Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels

Bone 76 (2015) 88–96

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Original Full Length Article

Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels Hyungjin Jung a, Makenzie Best c, Ozan Akkus a,b,c,⁎ a b c

Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA Department of Orthopedics, Case Western Reserve University, Cleveland, OH 44106, USA Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA

a r t i c l e

i n f o

Article history: Received 13 November 2014 Revised 25 February 2015 Accepted 17 March 2015 Available online 25 March 2015 Edited by: David Burr Keywords: Bone microdamage Intracellular calcium signaling Calcium efflux Voltage-gated calcium channel Calcium channel inhibitor

a b s t r a c t Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca2+]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca2+]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca2+]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca2+]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca2+]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca2+]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs. © 2015 Elsevier Inc. All rights reserved.

Introduction Bone cells work in concert to repair the damaged regions of bone matrix [1–3]. Alterations in matrix strain, local fluid flow patterns or osteocyte apoptosis have been postulated to trigger the repair function of bone cells [4–6]. While there is strong evidence for linear microcrack to induce osteocyte apoptosis and trigger bone remodeling, diffuse microdamage does not have an apoptotic effect [7,8]. It is not fully understood how the damaged regions of bone are targeted for repair, especially for diffuse microdamage. We have proposed mechanically induced calcium efflux ([Ca2+]E) from damaged regions of bone matrix as a potential stimulus which activates local bone cells [9]. Abundant amount of diffuse damage can be created in a controlled fashion in notched regions of devitalized cortical bone [10,11]. Ion-selective electrode measurements demonstrated increased calcium concentration concomitant with the inception of ⁎ Corresponding author at: Departments of Mechanical and Aerospace Engineering, 10900 Euclid Avenue, Cleveland, OH 44106, USA. E-mail address: [email protected] (O. Akkus).

http://dx.doi.org/10.1016/j.bone.2015.03.014 8756-3282/© 2015 Elsevier Inc. All rights reserved.

diffuse damage [12], providing evidence on the possibility of a mechanically induced [Ca2+]E from bone matrix at post-yield strain levels. When devitalized notched bone samples were seeded with MC3T3E1 preosteoblasts and diffuse damage was induced, increased intracellular calcium ([Ca2+]I) was observed among the cells on the damage process zone [9]. This cellular reaction was associated with calcium because the response vanished when the tests were repeated with cellsseeded on notched demineralized bone matrix. These results suggest that mechanically induced [Ca2 +]E triggers [Ca2 +]I signaling on MC3T3-E1 cells. However, it is largely unknown as to how [Ca2 +]I signaling occurs as a result of microdamage induction. Neomycinsensitive voltage-gated calcium channels (VGCCs) were implied in this extracellular calcium induced intracellular calcium response (ECIICR) [13]; however, the study did not identify the specific type(s) of VGCC involved in the response. The aim of this study is to affirm the involvement of VGCC on [Ca2+]E induced [Ca2+]I activation and to identify the type(s) of VGCC involved in the process. Non-selective VGCC inhibitor was used to affirm the general involvement of VGCC in [Ca2+]I activation, and specific blockers for known types of VGCC were applied to attain our aim.

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Materials and method Chemicals Minimum essential alpha medium (α-MEM), penicillin/streptomycin, trypsin/EDTA, HBSS, HEPES, and Fura-2AM were obtained from Invitrogen (Frederick, MD). Voltage-gated calcium channel blockers, Bepridil, (non-selective voltage-gated calcium channel inhibitor) Verapamil, (L-type calcium channel inhibitor) NNC55-0396 (T-type calcium channel inhibitor) and fetal bovine serum (FBS) were obtained from Sigma (St. Louis, MO). ω-Conotoxin MVIIC(N, P/Q type calcium channel inhibitor) and ω-Conotoxin GVIA(N type calcium channel inhibitor) were obtained from Tocris Bioscience (Ellisville, MO).

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in HBSS with 20 mM HEPES. Cells were prepared in cell culture insert, incubated for 90 min at 37 °C, 5% CO2, and transferred to the imaging system without rinsing. Cells were subjected to calcium efflux as elucidated. The media in the insert and in the well contained the same concentration of DIBAC4(3) to prevent the dye molecules from being washed-off from cell membrane during efflux exposure. Membrane potential change was measured under UV epifluorescent illumination using a × 20 waterimmersion objective lens with a UV microscope (BX51, Olympus America). The fluorescence changes were measured with fluorescence measuring module (Incyt™ Basic Im Fluorescence Imaging system, Intracellular Imaging Inc.). Membrane potential change measurements were performed in a dark room at room temperature. Application of pharmaceutical inhibitors of VGCCs

Well insert model for 1D calcium efflux from basal aspect of cells Well insert model is a practical way to emulate the calcium efflux from the bone matrix on which cells are seeded (Fig. 2-a) [13]. Cells were seeded on cell culture inserts (0.4 μm pore size culture plate insert, Corning) and the insert was placed in a culture well, creating a dual compartment. Mechanically induced calcium efflux from basal side of the cells was mimicked by applying higher concentration of calcium solution in the well than the concentration of calcium solution in the insert. Cell culture The murine pre-osteoblast MC3T3-E1 (passage 21, subclone 4, ATCC) were cultured in alpha-minimum essential medium (α-MEM; Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; Sigma-Aldrich), 1% penicillin–streptomycin (Gibco) under 37 °C, and 5% CO2. Cells were transferred to 6-well insert (0.4 μm culture plate insert, Corning), at a density of 15,000 cells/cm2, and cultured for 48 h, in the same culture medium, at 37 °C, and 5% CO2. Intracellular calcium concentration was measured by 10 μM Fura2AM solution prepared in HBSS with 20 mM HEPES. Cells were stained for 60 min at room temperature. After washing three times with HBSS solution, the cells were incubated for additional 30 min either in HBSS solution with the pharmaceutical inhibitor, or only HBSS solution (see Table 1 for concentrations of applied pharmaceutical inhibitors). 2.5 ml of 9.6 mM calcium solution in HBSS was loaded in the well while the HBSS in the insert had 1.5 ml of 0 mM calcium. At this concentration gradient, the concentration of the insert converges to 6 mM of Ca2+ efflux toward the cells at the steady state. The media in the insert and in the well contained same concentration of inhibitors. [Ca2+]I was measured under UV epifluorescent illumination using a ×20 water-immersion objective lens with a UV microscope (BX51, Olympus America). The system was equipped with a calcium imaging module for excitation and collection of fluorescence data (Incyt™ Basic Im Fluorescence Imaging system, Intracellular Imaging Inc.). Six inserts were measured in each of the control and inhibitor treated groups. Intracellular calcium fluorescence measurements were performed in a dark room at room temperature. Membrane potential changes in MC3T3-E1 cells by [Ca2+]E Membrane potential change in MC3T3-E1 cells under the influence of [Ca2+]E was measured by staining cells with 5 μM DIBAC4(3) prepared

Pharmaceutical blockers specific to different VGCCs were used according to the flowchart shown in Fig. 1. Bepridil, a non-specific blocker of all VGCCs, was applied at the first stage to evaluate and affirm whether VGCCs are involved in the ECIICR of MC3T3-E1. Subsequently, ω-conotoxin-MVIIC was used to verify the involvement N and P/Qtype VGCC. If ω-conotoxin-MVIIC showed inhibitory effect on [Ca2+]I, ω-conotoxin-GVIA was tested to conclude whether the effect of ωconotoxin-MVIIC was caused by N-type VGCC. If ω-conotoxin-MVIIC showed no effect on [Ca2 +]I, NNC55-0396 was used to test role of Ttype VGCC, followed by Verapamil to find out effect of L-type VGCC. The concentration of each pharmaceutical inhibitor used in this paper is listed in Table 1 along with the references of studies, which utilized these concentrations effectively in bone cells. Osteoblast-seeded bone loading test with calcium channel inhibitors Following the screening of various blockers using the insert model, the blockers which demonstrated a significant reduction in [Ca2+]I signaling were assessed in an in vitro bone damage model developed by our group (Fig. 2-b) [12,13]. Devitalized cortical bone wafers were sectioned from bovine femur (40 mm × 4 mm, 200 μm thickness). Blunt notches (1.5 mm in depth) were machined using methods described earlier [10]. Bone samples were photobleached under UV light for 30 min to decrease autofluorescent interference from bone substrate during intracellular calcium fluorescence measurements. The samples were sterilized by soaking the sample into 70% ethanol for overnight, washed with × 1 PBS and dried in the cell culture laminar flow hood for three times. MC3T3-E1 preosteoblasts were seeded on bone wafers at a density of 20,000 cells/cm2, and cultured overnight at 37 °C, 5% CO2. Bone slices seeded with cells were stained in 10 μM Fluo-8AM solution in HBSS with 20 mM HEPES for 90 min in the cell culture incubator (37 °C, 5% CO2). For treatment of cells with pharmaceutical blocker and dye de-esterification, the samples were incubated for additional 30 min either in HBSS solution with pharmaceutical inhibitor, or only HBSS solution. Cell-seeded bone samples were treated with Bepridil or a combination of Verapamil and NNC55-0396 to assess the involvement of L- and T-type calcium channels on the cell response. These blockers were selected for bone loading experiments because the prior insert experiments indicated these blockers to be effective. Mechanical loading was applied on V-notched bone samples for testing the effect of mechanically induced calcium efflux on ECIICR in

Table 1 Pharmaceutical inhibitors and their concentrations used in the study. Type of VOCC

Inhibitor

Concentration

References

Non-selective calcium channel blacker L-type calcium channel blocker T-type calcium channel blocker N, P/Q-type calcium channel blocker N-type calcium channel blocker

Bepridil hydrochloride Verapamil hydrochloride NNC55-0396 dihydrochloride ω-Conotoxin MVIIC ω-Conotoxin GVIA

30 μM 100 μM 5 μM 1 μM 2 μM

[37–39] [40–42] [43–45] [46–49] [49–51]

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H. Jung et al. / Bone 76 (2015) 88–96

Fig. 1. Experimental design for the application of pharmaceutical inhibitors of VGCC.

osteoblast. Samples were mounted on a miniaturized tensile loading device (Ernest F. Fullam Inc.), immersed in 50 ml of loading buffer and loaded in displacement control at a rate of 6 μm/s. The loading system was positioned under the microscope for in situ observation. The notch tip was observed during loading for the emergence of damage zone which is manifested by an opaque zone as we demonstrated earlier [10]. The loading is stopped upon damage initiation. Changes in intracellular calcium concentration were recorded as elucidated earlier in the methods and analyzed using ImageJ (NIH). Mechanical loading shifted individual cells out of the field of view; therefore, calcium fluorescence was not measured by tracing individual cells at high magnification as we did in the insert model. Rather, a lower magnification image

encompassing the entire damage zone was taken and the calcium related fluorescence was measured before and after damage induction. For bone loading tests, this calcium fluorescence intensity recorded from the population of cells is the only parameter we report. Data recording and analysis Fluorescence time histories of 180 cells from 6 inserts (30 cells from each insert) per each of the five inhibitors and controls were recorded. To avoid photo-bleaching and photo-toxicity, 25% neural density filter was used in UV source, and tofra wheel shutter was used to collect fluorescence data from the cells at every 6 s. During bone loading

Fig. 2. a. Experimental design for calcium exposure in well-inserts. Cells were seeded in inserts and placed in wells. The well had higher calcium concentration than the insert, resulting in a calcium efflux from the well to the insert. b. Experimental schema of mechanical loading test of MC3T3-E1 seeded V-notch bone specimen.

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tests, 5 samples per treatment group were analyzed (311 cells for control, 258 cells for Bepridil, and 244 cells for Verapamil + NNC550396). 50% neural density filter was used in UV source for avoiding photo-bleaching and photo-toxicity. The following variables were extracted from the fluorescence-time histories of insert experiments to quantify the effects of calcium exposure and VGCC inhibition on intracellular calcium activity (Fig. 3): baseline [Ca2+]I, number of [Ca2+]I peaks, maximum amplitude of [Ca2+]I peak, average amplitude of [Ca2 +]I peak, and full width at half maximum (FWHM) (as width of [Ca2 +]I peak) were measured for each calcium profile. Increases of baseline [Ca2+]I, number of [Ca2+]I peak, maximum amplitude of [Ca2+]I peak, and average amplitude of [Ca2+]I peak were considered as higher VGCC activation, while decrease of full width at half maximum (FWHM), more acute response of VGCC, was considered as higher VGCC activation. Normality testing of the samples showed that the data were not normally distributed. Data are presented as median and quartiles (Q1, Q3) because the distribution was non-normal. Also, a non-parametric analysis, Kruskal–Wallis test was used to test for significant differences. Mann–Whitney test was performed to address differences between control group and inhibitor treated groups, and Bonferroni correction was used for multi-comparison correction. Grubb's outlier test was used to identify the outliers which were less than 5% of the total number of observations. Results Pattern of [Ca2 +]I activation in MC3T3-E1 induced by [Ca2 +]E in insert model The results showed complex intracellular signaling patterns to emerge in response to extracellular calcium exposure. Some cells displayed an increase in the basal calcium levels with no evidence of oscillations in calcium intensity. Other groups of cells displayed calcium oscillations which were superimposed on the basal increase. The oscillations varied from a single peak to multiple peaks. In control samples, 46.3% of cells showed [Ca2 +]I oscillation patterns and 38.9% of cells displayed a single [Ca2 +]I peak in the middle, while 6.9% of cells did not demonstrate any [Ca2+]I peaks.

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Effect of VGCC blockers on response to extracellular calcium exposure The group treated with Bepridil showed significant decrease on VGCC activation (Fig. 4) (p b 0.001). Activation patterns of samples treated with Bepridil showed that number of cells with [Ca2+]I oscillation pattern was decreased from 46.3% to 11.7%, while cells with no [Ca2 +]I peak pattern were increased from 6.9% to 55.2%. Baseline [Ca2 +]I decreased 22.77% (from 78.6 ± 26.9 nM to 60.6 ± 22.9 nM), number of [Ca2 +]I peak decreased 74.54% (from 2.67 ± 2 peaks to 0.68 ± 0.94 peaks), maximum peak amplitude was decreased 45.42% (from 248.95 ± 96.80 nM to 135.63 ± 85.71 nM), average amplitude of peak was decreased 18.34% (from 141.19 ± 73.52 nM to 115.30 ± 80.19 nM) and full width at half maximum was increased 93.6% with Bepridil application (from 58.09 ± 40.26 s to 112.46 ± 83.28 s) (Table 2).

Identification of the type(s) of VGCC involved in ECIICR The increase in the baseline [Ca2 +]I level was not affected by any of the individual VGCC inhibitors (p N 0.1) (Table 2). Number of [Ca2 +]I peaks was significantly decreased by Verapamil (50%) and NNC55-0396 (50%). However, decrease on both inhibitors was not as low as decrease by Bepridil (p b 0.001), which means that these blockers contribute to occurrence of [Ca2 +] I peaks partially (Table 2). Maximum amplitude of peaks was decreased by Verapamil (16.3%) and NNC55-0396 (35.9%) treatment (p b 0.001). The extent of reductions due to these individual blockers was less pronounced than the effect of Bepridil (p b 0.001). ω-Conotoxin MVIIC and ω-conotoxin GVIA did not have any significant effects (Table 2). Average amplitude of [Ca2+]I peaks decreased by Verapamil (23.3%, p b 0.001) and to a comparable extent with that attained by Bepridil (p N 0.1) (Table 2). Average FWHM was increased by all four inhibitors (p b 0.001, except ω-conotoxin MVIIC (p b 0.01)). NNC55-0396 treatment caused greater increase of FWHM (90.7%) than samples treated with Verapamil (33.3%), ω-conotoxin GVIA (30.2%) and ω-conotoxin MVIIC (25.7%) (Table 2).

Fig. 3. Activation criteria extracted from the fluorescence-time histories to quantify [Ca2+]I activation in MC3T3-E1 (amp: amplitude).

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Fig. 4. Pattern of [Ca2+]I activation in MC3T3-E1 cells by [Ca2+]E. a) Average of [Ca2+]I activation in control group is showed by solid line and the gray region highlights the standard deviation, b) Average of [Ca2+]I activation in Bepridil treated group, c,d) typical appearance of cells in control group and Bepridil treated group, respectively. (Error bars represent standard deviation.)

Pattern of membrane potential changes in MC3T3-E1 induced by [Ca2+]E Accumulation of ionic species around the cell hyperpolarizes the cell membrane, followed by depolarization of the membrane by activation of the voltage activated ion-selective channels [14]. DIBAC4(3) membrane potential dye is displaced from cell membrane during hyperpolarization resulting in a decrease in fluorescence. During depolarization, there is an influx of the dye to the cell with an associated increase in fluorescence. DIBAC4(3) fluorescence was decreased from 100% to 96 ± 0.9% in the early stage (0–15 s) due to hyperpolarization, followed by an increase during depolarization in phase II (15–300 s, 96% to 113 ± 3%). Time history of membrane depolarization was synchronous with [Ca2 +]I response (Fig. 5). The membrane depolarization was associated with

an increase in intracellular calcium concentration during 0–300 s. Thereafter both curves did not change. Membrane depolarization did not occur when MC3T3-E1 cells were treated with Bepridil (Fig. 5b). Microdamage morphology and [Ca2 +]I fluorescence changes in cells on damaged bone Earlier studies from our group have shown that the damage process zone initiates at the notch tip and with further loading it grows in size with a flame-like morphology [10]. The average size of damage process zone was 0.33 mm2 ± 0.10 mm2. The amount of cells on the damaged area was estimated as 66 ± 20 cells, based on this damage zone size. The [Ca2+]I fluorescence within cells seeded on the V-notched loaded

48 96 64 91.55 60.33 62.51

(36.25–67) (39.5–174) (47.17–100) (62.25–172.9) (46.14–78.59) (47.47–89.00)

– p b 0.001 p b 0.001 p b 0.001 p b 0.01 p b 0.001

93

(63.41–91.48) (47.76–72.34) (66.08–90.01) (57.57–91.69) (62.31–114.9) (66.86–109.4)

– p b 0.001 p N 0.1 p N 0.1 p N 0.1 p N 0.1

2 0 1 1 2 2

(1–3) (0–1) (0–2) (0–2) (1–4) (1–4)

– p b 0.001 p b 0.001 p b 0.001 p N 0.1 p N 0.1

258 108 216 165.5 265.5 221.5

(170–319) (79–155) (140.25–247.75) (118–255.75) (143.75–310.75) (134.75–324.5)

– p b 0.001 p b 0.001 p b 0.001 p N 0.1 p N 0.1

123.83 93 95 104.75 125.46 104.15

(94.5–176) (52.5–148.3) (58.5–154) (74.33–148) (82.30–161) (74.28–157)

– p b 0.001 p b 0.001 p b 0.1 p N 0.1 p b 0.1

Average FWHM (s) Average amp of peak (nM) Maximum amp of peak (nM) Number of peak

Fig. 5. Comparison between [Ca2+]I increase (a.) and membrane depolarization (b.). Phase I showed membrane hyperpolarization by [Ca2+]E, and phase II showed membrane depolarization. Phase III showed stagnant phase. (Error bars represent standard deviation).

samples (12.1%, Q1 of 1.5%, Q3 of 23.4%) was significantly greater than the [Ca2 +]I fluorescence within cells seeded on V-notch no-load controls (0%, Q1 of −5.2%, Q3 of 3.4%) (p b 0.001). Effects of L- and T-type VGCC inhibitors on [Ca2 +]I response to microdamage L-type and T-type VGCCs were applied in combination in bone damage model because the insert model indicated these blockers to be effective in reducing [Ca2+]I activation in response to extracellular calcium efflux. The fraction of cells with increased fluorescence decreased with treatment of both Bepridil and NNC55-0396 + Verapamil (Fig. 6-a). Peak fluorescence of individual cells from both Bepridil treated groups and Verapamil + NNC55-0396 treated samples was significantly diminished (p b 0.001) (Fig. 6-b). When L type and T type inhibitors were applied alone, the effect was significant, but not as significant as the effect of Bepridil (Table 2). However, treatments of NNC-55-0396 and Verapamil together showed a comparable effect on [Ca2 +]I inhibition with that created by Bepridil treatment (p N 0.1). The results indicate that both L- and T-type VGCCs play key role together in ECIICR in MC3T3-E1 cells.

77.5 59.52 77.09 72.03 85.15 84.7

Discussion

Control Bepridil Verapamil NNC55-0396 ω-Conotoxin-MVIIC ω-Conotoxin-GVIA

Baseline [Ca2+]I (nM)

Table 2 Effect of different VGCC inhibitors on [Ca2+]I activation. Data presented with median (Q1–Q3). Inhibitors with significant effect (p b 0.001) represented with boldface type. p values were obtained between control groups and each inhibitor group.

H. Jung et al. / Bone 76 (2015) 88–96

[Ca2+]E plays a central role in activation of [Ca2+]I signaling and the corresponding downstream pathways related to damage repair. Such pathways include enhancing osteoblast proliferation and differentiation [15–17], up-regulating matrix protein synthesis and mineralization [17], and increasing osteoclast formation [18]. The previous paper from our group assessed excessively high concentrations of [Ca2 +]E (× 10 and × 100 of normal physiological level) [13]. In the current study, a steady state extracellular calcium concentration of 6 mM was

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Fig. 6. Effect of combined application of L- and T-type VGCC blockers on mechanically induced ECIICR. Both Bepridil treatment and Verapamil + NNC55-0396 treatment showed significant reductions in fluorescence increase (*p b 0.001).

used to trigger [Ca2 +]I response. This range was chosen because it is close to the physiological level in the basal level calcium concentration of 1.8 mM, and 6–8 mM [Ca2+]E range is reported to be optimal concentration for osteoblast differentiation and matrix mineralization [15,17]. The ionized calcium concentration in the body differs from the basal level of 1 mM [19]. For example, 40 mM concentration is reported around actively resorbing osteoclasts [20]. Our previous study reported amount of calcium efflux from damaged region of bone matrix under tensile loading as 2 pmol/cm2 s which drops sharply upon unloading [12]. However, it is very challenging to evaluate calcium concentration in very tight compartment between the basal cell membrane and the underlying bone matrix. Also, the amount of calcium efflux may vary according to the strain rate, for the reason that number of microcracks decreases when strain rate increases [21]. The calcium concentration during microdamage formation in vivo is still unknown which may differ from the range we employed. MC3T3-E1 preosteoblast was used in this study to evaluate ECIICR. Since majority of cells embedded in bone matrix are osteocytes which have been suggested as sensory cells for mechanotransduction [22], osteocyte may also play a role in sensing and repairing the damage by detecting mechanically induced [Ca2+]E. The effects of ECIICR on osteocytes need to be investigated. While damage is more likely to encounter the osteocytes, osteoblasts (bone lining cells) are not immune from damage. Bending and torsional stresses are the maximum on the periosteal surfaces of the cortex, and outer surfaces of the trabeculae. Therefore, microdamage may occur near osteoblasts, including osteoblast precursors in the periosteum. Upon extracellular calcium stimulation, intracellular calcium concentrations of cells increased from baseline levels and superimposed

on this increase were calcium oscillations in a significant fraction of cells. Five variables were extracted from time history curves to evaluate the degree of VGCC activation. Baseline [Ca2+]I was used for measuring overall increase of [Ca2+]I response, and increased number of [Ca2+]I peaks, which represents the channel gate opening, was considered as higher VGCC activation. Maximum amplitude of [Ca2+]I peak and average amplitude of [Ca2 +]I peak reflect the degree of the voltage gated channel opening, based on the assumption that the more the channel gates open, the more Ca2 + will be surged into the cell. Full width at half maximum (FWHM) (as width of [Ca2+]I peak) was used to assess the speed of reactivity. Narrow peaks reach and recover from maximum calcium levels more quickly. To address the role of VGCCs on [Ca2 +]I activation, non-selective VGCC inhibitor, Bepridil was used. Treating cells with Bepridil showed to suppress ECIICR successfully. Comparing activation patterns showed significant increase in a fully suppressed pattern (6.9% to 55.2%) and significant decrease in fully activated pattern (46.3% to 11.7%). This result signifies strong involvement of VGCC on [Ca2+]I modulation. Bepridil could not completely abolish baseline [Ca2+]I response, and this suggests that other underlying mechanisms exist in MC3T3-E1, such as intracellular calcium stores or calcium sensing receptor (CaSR) which can also respond to [Ca2+]E perturbations and release calcium ion into the cytosol [23,24]. However, the calcium oscillations were strongly dependent on VGCCs as they were close to completely abolished with Bepridil treatment. Results indicated the involvement of L-type VGCC and T-type VGCC in ECIICR in MC3T3-E1. Verapamil and NNC55-0396 treatment showed a significant decrease in number of [Ca2+]I peak, maximum amplitude of [Ca2+]I peak, and average amplitude of [Ca2+]I peak, and significant increase in full width at half maximum (p b 0.001). ω-Conotoxin-GVIA showed an increase in FWHM, but did not show a significant decrease in both amplitudes of [Ca2 +]I peak, indicating that the effect of ω-conotoxin-GVIA was limited. There was no significant decrease in baseline [Ca2 +]I by four different VGCC inhibitors, and this can be explained by inhibition effect of Bepridil both VGCC and intracellular calcium store [25], or complementary effect of L- and T-type VGCCs on baseline [Ca2+]I increase. Cell membrane becomes hyperpolarized with increasing [Ca2 +]E and depolarization occurs by influx of ions. It was observed that depolarization was concomitant with an increase in intracellular calcium levels, indicating that extracellular calcium hyperpolarizes the cells and that depolarization occurs via the Ca2+ influx through the VGCC. Mechanical strain or fluid shear is known to evoke [Ca2+]I response in bone cells [26,27]. The experimental design controlled these variables to address their effects. Mechanical strain is inherently absent in the insert model. Mechanical strain occurs in bone loading model inevitably, however, our earlier studies had demonstrated the absence of ECIICR in loading of cell-seeded demineralized notched bone samples [12]; therefore, the contribution of mechanical strain to reported outcomes is expected to be negligible. Some fluid flow may have occurred during sample preparation a priori to calcium stimulation (staining and rinsing, transferring and mounting the samples under the microscope). Sufficient time was provided for such effects to subside before calcium exposure. We used calcium-free HBSS in the staining solution, washing buffer, and bath media for experiment to exclude the possibility of calcium stimulation a priori to experiments. L- and T-type blockers were applied individually in cell-insert experiments. Both agents reduced intracellular calcium response; however, to a lesser extent than a general VGCC blocker Bepridil. During boneloading experiments, L-type and T-type VGCC blockers were applied in combination and their effectiveness in blocking the response was comparable to that of Bepridil. This outcome indicates that L- and T-type VGCCs have an additive effect in blocking the observed response because their combined application equals the effect of the general VGCC blocker bepridil. VGCCs other than L- and T-type are unlikely to contribute to the reported outcome because utilization of blockers

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specific to P-, Q- and N/R VGCCs did not inhibit the activation of intracellular calcium signaling. Other mechanisms have been reported to trigger intracellular calcium response from bone cells. Fluid shear triggers [Ca2+]I increase by activating L-type calcium channel [28], intracellular calcium storage [29] or stretch-activated calcium channel [27]. Also, PTH can sensitize osteoblasts to changes in [Ca2+]E, enhancing [Ca2+]I by via involvement of mechanosensitive cation-sensitive channels [30]. In the insert experiment of this study where fluid shear and PTH are absent, it has been shown that high concentration of [Ca2+]E can trigger [Ca2+]I responses. In experiments which involved damage zone induction on notched bone samples, we have observed intracellular calcium increase in the cells in the damaged region of loaded bone specimen. Substrate strains alone did not trigger [Ca2+]I response because there were no changes in [Ca2+]I from the cells away from damaged area of stretched bone wafer. Therefore, we have concluded that high concentration of calcium was released from damaged area of loaded bone sample, and released calcium triggers intracellular calcium increase on V-notched bone specimens used in this study. The downstream calcium signaling pathways triggered by the process we report remain to be investigated. [Ca2+]I signaling triggered by [Ca2+]E is deeply involved in regulating cell function for matrix damage repair by controlling a variety of downstream pathways. It has been reported that extracellular calcium controls osteoblast viability and proliferation, differentiation and matrix synthesis, in dose dependent manner [15,31,32]. Osteoclast formation, differentiation, and resorption activity were stimulated by extracellular calcium in co-culturing with osteoblast [33,34], and nuclear factor of activated T cell (NFATc1), the master transcription factor for osteoclastogenesis, is regulated by calcium signaling pathway [35]. In osteocytes, calcium influx triggers PKA signaling pathway, which finally activates IGI-I and osteocalcin production [36]. Therefore, future studies will be focused on the effect of mechanically induced [Ca2+]E on matrix repair and bone formation. Conclusion In conclusion, L-type and T-type voltage-gated calcium channels (VGCCs) play central role in response of MC3T3-E1 cells to elevated levels of calcium due to bone microdamage. These VGCCs determine how cells detect changing calcium concentration in the pericellular niche in association with mechanical damage (Fig. 7). Further research will be performed for investigating downstream pathways of the bone cells triggered by mechanically induced ECIICR for anabolic

Fig. 7. Calcium sensing mechanism of osteoblast in Extracellular Calcium Induced Intracellular Calcium Response (ECIICR). Intracellular calcium signaling in the cell is activated by mechanically induced calcium efflux through voltage-gated calcium channel.

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