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for healthy bone formation. However, how exposure to nano-

particles (NPs) by osteoblasts affects the deposition and

organization of such ECM proteins is poorly understood.

Previous studies of natural bone suggest that the mineral

component of bone is composed of apatite platelets with

a thickness of 27 nm, length of 15200 nm and width

of 1080 nm. These particles have also been described as

being needle-shaped [3]. CaP NPs, such as hydroxyapatite

(Ca10(PO4)6(OH)2, HA), can be artificially synthesized to be

as small as the nanosized-HA plates found in native bone

tissue (approx. 50 nm) [8]. These NPs potentially have an

important role in bone tissue engineering, forming nanosized

building blocks for bone tissue repair and/or replacement.

Thian et al. evaluated the bioactivity of electrohydrodynamic

atomized nanoapatite [9], as well as studying nanostructured

HA coatings produced using magnetron sputtering and elec-

trohydrodynamic spraying [1013]. Nanoapatite has also

been observed to form in various shapes, and with different

aspect ratios, when synthetic HA has been incubated in simu-

lated body fluid that contains similar ion levels to blood

plasma [14]. The aspect ratio of the particles may also influ-

ence the dissolution of a degradable ceramic such as HA.However, the stimulatory effect of differently shaped CaP

NPs on active osteoblasts, and the effect of uptake on intra-

cellular ion levels has not previously been studied. In

addition, a thorough, ultrastructural investigation into the

response of osteoblasts to HA NPs of different shapes, as

well as their influence on subsequent ECM formation, is

also lacking. The idea that different NP shapes may differen-

tially influence cell behaviour has been measured using

apoptosis and proliferation assays [15], as well as protein

expression analysis [16] and stains for NP uptake in a macro-

phage cell line [1619]. An experiment testing two differently

sized rod-shaped HA NPs on an osteoblast-like (osteosar-coma) cell line found that although the smaller particles

caused a significant amount of cells to apoptose, there was

no difference in alkaline phosphatase activity [20]. Another

study found that rod-shaped particles caused primary osteo-

blasts to decrease their proliferation after 3 days of culture

compared with spherical-shaped. A change of morphology

was also visible under scanning electron microscopy (SEM)

[21]. The effect of different-shaped NPs on the intracellular

ion content of primary osteoblasts, as measured using

TEM-based microanalysis, as well as ultrastructural and

ECM changes other than NP internalization [22] have not,

to the authors knowledge, been previously investigated

[18]. Whether NP treatment enhances MV formation and

release is currently unknown. With bone regeneration being

the ultimate goal of using HA NPs, our hypothesis was

that the osteoblast proliferation, function and ECM pro-

duction would respond to HA NPs, and that this response

would be sensitive to HA NPs having two different shapes

and aspect ratios. In addition to traditional biochemical

assays for measuring cell proliferation, we also used a

highly sensitive, TEM-based microanalysis technique that

measures cytoplasmic ion concentrations, including

potassium/sodium (K/Na) ratio [23,24]. To gain further

information of the effects of HA NP exposure on osteoblast

physiology and differentiation, microanalysis was also usedto measure intracellular calcium (Ca) and phosphorus (P)

levels, as a complement to other measures such as alkaline

phosphatase (ALP) activity. The effects of HA NPs on the

ultrastructural composition of osteoblasts (MVs and other

intracellular compartments), and early matrix and MV release

was studied.

2. Material and methods

2.1. Nanoparticle productionHA NPs were synthesized using the precipitation method. A

total of 200 ml 0.3 M phosphoric acid (Aldrich) was slowly

dripped into in 200 ml 0.5 M Ca hydroxide suspension under

shear stress (250 and 4500 r.p.m.). The pH was adjusted to

above 11 by adding ammonium hydroxide. The resultant

precipitate was then left at room temperature and aged for

7 days [25].

2.2. Nanoparticle characterization and analysis2.2.1. Fourier-transformed infrared spectroscopyFourier-transformed infrared spectroscopy was carried out on a

Perkin Elmer Spectrum One FTIR spectrometer, with a Perkin

Elmer Universal ATR sampling accessory. Perkin Elmer Imaging

Suite version 4.1 was used to collect the data, between the range

of 6000250 cm21, and the background signal was subtractedfrom sample data.

2.2.2. Inductively coupled plasma mass spectroscopyQuantification of media ion concentrations (Na, K, Ca and P),

using inductively coupled plasma mass spectroscopy (ICP-MS),

was carried out to obtain an approximate measure of the concen-

tration of ions released by HA NPs of different shapes, which

osteoblasts would be exposed to during in vitro tests. For this,

10 mg HA NPs (rice- and round-shaped, abbreviated as AR2 and

AR4) were incubated in 10 ml supplemented osteoblast media

(see below) at room temperature for 24, 48, 96 and 144 h

(7 days), in polypropylene tubes. Samples were kept rotating for

the entire incubation time (Denley Spiramix 10 roller system).

The supernatant media was removed to fresh tubes and diluted

1/20 with distilled water and analysed using ICP-MS. The ICP-

MS instrument used was a Perkin Elmer/Sciex Elan 6100DRC,

with a Perkin Elmer AS93 autosampler using Perkin Elmer

Elan v. 3.3 software. Argon was used as the plasma gas. The

elements and isotopes monitored were: 39K, 23Na, 44Ca and 31P.

For all elements except Ca, the most abundant isotope was moni-

tored, and standard curves made up with the same isotope. For

Ca, the most abundant isotope, 40Ca matches the mass of the

plasma gas, argon (40Ar). In order to distinguish between Ca

and Ar, the 44Ca isotope was monitored instead. A reaction cell

was used for Ca, with ammonia flowing at 0.7 ml min21. Each iso-

tope was monitored for 4 s, and five such replicates were averagedto produce the measured intensity reading quoted in the results.

Standards were prepared by spiking the cell culture medium with

the elements to be measured at concentrations of 0.1, 1, 10, 100,

1000 and 10 000 mg l21.

2.3. Osteoblast cell culturePrimary human osteoblasts (HOBs) were isolated from bone

discarded during elective orthopaedic surgery, and cells grown

using an explant outgrowth technique, as described previously

[26]. Cells were plated onto tissue culture plastic and passaged

when confluent. For proliferation, alkaline phosphatase (ALP)

activity and lactose dehydrogenase (LDH) assays, cells were seeded

onto tissue culture plastic according to standard culture protocolsin osteogenic media (1027 M water-soluble dexamethasone, 10 mM

b-glycerophosphate and 0.02 mM ascorbic acid (all Sigma)). Briefly,

40 000 HOBs were seeded per plate and flushed with 2 ml media

per well, either control media (no NPs) or with AR2 or AR4 NPs

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(0.1 mg ml21). For electron and fluorescence microscopy, cells were

seeded on glass coverslips for 1 h in a 100 ml droplet, before flushing

with 2 mlmedia, which eithercontained HANPs or noHA NPs. For

cell assays, cells were grown for 3 and 7 days, after which they were

lysed with DNase/RNase-free water (Sigma, UK). For TEM, cells

were cultured on glass coverslips for 7 and 28 days, after which

cells were fixed and processed for transmission electron microscopy

(TEM) as described below.

2.4. Cell proliferation: DNA (Hoescht) assayTotal cellular DNA was measured using the Hoechst 33258 dye

(B2883, Sigma-Aldrich, Dorset, UK). Briefly, this was diluted

from 1 to 1.0 mg ml21 in saline sodium citrate (SSC) buffer added

to each standard or sample well in the Fluoronunc plate at a 1 : 1

ratio with sample volume. Samples were read in an Opsys

MR plate reader (Dynex Technologies, UK) at 460 nm, using

REVELATION QUICKLINK software (v. 4.24). Adenine thymidine

Hoechst 33258-specific fluorescence was read at 460 nm.

2.5. Alkaline phosphatase activityThe ALPactivity was used as a phenotypic marker of osteogenesis

[26]. For the assay, 10 ml of substrate reagent was mixed (40 mgp-nitrophenyl phosphate, 34 mg MgCl2.H2O, 80 ml Triton X-100

12.5% v/v (Sigma, UK) in 0.1 M glycine (pH 10.3) and a

p-nitrophenyl standard (Sigma, UK, 100 mg ml21). After 3 and

7 days of culture, samples were washed in phosphate-buffered

saline (PBS), and cells lysed by adding DNase/RNase-free water

followed by three freezethaw cycles, then transferred to Eppen-

dorf tubes and spun in an ultracentrifuge at 10 000 r.p.m. for

10 min. Samples were reacted with substrate reagent in 1 : 1 ratio

in black 96-well plates (Cliniplate, Thermo Fisher Scientific),

sealed (pre-cut transparent microplate sealers, Greiner Bio-One,

Stonhouse, Glous, UK) and shaken for 2 min (Titertek, Flow

Laboratories, UK) and measured at 410 nm (reference wavelength

630 nm) on a Hidex Plate Chameleon multilabel detection plat-

form (Hidex, UK) with Mikrowin 2000 software (MikrotekLaborsysteme GmbH). A standard curve using p-nitrophenyl

(the cleavage product ofp-nitrophenyl phosphate detected using

the assay) was used to determine ALP activity. To compare results

within groups, ALP activity readings were normalized for the

numberof cells, by measuring the amount of DNAin each sample.

2.6. Preparation of cells for transmission electron

microscopy and immuno-transmission electron

microscopyFor imaging, microanalysis and immune-TEM of HOB cultures,

cells on coverslips were fixed in 2.5% glutaraldehyde in phosphatebuffer, rinsed several times and then treated with 1% (w/v)

osmium tetroxide in phosphate buffer. The coverslips were

rinsed and dehydrated in a graded ethanol series. The coverslips

were then infiltrated with epoxy resin (TAAB, UK) and then sec-

tioned. Sections in the plane of the monolayers, as well as cross

sections through the sample (profile view of the cell culture

layers) were made. Ultrathin sections (7090 nm) were cut using

a Reichert-Jung Ultracut E ultramicrotome, mounted on mesh

copper grids and contrasted using uranyl acetate and lead citrate.

For TEM immunogold labelling, cells grown on coverslips

were fixed in 0.05% glutaraldehyde in phosphate buffer, washed

in 50 mM glycine in phosphate buffer, rinsed briefly in phosphate

buffer and water, and dehydrated through a graded series of etha-

nol. The coverslips were flooded with resin and left to infiltrateovernight at room temperature. Resin embedding was carried

and resin polymerized at 508C. Ultrathin resin sections were cut

(7090 nm) and mounted onto nickel grids. To block non-specific

reactions grids were floated on drops of 1% BSA in PBS. After the

blocking step, grids were incubated in primary antibody in 1%

BSA-PBS for 2 h, 1 : 100 for rabbit anti-human matrilin-3

(Abcam, UK), or 1 : 400 for rabbit anti-human FN (Dako, UK).

Grids were washed with 1% BSA-PBS, incubated on drops of

12 nm gold NP-conjugated secondary antibody (Jackson Immu-

noResearch Europe, UK) in 1% BSA-PBS for 1 h at a 1 : 40

dilution. Grids were then washed in 1% BSA-PBS and then PBS.

After rinsing in distilled water, the grids were allowed to air dry,

and finally stained for contrast with uranyl acetate and lead citrate

as previously described.

2.7. Transmission electron microscopy, immuno-

transmission electron microscopy and electron

dispersion of X-rays spectroscopyHA NPs were imaged using an FEI Tecnai T20 electron micro-

scope, and images captured using a Gatan ultrascan camera. For

TEM and immuno-TEM imaging, sections were examined on a

FEI Tecnai 12 transmission microscope operated at 120 kV and

images acquired with an AMT 16 000 M digital camera. For

elemental analysis (electron dispersion of X-rays spectroscopy

(EDS)), a Tecnai T12 microscope equipped with an EDAX X-raydetector and GENESIS software was used. HA NP aspect ratios

were calculated based upon measurements of length versus

width of a minimum of 25 samples pergroup. For both side (trans-

verse) and top views of HOB TEM sections, MVs were quantified

for each group (n 510). MVs released by cells at the top of cell

cultures or between cell layers were quantified and averaged for

each group (control, AR2 and AR4). MVs which had clustered at

the plasma membrane of cells were also quantified ( figure 8a).

Intracellular ion concentrations can be measured using TEM-

EDS microanalysis [23]. In this study, the use of the microanalysis

technique was applied as a novel measure of osteoblast response

to HA NPs, over a time course. Intracellular concentration of

sodium and potassium were estimated using TEM X-ray microana-

lysis. Cells were grown on gold TEM grids as described in Warleyet al.[27]. At each time point (24 h, 48 h and 7 days), the grids were

removed from the culture and washed briefly by dipping into ice

cold distilled water, then cryofixed by plunging into liquid nitro-

gen. The grids were then freeze-dried and coated with a thin

layer of carbon before analysis. Point analysis of Na, K, Ca and P

in the cell cytoplasm was carried out, avoiding internalized HA

NPs. Quantification was carried out using the Hall method.

Elements were quantified as mmol/kg by reference to previously

prepared internal calibration standards [23]. A minimum of

10 cells per group were analysed at each time point.

2.8. StatisticsA minimum of four samples were tested per group for all assays

unless otherwise stated, apart from microanalysis where a mini-

mum of 10 cells were analysed per group for each time point. For

statistical analyses, SPSS v. 17.0 was used. The MannWhitney U

non-parametric test was used to compare between two sam-

ples, and the KruskallWallis non-parametric test was used to

compare groups of more than two samples.

3. Results

3.1. Nanoparticle characterization and analysis3.1.1. Fourier-transformed infrared spectroscopyFTIR showed that both AR2 and AR4 NPs had similar spectra

(figure 1). The peaks at 1023 cm21 (AR4) or 1024 cm21 (AR2)

are most likely PO43

n3, confirming the presence of HA. The


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bands at 874 cm21 (both groups), and at 1416 cm21 (AR4) or

1421 cm21 (AR2) suggest the presence of CO32 [28].

3.1.2. Transmission electron microscopy and electron dispersiveX-ray spectroscopyTEM of the HA NPs showed a size of approximately 50

20 nm for AR4, and a rice-grain morphology ( figure 2a,b),

and an approximately 20 nm diameter and spherical shape

for AR2 (figure 2c,d). Aspect ratios are listed intable 1. High-

angle annular dark-field-scanning TEM (HAADF STEM)

showed the presence of elements of high atomic number (for

example, higher than H, O or C) within the HA NPs. EDS

was then used to confirm the presence of Ca and P ( figure 3).

3.1.3. Inductively coupled plasma mass spectroscopyMeasurement of Ca, P, Na and K concentrations in cell culture

media, using ICP-MS, showed that following incubation with

0.9

0.8

0.7

0.6

0.5

0.4ab

sorbance

0.3

0.2

0.1

01700 1500 1300

wavenumber (cm1)

1100 900 700

Figure 1. FTIR showing similarity of AR4 and AR2 HA NPs. Grey line, AR4; black line, AR2.

100 nm 50 nm

20 nm 20 nm

(a) (b)

(c) (d)

Figure 2.TEM of (a,b) rice-shaped (AR4) HA NPs and (c,d) round-shaped (AR2) HA NPs. (a,c) Show NPs in clusters (100 and 20 nm scale bar, respectively); (b,d)

show singular HA NPs (20 and 50 nm scale bar, respectively). One side of AR4 was similar in length to the diameter of the AR2 (20 nm), the long side of the AR4

was larger (approx. 50 nm).


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HA NPs, Ca, P, Na and K ion levels decreasedat all time points,

for both AR2 and AR4 (figure 4). The decrease in Ca and P

concentration was the highest in AR2 samples, whereas for

Na and K, although after 24 h in the AR2 group had a lower

concentration of both elements than AR4, there was less of a

notable difference in Na and K levels between the two

groups at the other time points. However for both NPs, the

levels of all elements measured were lower at every time

point than in the control (NP-free) media.

3.2. Proliferation assayCell proliferation, as measured by total DNA, was lower

than controls in the presence of both groups of HA NPs, at

3 and 7 days compared with controls ( figure 5), although

this was only statistically significant for AR2, when compa-

red to the control group ( p

0.043 and p

0.034 at 3 and7 days, respectively).

3.3. Alkaline phosphatase activityALP activity increased in the presence of NPs at 3 days, and

in the AR2 group at 7 days (figure 6). This was found to be

statistically significant ( p 0.034) for both AR2 and AR4 at

3 days, and for AR2 at 7 days ( p 0.034).

3.4. Transmission electron microscopy-based ultrastructural

observations and matrix vesicle release, microanalysis

of cytoplasmic ion concentrations and immuno-transmission electron microscopy

Transverse sections, looking at a side view of monolayer

cultures revealed a 5 10 cell thick cell layer. Osteoblasts

appeared to self-organize in culture. In all groups, cells at the

top of the 5 10 cell-layer structure (media-facing side, asopposed to tissue culture plastic-facing)contained higher num-

bers of vesicles and higher MV release was observed (figure 7),

suggesting that these cells may have a greater potential for

promoting bone/matrix mineralization. After 7 and 28 days,

cells displayed normal morphology. NP-stimulated cell cul-

tures appeared more active, containing a greater number of

Golgi bodies, mitochondria and MVs (figure 7b), compa-

red with controls (figure 7a). In some cells, NP-like clusters

were observed at the plasma membrane, especially at the

top layer of cultures (figure 8a). In addition, intra-

cellular osmium-stained vesicles were observed to a greater

extent in NP-stimulated samples following 7 days culture(figure 8b,c), especially at the top layer of cells. Counting of

the released MVs, at the top of the cell layers or between cell

layers, found an overall decrease in vesicles released post-HA

NP stimulation, although this was not statistically significant

(figure 8c,p 0.10). There were more MVs observed in AR2-

stimulated HOB cultures than in the AR4 group, although

there was no statistically significant difference observed

between the two groups ( p 0.12).

Random, disorganized fine threads, which did not show

typicalcollagen banding patterns (figure 7c), were observed fre-

quently within the extracellular space of control cultures, but

not in either type of HA NP cultures (figure 7d). There were

also fewer gaps visible between cells following NP stimu-lation. Immuno-TEM identified the thread-like fibrils as FN

(see arrows,figure 9a). FN-positive staining fibrils were not as

apparent in the HA NP-treated groups and did not show the

banding pattern associated with collagen (figure 9b). Following

rice-shaped HA NP exposure, matrilin-3 was found to form

larger polymeric units [7] as identified by clusters of the

immunogold secondary antibody ( figure 9d ). These large clus-

ters were not visualized in the control HOB samples (figure 9c).

Measuring cytoplasmic Ca and P concentrations showed

highly elevated levels in both AR2 and AR4 groups at 24 h.

This was statistically significant for AR4 ( p 0.05 for Ca and

P, table 2). The Ca and P concentration in AR4 seemed todecrease between 24 and 48 h, whereas it increased in AR2.

The reverse was observed at 7 days, with the AR4 group

having the highest Ca and P levels (although Ca levels were

still elevated in the AR2 group).

500 nm

1

30

25

20

counts

15P

Ca

CaCu

Cu

EDX HAADF detector area 1

10

5

02 4 6 8

energy (keV)

10 12

(a) (b)

Figure 3. (a) HAADF STEM image and (b) EDS of agglomerated AR4 NPs, confirming the presence of Ca and P in the sample.

Table 1. Length and width measurements of rice- and round-shaped HA

NPs, as well as corresponding aspect ratios (n 25).

HA NP

shape length (nm) width (nm)

aspect

ratio

rice (AR4) 107.8+ 27.0 28.4+ 9.1 3.79

round (AR2) 39.7+ 6.6 18.7+ 5.0 2.12


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K/Na ratios measured with TEM microanalysis have been

used as a sensitive indicator of cell viability and proliferation

[29]. Following treatment with HA NPs for 24 and 48 h, both

types of HA NPs had lowered K/Na ratios compared with con-

trols, suggesting that they had lower viability. The change in

K/Na ratio was significantly lower for AR4 group ( p 0.05),

indicating lower cell viability. These data did not correlate

with the DNA assay results, which showed that the AR2

group had the lowest cell proliferation.

4. DiscussionPrevious work in the literature has described initial NP

uptake by cells and their effects on cell proliferation and

protein expression [16]. However, a thorough ultrastructural

investigation into the effects of HA NPs on osteoblasts,especially of different NP shapes, and MV release has not

previously been carried out, to the authors knowledge. Simi-

larly, the use of microanalysis to understand how exposure to

NPs alters the intracellular ion balance has not previously

been tested in osteoblasts, which we have shown here. Fur-

thermore, we have been able to demonstrate that exposure

to NPs may be influenced by NP shape, in terms of changes

to osteoblast proliferation, ALP activity and intracellular ion

levels. A previous study compared ALP activity following

NP exposure to two rod-shaped NPs of different sizes and

did not find any significant difference between them

[19,21]. Another study looking at an osteosarcoma-derived

cell line found that cell proliferation was decreased in the

presence of spherical-shaped HA NPs [21]. The group also

reported a change in cell morphology, as observed via

SEM, although this was not based on ultrastructural analysis

of ECM proteins or intracellular ion concentration. This study

provides additional in-depth information to these initial

reports that tested HA NPs of different shapes. Two ECM

proteins, which were affected by the exposure of cells to

NPs, FN and matrilin-3, were identified using immuno-

TEM. This work has shown that osteoblast cell layersappear to self-organize, such that the top layer of cells is

highly active and contains numerous MVs. MV release

by HOBs into the extracellular space was altered in the

NP-stimulated groups.

3000

2500

2000

1500

1000

24 48time (h) time (h)

control AR4 AR2

96 144

mgl1

mgl1

mgl1

mgl1

14000

13000

12000

11000

10000

9000

8000

140000

150000

160000

170000

130000

120000

3000

2500

2000

1500

1000

24 48 96 144

24 48

time (h) time (h)

96 144 24 48 96 144

(b)(a)

(c) (d)

Figure 4. Graphs comparing release of (a) Ca, (b) P, (c) K and (d) Na by HA NPs following 24, 48, 96 and 144 h (7 days) incubation in cell culture media, as

measured using ICP-MS. Note that the media ion concentrations decreasedafter incubation with HA NPs at all time points.

0

3 days

*

*

7 daystime (days)

control AR4 AR2

0.2

0.4

0.6

0.8

1.0

1.2

mgDNA

Figure 5.Graph representing results of DNA (Hoescht) assay at 3 and 7 days

post-incubation of HOBs with HA NPs (AR4 and AR2). Cells exposed to AR2

showed the lowest proliferation over the 7-day period. Statistical significance

is shown with an asterisk (*) where p 0.05.

0

3 days

**

*

7 daystime (days)

control AR4 AR2

1

2

3

4

5

6

ALP/DNA

Figure 6.Results of ALP assay, showing a change to ALP levels following 3

and 7 days treatment with AR4 and AR2. Statistical significance is shown

with an asterisk (*) where p 0.05.


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1mm 1mm

500 nm 500 nm

(a) (b)

(c) (d)

Figure 7.Images from TEM, showing in (a) and (b) a transverse view of HOB cell layers following 7 days culture, ( a) controls (no NPs) and (b) with AR2, which

showed higher levels of MVs towards the top of the culture, compared with controls (see arrow); ( c) thin threads in the extracellular space of control osteoblast

cultures (see arrow); (d) a lack of such ECM structures in the round HA NP-stimulated group. The scale bars in ( a) and (b) are 1mm; in (c) and (d) they are 500 nm.

(a) (b)

120

100

80

60

40

no.vesiclesreleasedpercell

20

0control AR4

group

AR2

(c)

Figure 8.TEM images of MVs release by round-shaped HA NP-stimulated osteoblasts, including (a) a growing cluster of MVs at the cell surface and (b) chains of

MVs being released into the extracellular space (scale bar, 100 nm for all images). ( c) Number of MVs released by primary HOBs under control conditions and after

stimulation with rice- or round-shaped HA NPs. There was no significant difference between groups, although AR2 had higher MV release than AR4.


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Despite extensive research into ceramic NPs and their

in vitro biocompatibility, as well as the effect of HA NPs

on other cell types [18,30], this is the first work to speci-

fically document the changes in osteoblast ultrastructure

and ECM following HA NP exposure. Furthermore, it

attempts to quantify cytoplasmic Ca and P, based on a micro-

analysis technique which has not been used with cultured

osteoblasts. In terms of cultured cells, microanalysis hasbeen limited to use in other cell types, such as red blood

cells and monocytes [15,24]. In the first description of MVs,

Anderson [4] used TEM, EDS and XRD techniques to detect

Ca and phosphate within sections of cartilage and bone. Mot-

skinet al.[15,30] used microanalysis to measure the Ca and P

content of HA NPs engulfed within monocyte-macrophages

in vitroand to study NP dissolution, as opposed to the specific

measurement of cytoplasmic Ca and P concentrations. Relating

the increase in ALPactivity to the different intracellular Ca and

P levels, we suggest that a moderate increase in Ca and P levels

for 24 h, as experienced following AR2 HA NP exposure, may

be stimulatory for increasing osteoblast function, for example,

via increased MV CaP accumulation and release. Xuet al.[16]showed an increase in expression of Ca-regulating proteins

after stimulation with needle-shaped or round-shaped HA

NPs. By contrast, higher Ca and P ion levels may not be as

effective, or indeed detrimental, as observed with AR4 HA

NPs. The decrease in cell proliferation in the AR2 group, as

detected using DNA quantification, may be a result of

increased cell differentiation, as suggested in an osteoblast

study by Robinson et al. [31]. A similar result was observed

by Xuet al. [16] except between sharper, needle-shaped HA

NPs and round-shaped HA NPs.

Intracellular Ca levels have an important role in osteoblast

homeostasis, regulation and apoptosis [3234]. Interestingly,a time-dependent change in Ca2 levels was observed over

7 days, when cells were exposed to HA NPs. Whether this

change was due to HA NPs entering into the cell cytoplasm

followed by NP dissolution is yet to be determined. Motskin

et al. [15] showed that microparticles and NPs made via

different materials synthesis techniques had different effects

on human monocyte-derived macrophages, with some show-

ing significantly greater uptake and/or toxicity. TEM images

demonstrated that HA NPs were dissolved by cells over time,

whereas microparticles broke into NPs before they were dis-

solved by cells [15]. Using two complimentary methods of

assessing cell viability and/or proliferation (DNA quantifi-cation and Na/K microanalysis), we have shown that cell

viability was affected by treatment with HA NP over 24 h,

48 h and 7 days, and that HA NPs with two different

shapes resulted in different microanalysis results. The effects

of four differently shaped NPs (needle, spherical, long rod-

like and short rod-like) on the proliferation, ALP activity,

intracellular reactive oxygen species and apoptosis, has also

been carried out by Xuet al. [18].

Although extracellular phosphate is essential to the regu-

lation of mineralization [35], in this experiment we have

actually observed adecrease in phosphate-media levels follow-

ing incubation with HA NPs, rather than increase. Differences

in,or changes to surface chemistry, with or without ionadsorp-tion/de novo apatite formation, may have contributed to

changes in NP toxicity [15], uptake, intracellular distribution

and dissolution. Differences in protein and/or ion adsorption

and apatite formation on NP surfaces, which have been

linked to NP morphology [36,37], may have also contributed

to the microanalysis results.

Another observation in this study was the apparent self-

organization of osteoblasts following cell culture for 7 days,

as demonstrated by transverse TEM sections showing that

HOBs formed a four- to five-cell thick layer on tissue culture

plastic. MVs, which were visualized using TEM, were much

more prevalent in the top osteoblast layer than at thebottom of the cell-layer structure. Budding and release of

MVs into the extracellular space were also located at the

top layer of the culture. These TEM images show some evi-

dence that osteoblasts are able to self-organize in vitro, such

(a) (b)

(c) (d)

Figure 9.Immuno-TEM of HOBs for (a) FN (control group), (b) FN (AR4), (c) matrilin-3 (control group) and (d) matrilin-3 (AR4). Gold labelling showed that FN was

shown to co-localize with the fine fibrils that were decreased in the presence of HA NPs (fibrils with no apparent collagen double-banding pattern). Matrilin-3,

which is important for normal endochondral ossification during skeletal development, was found to have different staining patterns following HOB treatment with

HA NPs, showing increased oligomerization and/or polymerization. Arrows indicate positive immunogold labelling.


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that the most active cells are located at the top layer of the

culture. In this five-cell thick in vitro culture, we have

shown that by self-organizing, the HOBs have exposed their

MV-releasing region of the outer membrane to the extracellular

environment, especially at the top of the culture where

MV release is most apparent. By observing cryo-electron

microscopy of developing mouse calvaria and long bones,

and using elemental analysis (EDS), Mahamid et al.[5] hypo-

thesized that phosphate first concentrates within MVs,

followed by the sequestration of Ca, forming an amorphous

CaP. We have also shown the deposition of osteoid/MVs

by these cells from the top of the cultures and within the

extracellular space, similar to that imaged by Ecarot-Charrier

et al. [38], and that their release into the extracellular space

appeared to be altered by HA NPs of either morphology. The

statistically significant increase in ALP activity in the round-

shaped HA NP (AR2) group suggests that osteoblasts in this

group may be more active compared to those stimulated

with AR4 [39].

In terms of changes observed in the ECM, which plays a

crucial role in bone formation, we have shown that thin fibrils

observed via TEM stain positively with anti-FN antibody, andappear to be altered in the presence of HA NPs. FN is required

for collagen assembly and maintenance, as well as bone devel-

opment and repair [4042]. HA has been shown to avidly bind

proteins and other ions and molecules, including FN [4345].

It may be that the FN-binding ability of HA may have inter-

fered with thein vitroformation of normal FN networks. It is

also not known whether the changes to the ECM are linked

to matrilin-3 oligomerization. Matrilins-1 and 3 have been

shown to be expressed temporally within the growth plate

and also have been observed to formextended filamentous net-

works [7]. Matrilin-3 expression has not previously been

observed in cultured osteoblasts [7] and is reported here forthe first time. The role of biomaterials in regulating such

ECM proteins is an area of current investigation.

5. ConclusionIn conclusion, HA NPs of two different shapes, rice-shaped

(AR4) and round-shaped (AR2), were shown to influence

HOB proliferation, cytoplasmic ion levels, ultrastructure

and ECM assembly. AR2 and AR4 were shown to adsorb

Ca, P, Na and K at different levels, from cell culture media.

A sensitive TEM-based microanalysis technique was used to

detect subtle changes in cell viability and cytoplasmic ion

levels, by measuring the K/Na ratio and Ca and P concen-

trations. ECM proteins FN and matrilin-3 were shown to

change in organization following culture with HA NPs, and

expression of matrilin-3 in primary HOBs was confirmed

using immuno-TEM. AR2 had a greater osteogenic effect

when compared with AR4, as measured, suggesting that HA

NPs of this shape and/or aspect ratio may have a positive

effect upon new bone formation. AR2 caused a moderate

increase in intracellular Ca and P levels, while AR4 caused a

larger increase in ion levels. Interestingly, when primary

HOBs were cultured in vitro, they were observed to self-

organize into a layered structure four to five cells thick, withthe most active cells located at the top of the culture. The rel-

evance and consequences of changes to cell viability,

cytoplasmic ion levels, cell self-organization to new bone for-

mation in vivo requires further investigation. In addition,Table

2.

CytoplasmicHOBCa,

P,

NaandKconcentrationsfollo

wing3and7daysculture,withandwithout

HA

NPs(AR4andAR2).DifferencesinionlevelswerefoundfollowingHA

NPtreatment,whichcorrespondedtoassaysforcell

proliferation(DN

A)andosteoblastfunction(ALP).

K/Na

Ca

P

control

AR4

AR2

control

A

R4

AR2

control

AR4

AR2

24h

16.4

7+

4.5

0

2.8

1+

1.4

1

6.7

6+

4.5

0

22.5

4+

11.1

8

287.2

2+

119.4

1

156.2

5+

179.1

2

846.0

5+

68.6

1

1350.6

1+

147.3

0

908.3

0+

129.8

8

48h

5.9

4+

0.0

7

1.2

0+

1.1

4

3.8

1+

3.2

4

62.3

0+

88.2

0

50.9

5+

26.4

8

137.5

1+

125.2

1

962.0

7+

141.7

2

928.3

3+

183.8

5

1051.9

3+

365.7

1

7days

5.3

3+

3.0

2

6.6

0+

4.9

3

4.8

2+

1.0

5

19.6

0+

17.9

4

135.1

9+

151.0

4

53.7

2+

61.5

5

966.1

6+

416.5

3

912.2

4+

253.5

5

803.3

6+

161.0

3


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further analysis of NP effects on osteoblast ultrastructure and

ECM production is an important and critical biological

consideration for future work.

Acknowledgements. The authors would like to thank OrthopaedicResearch UK (ORUK) for their generous support and Dr GiuseppeCama for assistance with FTIR analysis of HA NPs.

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