There is diverse opinion about the mechanism of bone mineralization with only intermittent reports of any direct organellar role played by the golgi apparatus (juxtanuclear body). Light and laser confocal microscopy was combined with electron microscopy and elemental EDX (energy dispersive X-ray microanalysis) in comparing “young” osteocytes in situ in fresh and “slam” frozen developing mouse calvarium, with similar cells (MC3T3-E1) maintained in vitro. The distribution of “nascent” electron dense mineral was examined histochemically (von Kossa, GBHA), including tetracycline (TC) staining as a fluorescent complex with bone salt, while golgi body activity was demonstrated by transfection with a specific green fluorescent construct (GFP/mannosidase II). In tissue culture golgi body activity and mineralization were both blocked by brefeldin A (an established golgi inhibitor) and restored by forskolin, enabling an association with mineral fabrication to be quantified as changing fluorescence intensity (AU) of GFP or TC markers. Results from osteocytes in situ supported previous descriptions of intracellular electron dense objects (microspheres and nanospheres) in a juxtanuclear pattern, containing Ca, P and transitory Si, in a spectrum recapitulated in the calcifying culture after 10 days, when GFP fluorophore surged from 71.7 ± 1.4SD to 133.7 ± 2.7SD AU by 14 days (p < 0.0001). At this stage TC fluorophore mean intensity was 23.8 ± 3.7SD AU (14 days) rising to 45.0 ± 5.1SD AU by 17 days, compared to its stationary 21.7 ± 3.6SD when treated 3 days previously with BFA golgi inhibitor (p < 0.0001), until forskolin reversal. It was concluded from the changing juxtanuclear morphology, Si mineralization mediation and the variably controlled activity versus stasis that the inorganic phase of bone is a complex golgi-directed fabrication with implications for bone matrix biology and evolution.
In examining events at the calcification front of bone it has been long recognized that the first-formed (i.e. nascent) bone mineral is soluble, readily transformed by exposure to chemicals and is easily lost during processing, resulting in a diversity of views in the literature about whether the mineralization process commences extracellularly or intracellularly. High velocity impact (slam) freezing is an instantaneous cryo-preparative technique considered to conserve tissue chemistry optimally in small bone specimens which risk losing some of their nascent bone mineral with more prolonged processing [
It has been previously reported histochemically [
It is proposed to expand upon the previous static bone histology which used the von Kossa stain for phosphate/carbonate and Kashiwa’s GBHA for calcium [
Transverse, unstained, ultrathin sections of slam-frozen neonate mouse calvarium (1 - 5 days old) were prepared as previously reported in detail [
MC3T3-E1 cells (from neonate murine calvarium; a gift from Dr. P. Genever, University of York) were seeded (density 5 × 104/mL) onto acetate coverslips, placed in a welled plastic plate and grown for 6 - 7 days in α-MEM (minimum essential medium) containing 10% FCS (foetal calf serum), penicillin (100 units/mL) and streptomycin (100 µg/mL), together with supplement (5 mM β-glycerophosphate and 25 µg/mL ascorbic acid phosphate [
MC3T3-E1 cells were seeded as above, fixed, rinsed and stained in tetracycline hydrochloride (10 µg/mL) for 30 min and examined under the laser confocal microscope (excitation laser 360 nm ± 40, emission filters to collect 528 nm ± 38). TC fluorescence quantification was performed using the Imaris 4.1 (Bitplane) programme.
MC3T3-E1 cells were cultured in flasks containing α-MEM and 10% foetal calf serum (FCS), with penicillin, streptomycin and calcifying supplement in a humidified atmosphere as above. At 3 days prior to analysis the cells were rinsed in calcium/magnesium-free phosphate buffered saline (PBS) and sub-cultured using 0.05% trypsin to cause cell detachment. As a BFA toxicity assay, cells were seeded on acetate coverslips as above and grown for 14 days when BFA (10 nM) was added followed by cell growth for 3 - 4 more days before sampling for confocal microscopy. [Experimentation at higher BFA doses (200, 100 and 50 nM) compared with untreated controls resulted in cell death at approximately 4, 12 and 36 hr respectively]. Subsequent supplementation with forskolin (100 µM for 30 min, 1, 2 and 3 hours respectively) in the continued presence of BFA followed. Also included was a test for apoptosis by adding propidium iodide (1 µl/5mL) to cultures for the remaining 20 min of the investigation (excitation laser 536 nm, emission filters for 620 nm). TC and GFP fluorescence quantification was performed throughout using the Imaris 4.1 (Bitplane) programme above. The statistical analysis of the data as two sample t-tests and ANOVA was performed using Minitab Version 14 and Genesis software.
Electron dense calcified objects (
MC3T3-E1 cultured control cells had well-defined nuclei with many nucleoli as shown by TOPRO-3 stain and those transfected with GFP/mannosidase II construct showed fluorescence located in the juxtanuclear region (
Cell-free control incubation of the supplemented medium showed no sign of mineral deposition, neither did cell
Bone cells and mineral particles in situ in the 5-day-old mouse calvarium. (a) Intact osteocyte stained histochemically for calcium (GBHA stain) and phosphate/carbonate (von Kossa stain) showing the nucleus (N) and a juxtanuclear crescent (large arrow) composed of dark microspheres (<1 µm), together with peripheral groups (small arrow); H. P. incident light inter- ference contrast (Nomarski) optics scale bar 4 µm. (b) Similar osteocyte in section, “slam” frozen and unstained showing a juxtanuclear crescent and peripheral groups of white microspheres (arrowed), each composed of dense bodies (30 - 50 nm); L. P. TEM (unstained, negative image, i.e. electron dense mineral white for maximum resolution), scale bar 3 µm. (c) Juxtanu- clear microspheres (1 µm in diameter) in detail as clusters of nanospheres (30 - 50 nm in diameter), with (e) a corresponding EDX spectrum showing Ca and P peaks together with Si (below star); H. P. TEM, scale bar 1 µm. (d) Electron dense microsphere populations (1µm diameter) in the calcified extracellular matrix (some with less dense centres, arrowed) and (f) with a corresponding EDX spectrum showing Ca and P peaks and no S
cultures without the β-glycerophosphate/ascorbic phosphate supplement. MCT3TC-E1 cultured control cells showed only a dull autofluorescence at the outset i.e. they were apparently not fabricating fluorescent mineral particles for several days, as monitored by von Kossa and alizarin red (calcium) stains. Inspection of supple
Substructure of intracellular (a) and extracellular (b) mineral particles in situ in undecalcified bone. Showing micron-sized electron dense objects as clusters (yellow arrowheads; 1 µm in diameter) of beaded (black arrows) filaments (5 nm wide). TEM, stained for contrast (negative image i.e. mineral white)
Fluorescent markers of the golgi apparatus (arrowed) in individual bone cells (MC3T3-E1) in vitro. Showing examples of the crescent-shaped juxtanuclear fluorescence (left) with GFP/mannosidase II construct and (right) with TC stain. Laser confocal microscope
. Cultured bone cell (MC3T3-E) mean fluorescence intensity of GFP mannosidase II construct for golgi complex activity and of TC (tetracycline) for bone salt fabrication in 6 assays (n = 6) after 7, 9 and 14 days showing the time-related fluorochrome surge in both. A specific TC filter removes the autofluorescence component (13.5 AU) and columns in brackets were below the 13.5 threshold until after 9 days when previous engagement in collagen precursor synthesis switches to mineral fabrication. Laser confocal microscopy (AU arbitrary units). (ANOVA p < 0.0001)
Culture | Fluorescence Intensity (AU) | |||||
---|---|---|---|---|---|---|
Assay | GFP | TC | ||||
7 days | 9 days | 14 days | 7 days | 9 days | 14 days | |
1 | 32.7 | 69.3 | 132.2 | (11.3) | (12.6) | 21.5 |
2 | 36.1 | 72.7 | 131.0 | (9.9) | (13.1) | 28.6 |
3 | 34.7 | 71.9 | 133.4 | (10.1) | (12.9) | 32.5 |
4 | 33.1 | 72.8 | 137.6 | (8.8) | (10.8) | 20.3 |
5 | 35.2 | 70.4 | 136.3 | (8.3) | (11.9) | 18.1 |
6 | 35.9 | 72.6 | 131.8 | (7.6) | (11.5) | 31.6 |
Mean | 34.6 | 71.7 | 133.7 | 0.0 | 0.0 | 25.4 |
SD± | 1.4 | 1.4 | 2.7 | 0.0 | 0.0 | 6.2 |
Fluorescent markers of golgi apparatus activity in bone cells (MC3T3-E1) in vitro after 14 days culture when their shape is typically irregular. Showing (left) the prominent intensity of the condensed jux- tanuclear golgi apparatus and also its more extensive dispersion throughout the cytoplasm, GFP/mannosidase II construct; (right) the similar intracellular golgi-related distribution of the TC-tagged mineral (arrowheads), with some extracellular labelling (arrowed). Topro-3 nuclear stain (red). Laser confocal microscope
mented cultures at intervals of 7 to 10 days showed an initial shape transformation from fusiform to cuboidal had taken place with continued negativity for TC-mineral binding. It was not until 10 days had elapsed when cell elongation to ovoid and stellate was recorded (
MCT3TC-E1 cultured control cells exposed to BFA at 14 days was followed by the retraction of cytoplasmic processes during the subsequent 3 days, while the nuclei and nucleoli remained well defined and the propidium iodide stain for apoptosis was negative. However, more prolonged exposure beyond a further 4 days was associated with the appearance of features consistent with programmed cell death i.e. pyknotic nuclei and positive staining with propidium bromide for apoptosis. Observations were therefore confined to the 3 day window i.e. inspection of supplemented cultures at 17 days. At this stage, untreated cells stained intracellularly for mineral with von Kossa stain, with some extracellular stained particles bordering the cells which showed an increase in cell processes and a general confluence. At the same time histochemically stained juxtanuclear bodies were a prominent feature. In contrast, cells exposed continuously to brefeldin after 14 days optimal culture showed retraction of their processes, shrinkage and fragmentation of the juxtanuclear body and fewer mineral particles equivalent only to those present at 14 days, suggesting the arrest of mineral fabrication coincident with the BFA golgi “switch off.”
To enable quantification of these subjective observations TC stain was applied to untreated and BFA-treated cells cultured optimally to 14 days and subsequently exposed to BFA for 3 days as above and the mean fluorescence intensity of the TC–mineral complex measured (
Bone cell (MC3T3-E1) mineral particle trafficking in vitro. Showing (top) a typical irregular cell with 1 µm intracellular mineral particles and (bottom right) elongated, slender cytoplasmic processes with mineral particles inside and outside (LM), and with one enlarged cell process (bottom left) bulging with a single calcified microsphere about 1 µm in diameter (SEM)
EDX microanalysis of bone mineral fabricated in vitro by 49 day cultured bone cells (MC3T3-E1). (Left) A typical intact stellate cell with a central nucleus (Nu) and cytoplasmic mineral particles with extracellular assemblies and dispersed particles. Von Kossa stain. (Right) Typical elemental spectrum of cell isolates showing Ca and P peaks and also Si and S
for further details and references).
The absence of mineral particles from incubated cell-free, supplemented medium (β-glycerophosphate and ascorbic acid phosphate) suggests that the production observed was dependent upon the input of biological energy and not confined to physicochemical precipitation from a metastable solution. At the same time, the failure of cells grown without supplement either to fabricate calcified objects or to develop a related prominent golgi complex, implies that β-glycerophosphate/ascorbic phosphate supplement was essential for this particular phe-
Golgi apparatus inhibition in vitro in 14 days cultured bone cells (MC3T3-E1) with- out and with added brefeldin A (BFA). Showing (a) the juxtanuclear TC-tagged mineral at 14 days and (b) its organellar expansion throughout the cytoplasm by 17 days, in contrast to (c) its organellar stasis with BFA. TOPRO-3 nuclear stain (blue). Laser confocal microscope
Histogram of tetracycline (TC)-tagged mineral inhibition in cultured bone cells (MC3T3-E1) by 17 days, with and without the golgi apparatus inhibitor brefeldin A (BFA) administered at 14 days. Mean fluorescence intensity with highest and lowest readings in- dicated (whiskers). Respective means 23.8 ± 3.7SD, 45.0 ± 5.1SD and 21.7 ± 3.6SD. (Each column represents 6 culture assays, n = 6; t test p < 0.0001)
notype expression. Just as the presence of silicon seems to be a key early intermediary of calcification with phosphate, featuring regularly from mineralizing bacteria [
By 35 days of optimal culture, nodular accretions about 10 microns in diameter resembled those described [
Green fluorescent protein (GFP) is a natural jellyfish bioluminescent where it functions as an energy-transfer acceptor changing blue light emitting photoprotein aequorin into green and its use as a golgi marker in live cells is well established [
In summary there is now available a bone biomineralization system that is apparently responsive to the BFA golgi apparatus “on/off” switch and three main conclusions can be drawn from the results. First, there are close similarities between the mouse cells in situ in the developing calvarium and those maintained in vitro in terms of intracellular and extracellular calcified particles, their elemental spectra including a significant Si peak [
We are grateful to Dr. Roger C. Shore, Department of Oral Biology, University of Leeds , for guidance with EDX microanalysis, to Prof. Michelle Peckham for valuable advice about tissue culture procedures and together with Ruth Anderson-Beck for use of their facilities, and finally to Dr. F. G. E. Pautard for inspiration.