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

doi:10.3969/j.issn.2095-4344.2014.11.006

Abstract

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

CLC Number:

R318

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.

Figures/Tables 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, A490 nm value rose also. Except the differences between 30 times and control group, 90 and 150 times group, the differences of A490 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 A490 nm value significantly as compared with controls and constant stimulation groups (P < 0.05). The A490 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 10-11 mol/L intermittent rhPTH1-34 stimulations’ effect on cell proliferation A490 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). A490 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). "

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 10-11 mol/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)."

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