormone. Phytomedicine. 2013;20(14):1256-1266.

[19] Hamedifar H, Salamat F, Saffarion M, et al. a novel approach for high level expression of soluble recombinant human parathyroid hormone (rhPTH 1-34) in Escherichia coli. Avicenna J Med Biotechnol. 2013;5(3):193-201.

[20] Pereira Vasconcelos DF, Marques MR, Benatti BB, et al. Intermittent PTH administration improves periodontal healing in rats. J Periodontol. 2013.

[21] Klein-Nulend J, Semeins CM, Burger EH. Metabolism in primary mouse osteoblastic Prostaglandin mediated modulation of transforming growth factor-β metabolism in primary mouse osteoblastic cells in vitro. J Cell Physiol. 1996;168:1-7.

[22] Reich KM, Mcallister TN, Gudi S. Activation of G protein mediates flow-induced prostaglandin E2 production in osteoblast. Eradocranology. 1997;138:1014-1018.

[23] Ajubi NE, Klein-Nulend J, Nijweide PJ, et al. Pulsatile fluid flow increases prostaglandin production by cultured chicken osteocytes--a cytoskeleton-dependent process. Biochem Biophys Res Commun. 1996;225:62-68.

[24] Ueberle F, Rad AJ. Characterization of unfocused/weakly focused pressure pulse sources for extracorporeal pain therapy (“Radial Shock Wave Therapy” Sources). Biomed Tech (Berl). 2013.

[25] Zhang X, Yan X, Wang C, et al. The dose-effect relationship in extracorporeal shock wave therapy: the optimal parameter for extracorporeal shock wave therapy. J Surg Res. 2014;186(1):484-492.

[26] Moon SW, Kim JH, Jung MJ, et al. The effect of extracorporeal shock wave therapy on lower limb spasticity in subacute stroke patients. Ann Rehabil Med. 2013;37(4): 461-470.

[27] Schmitz C, Császár NB, Rompe JD, et al. Treatment of chronic plantar fasciopathy with extracorporeal shock waves (review). J Orthop Surg Res. 2013;8(1):31.

[28] Troncati F, Paci M, Myftari T, et al. Extracorporeal shock wave therapy reduces upper limb spasticity and improves motricity in patients with chronic hemiplegia: a case series. NeuroRehabilitation. 2013;33(3):399-405.

[29] Qin L, Fok P, Lu H, et al. Low intensity pulsed ultrasound increases the matrix hardness of the healing tissues at bone-tendon insertion-a partial patellectomy model in rabbits. Clin Biomech (Bristol, Avon). 2006;21(4):387-394.

[30] Vulpiani MC, Vetrano M, Conforti F, et al. Effects of extracorporeal shock wave therapy on fracture nonunions. Am J Orthop (Belle Mead NJ). 2012;41(9):E122-E127.

[31] Fovargue DE, Mitran S, Smith NB, et al. Experimentally validated multiphysics computational model of focusing and shock wave formation in an electromagnetic lithotripter. J Acoust Soc Am. 2013;134(2):1598-609.

[32] Speed C. A systematic review of shockwave therapies in soft tissue conditions: focusing on the evidence. Br J Sports Med. 2013.

[33] Ha CH, Kim S, Chung J, et al. Extracorporeal shock wave stimulates expression of the angiogenic genes via mechanosensory complex in endothelial cells: mimetic effect of fluid shear stress in endothelial cells. Int J Cardiol. 2013;168(4):4168-4177.

[34] Császár NB, Schmitz C. Extracorporeal shock wave therapy in musculoskeletal disorders. J Orthop Surg Res. 2013;8:22.

[35] Suhr F, Delhasse Y, Bungartz G, et al. Cell biological effects of mechanical stimulations generated by focused extracorporeal shock wave applications on cultured human bone marrow stromal cells. Stem Cell Res. 2013;11(2): 951-964.

[36] Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc Nad Acad Sci USA. 1998;95: 3597-3602.

[37] Saini V, Marengi DA, Barry KJ, et al. Parathyroid hormone (PTH)/PTH-related peptide type 1 receptor (PPR) signaling in osteocytes regulates anabolic and catabolic skeletal responses to PTH. J Biol Chem. 2013;288(28): 20122-20134.

[38] Dossing DA, Radeff JM, Sanders J, et al. Parathyroid hormone stimulates translocation of protein kinase C isozymes in UMR-106 osteoblastic osteosarcoma cells. Bone. 2001;29(3):223-230.

[39] Lee SJ, Kang JH, Kim JY, et al. Dose-related effect of extracorporeal shock wave therapy for plantar fasciitis. Ann Rehabil Med. 2013;37(3):379-388.

[40] Raabe O, Shell K, Goessl A, et al. Effect of extracorporeal shock wave on proliferation and differentiation of equine adipose tissue-derived mesenchymal stem cells in vitro. Am J Stem Cells. 2013;2(1):62-73.

[41] Rao DS, Parikh N, Palnitkar S, et al. The effect of endogenous parathyroid hormone in iliac bone structure and turnover in healthy postmenopausal women. Calcif Tissue Int. 2013;93(3):288-295.

[42] Tamura Y, Kaji H. Parathyroid hormone and Wnt signaling. Clin Calcium. 2013;23(6):847-852.

引言

Extracorporeal shock wave (ESW) and parathyroid hormone (PTH) have been employed to promote healing of bone in recent years. Fracture healing, especially osteoporotic fractures in the elderly, represents a health concern as they are associated with increased morbidity and mortality together with an increased economic burden for the society. It is widely known that a variety of physical and chemical factors may be involved in modulation of bone formation in different stages. During the past 20 years, a great effort has been done in order to reduce the risk of fracture and many drugs and therapies are now available for this purpose, but osteoporosis is still regarded as an inevitable consequence of the aging process. They are associated with significant morbidity and those of the hip and spine are also associated with excess mortality. Until now, fracture healing is still a challenge remained to be solved. During this process, different factors cooperate in different periods to affect bone metabolism. The effects of ESW and PTH on differentiation and proliferation of osteoblasts are interested in recent years^[1-10].

PTH is an important regulating factor of bone metabolism. Although in primary hyperparathyroidism bone catabolism prevails on bone anabolism, PTH functions as a potent stimulator of osteoblasts and its anabolic properties can be seen when it is given at a low dosage and intermittently. Intermittent PTH can stimulate bone formation to a greater extent and earlier than bone resorption, thus creating the so called “anabolic window”. Together with well known anti-resorptive agents (like bisphosphonates, estrogen and selective

estrogen receptor modulators) in the past few years, anabolic therapy with PTH has become available for the treatment of severe osteoporosis. Human recombinant PTH peptide 1-34 (Teriparatide) belongs to this group of agents. Depending on a higher concentration of PTH in vivo, the number and activity of osteoclasts can be improved, which will lead to bone absorption. However, stimulation of low-dose intermittent PTH can promote bone formation in vivo^[11-12]. All the PTH concentrations adopted in existing studies are much higher than the physiological concentration (1-5×10^-¹² mol/L). In this study, we studied the effects of low-dose intermittent recombinant human parathyroid hormone 1-34 (rhPTH1-34; simulating the physiological concentration) stimulation on cultured osteoblast proliferation and differentiation.

Bone tissue is sensitive to high-frequency and low-density mechanic energy. As a kind of suitable mechanic stimulation, low-density ESW has been proved to be able to improve healing of fracture^[13-14]. The mechanism of ESW therapy is not fully understood. The most important physical parameters of ESW therapy for the treatment of orthopedic disorders include the pressure distribution, energy flux density and the total acoustic energy. In contrast to lithotripsy in which shock waves disintegrate renal stones, orthopedic shock waves are not being used to disintegrate tissues, but rather to microscopically cause interstitial and extracellular responses leading to tissue regeneration. It is widely known that the effectiveness of shockwave therapy on bone mass and bone strength appears to be in a dose- and time-dependent manner. Fracture healing is a complex physiologic process that involves the coordinated participation of several cell types. ESW has been indicated as an alternative, non-invasive but promising method for treatment of bone fractures, effective even in delayed fracture healing, nonunion and bone defects. ESW used is generated by an electron hydraulic shock wave generator, which can cause an explosive evaporation of water and produce high-energy acoustic waves. The acoustic waves are focused on a fluid-filled head with a silicone-type membrane and therefore can be transmitted into a targeted site. ESW plays a role in the management of non-union because they produce microfractures of the bone, and stimulate neovascularization, osteoblast proliferation and activation, and synthesis of bone tissue. Shock waves can produce radiographic lucencies in the bone marrow, intense formation of new cortical bone, and minor trabecular remodeling but did not cause gross fractures. Osteoblasts play a crucial role in this process. So, in this study, we try to observe the interaction mechanism of ESW and osteoblasts during the process of bone formation.

Bone healing is a complex procedure. In vivo, except chemical stimulations such as PTH, osteoblasts are also under a continual mechanic stress such as fluid shear stress, extension and pressure, etc. Therefore, not only can we observe stress stimulation, ESW effects on the proliferation and differentiation of osteoblasts, but also we can study the cooperation of ESW and low-dose intermittent rhPTH1-34 on the proliferation and differentiation of osteoblasts, thereby demonstrating the cooperation of ESW and rhPTH1-34 on osteoblasts, and trying to a find new way of bone healing.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

材料方法

Design

Two-factor, completely randomized study.

Time and setting

The study was completed at the Central Laboratory of Dalian Medical University, from October 2007 to October 2011.

Materials

Totally 190 neonatal Wistar rats, class A, 95 male and 95 female, were selected from the SPF Test Animal Centre of Dalian Medical University, China. License number was SCXK(Liao)2004-0017.

Methods

Cell culture

According to Zhong’s method^[10], osteoblasts from the rat’s skull were isolated and cultured by type I collagenase. The cells were cultured in vitro and identified. The growth curve was plotted to observe the proliferation of cells. Cell morphology was observed and recorded by microscope.

Different times of ESW stimulations

The ESW energy density was adjusted to 0.18 mJ/mm², and the 3^rd generation of osteoblasts were cultured in medium without FBS for 24 hours to be synchronized. Then cells were placed into little freezing bottles that were located by C-arm X-ray machine to the 2^nd focus point of ESW machine. Hereafter, 30, 60, 90, 120, 150 times of ESW (0.18 mJ/mm²) were employed to treat osteoblast suspension in the little frozen bottles.

Different patterns of rhPTH1-34 stimulations

The 3^rd generation of osteoblasts at 2×10⁷/L were seeded into hole plates with high-glucose DMEM medium containing 10% FBS. After the cells adhered to the plate, they were cultured in medium without FBS for 24 hours to be synchronized. Then osteoblasts were cultured in the medium containing 10% FBS freed of steroid, and different rhPTH1-34 stimulations were added as follows:

Combining different times of ESW stimulations with low-dose intermittent rhPTH1-34 to act on cultured osteoblasts

The 3^rd generation of osteoblasts were cultured in medium without FBS for 24 hours to be synchronized. Then the cells were cultured in the medium containing 10% FBS freed of steroid, and 10^-¹¹ mol/L rhPTH1-34 acted in the first 8 hours per 24 hours as a cycle, acting for two cycles. Then cells were collected and stimulated with 30, 60, 90, 120, 150 times of 0.18 mJ/mm² ESW.

Establishment of two control groups

One group was cultured with test groups at the same condition, and the other was cultured not only at the same condition with test groups, but also changed the medium the same as intermittent rhPTH1-34 stimulation groups.

Evaluation of cell proliferation by cell counting with trypan blue dye

The 3^rd generation of osteoblasts were cultured in medium without FBS for 24 hours to be synchronized. Then cells were cultured in the medium containing 10% FBS freed of steroid. After treated with different groups of ESW and rhPTH1-34 stimulations, every sample (500 μL cell suspension) was added with 500 μL of 0.4% trypan blue dye 5 minutes. Consequently, cell numbers were observed in cell counters by microscope.

Evaluation of cell proliferation by cell counting with thiazolyl blue tetrazolium bromide (MTT)

The 3^rd generation of osteoblasts were cultured in medium without FBS for 24 hours to be synchronized. Then cells were cultured in the medium containing 10% FBS freed of steroid. After treated with different groups of ESW and rhPTH1-34 stimulations, every sample was added with 20 L thiazolyl blue for 4 hours. Then, after 150 L dimethyl sulfoxide was added for 20 minutes, the absorbance value of every hole at 490 nm (A_{490 nm}) was detected.

Cell cycle analysis and proliferation index detected by flow cytometry

After being stimulated as grouped above, the 3^rd generation of osteoblasts were cultured in high-glucose DMEM containing 10% FBS freed of steroid for 24 hours. Then, collected cells were fixed by 70% alcohol for more than 12 hours. Before detection, RNase was added to reduce RNA segments. After PI dye was added and incubated together with osteoblasts in the dark for 30 minutes, samples were detected. The software of Modfit was used to analyze the data. Value of proliferation index (PI) was computed as follows: PI=S%+G₂/M%.

Osteoblast differentiations detected by ALP activity detection through a microplate reader

After being treated as grouped above, the 3^rd generation of osteoblasts were cultured in high-glucose DMEM containing 10% FBS freed of steroid for 72 hours. Then, cells were treated as steps of ALP activity kit and A_{490 nm} value were detected through the microplate reader. ALP activity was computed as follows:

ALP activity (kat/100 mL)=(Absorbance value of each sample/standard absorbance value)×0.005×(100/0.05)× 16.67.

Osteoblast differentiations detected by immunocytochemistry detection of type I collagen expression

After being treated as grouped above, the 3^rd generation of osteoblasts were cultured together with glass slices in high-glucose DMEM containing 10% FBS freed of steroid for 4 days. After samples fixed, citrate buffer was added to repair antigen protein in water bath of 95 ℃ for 10 minutes. Samples were incubated with the 1^st antibody (rabbit antibody of rat’s type I collagen, Nanjing Keygen, 1:100 dilution) 4 hours in 37 ℃. Then, the samples were incubated with the second antibody (goat antibody of rabbit labeled with biotin, Wuhan Boster, 1:100 dilution) in 37 ℃ for 30 minutes. Then, DAB chromogenic reagent kit was used. Three random fields of every sample were selected to be observed under microscope and positive cell ratios were calculated.

Statistical analysis

All data were processed by SPSS 13.0 statistic software. All data were expressed as mean±SD. According to Standard Deviation being similar or not, one-way analysis of variance or repeated measures analysis of variance was adopted to compare difference among groups. Least significant difference analysis was adopted to analyze intra-group difference. If a P value was less than 0.05, the difference was significant.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

PTH is one of the key factors which are involved in regulation of bone formation in vivo. Present studies have shown that the secretion of PTH in human body abide by two different rules. One rule is that the concentration of PTH in vivo remains stable through frequent secretion regulation and this would contribute to keep concentration of Ca²⁺ in serum; the other rule is that the secreting times and quantity of PTH is fixed and this would be beneficial to bone formation. Alternations of these two kinds of secretion patterns are controlled by some kinds of physiological switches and the two states can be converted mutually when necessary. So it would be easy to understand why different patterns of PTH stimulations result in different effects on bone formation or bone absorption^[15-20]. In this study, constant stimulations of 10^-¹², 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 had no effects on cell proliferation, but intermittent stimulations of 10^-¹¹ and 10^-¹⁰ mol/L rhPTH1-34 could promote cell proliferation significantly. It proves that the variation of PTH concentrations do accurately regulate differentiation and proliferation of cultured osteoblasts. Toshinori drew the conclusion that, when hPTH1-34 was given in the first 6 hours per 48 hours as a cycle, the effect of hPTH1-34 on promoting human osteoblast proliferation was the strongest^[16], which is consistent with our findings.

It has been widely known that suitable stress stimulations contribute to bone formation in vivo and in vitro, so the methods of stress construction and continual irrigating construction are advocated in bone tissue engineering research in recent years^[21-28]. Some studies have proved that, as a mechanic stimulation, ESW can change the fluid mechanics state of target region and act on cells included through fluid shear stress and extension stress^[29-35]. Osteoblast and bone cells have always been identified as two kinds of stress sensitive cells. Our study showed that 60-150 times of 0.18 mJ/mm² ESW could promote proliferation and differentiation of cultured osteoblasts significantly.

In vivo, osteoblasts grow in the environment containing physiological concentration of PTH and continual stress stimulation. To observe cooperative effects of these two factors on cultured osteoblasts, we combined 30-150 times of ESW with 10^-11 mol/L rhPTH1-34 intermittent stimulation. The result showed that 60-150 times ESW+10^-11mol/L rhPTH1-34 intermittent stimulation groups were more effective than only ESW stimulation groups (P < 0.05) and only 10^-11 mol/L rhPTH1-34 intermittent stimulation group. In the physiological state, bone absorption and remodeling are in a balance which depends on the balance between osteoblast’s and osteoclast’s functions. It is accurate and subtle to regulate this balance by PTH. In a range of low concentrations of PTH, the balance between functions and differentiation of osteoblasts and osteoclasts is maintained, and thereby the balance of bone metabolism is kept. If the concentration of PTH rises out of the suitable range, osteoclast’s effect on bone absorption is promoted at the same time of weakening osteoblast’s effect on bone formation. These come together to result in bone absorption^[36-42]. Our findings show that ESW and 10^-11 mol/L rhPTH1-34 intermittent stimulations could amplify the effects on promoting osteoblast proliferation and differentiation. This may contribute to explain why exercising and low dose intermittent PTH injection can promote bone formation. Therefore, we can conceive that, in vivo, if factors which inhibit bone formation and improve bone absorption, such as hyperthyroidism, are presented, the balance would incline to bone absorption, and vice versa. Although ESW and low-dose rhPTH1-34 intermittent stimulations have been found to be able to promote osteoblast proliferation and differentiation, the mechanisms are far from illuminated and relevant studies need to be furthered.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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低能体外冲击波和低剂量间歇甲状旁腺素干预成骨细胞的增殖和分化

Low-density extracorporeal shock wave and low-dose intermittent recombinant human parathyroid hormone 1-34 influence proliferation and differentiation of osteoblasts

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