back-scattered electron imaging and elemental microanalysis of retrieved bone tissue following...

Back-scattered electron imaging andelemental microanalysis of retrievedbone tissue following maxillary sinusfloor augmentation with calciumsulphate

Nicola SlaterAmir DasmahLars SennerbyMats HallmanAdriano PiattelliRachel Sammons

Authors’ affiliations:Nicola Slater, Rachel Sammons, School ofDentistry, University of Birmingham, Birmingham,UKAmir Dasmah, Lars Sennerby, Department ofBiomaterials, Institute for Clinical Sciences,Goteborg University, Goteborg , SwedenMats Hallman, Department of Oral & MaxillofacialSurgery, Gavle Hospital, Gavle, SwedenAdriano Piattelli, School of Dentistry, University ofChieti-Pescara, Pescara, Italy

Correspondence to:Rachel Sammons, BSc, PhDSchool of DentistryUniversity of BirminghamSt Chad’s QueenswayBirminghamB4 6NNUKTel.: þ 44 121 237 2910Fax: þ 44 121 237 2932e-mail: [email protected]

Key words: back-scattered electron imaging, calcium sulphate, energy dispersive X-ray

analysis, maxillary sinus augmentation, resorption

Abstract

Objectives: To investigate the presence and composition of residual bone graft substitute

material in bone biopsies from the maxillary sinus of human subjects, following

augmentation with calcium sulphate (CaS).

Material and methods: Bone cores were harvested from the maxillary sinus of patients who

had undergone a sinus lift procedure using CaS G170 granules 4 months after the initial

surgery. Samples from seven patients, which contained residual biomaterial particles, were

examined by field emission scanning electron microscopy and energy dispersive X-ray

spectroscopy was used to determine the composition of the remaining bone graft

substitute material.

Results: Residual graft material occurred in isolated areas surrounded by bone and

consisted of individual particles up to 1 mm in length and smaller spherical granules. On the

basis of 187 separate point analyses, the residual material was divided into three categories

(A, B and C) consisting of: A, mainly CaS (S/P atomic% ratio � 2.41); B, a heterogeneous

mixture of CaS and calcium phosphate (S/P¼0.11–2.4) and C, mainly calcium phosphate

(S/P�0.11; C), which had a mean Ca : P ratio of 1.63 � 0.2, consistent with Ca-deficient

hydroxyapatite. Linescans and elemental maps showed that type C material was present in

areas which appeared dense and surrounded, or were adjacent to, more granular CaS-

containing material, and also occurred as spherical particles. The latter could be

disintegrating calcium phosphate in the final stages of the resorption process.

Conclusions: CaS resorption in the human maxillary sinus is accompanied by CaP

precipitation which may contribute to its biocompatibility and rapid replacement by bone.

Calcium sulphate hemihydrate (CaS) is a

simple, easy to use, inexpensive bone graft

substitute material, which has been used

clinically for more than a century as a

resorbable biomaterial, which is gradually

replaced by bone (reviewed by Pietrzak &

Ronk 2000).

The rate of adsorption of CaS varies

depending on the in vivo site, chemistry,

and particle size (Tay et al. 1999; Guarnieri

et al. 2004). While resorption may occur at

the same rate as bone formation, it may

also occur faster than bone can form and

this may be clinically disadvantageous in

some sites. Applications of CaS in human

dental surgery include treatment of perio-

dontal defects (Bier & Sinensky 1999;

DiBaltista et al. 1995), repair of fenestra-

tions and bifurcations (Pecora et al. 1998;

Maragos et al. 2002) and alveolar and

maxillary sinus augmentation (de Leonar-

dis & Pecora 1999 & 2000; Guarnieri &

Date:Accepted 3 December 2007

To cite this article:Slater N, Dasmah A, Sennerby L, Hallman M, PiattelliA, Sammons R. Back-scattered electron imaging andelemental microanalysis of retrieved bone tissuefollowing maxillary sinus floor augmentation withcalcium sulphate.Clin. Oral Impl. Res. 19, 2008; 814–822doi: 10.1111/j.1600-0501.2008.01550.x

814 c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard

Bovi 2002; Guarnieri et al. 2004, 2006). It

can also serve as a barrier in guided tissue

regeneration, preventing the ingress of soft

tissue (Pecora et al. 1997a & b; Orsini et al.

2001; Yoshikawa et al. 2002).

As CaS resorbs, it acts as a direct source

of calcium for new bone formation, while

possibly permitting an earlier ingress of

osteoprogenitor cells in comparison with

less resorbable calcium-containing ceramic

graft materials (McNeill et al. 1999). It has

been shown to evoke a minimal foreign

body response and to result in normal

regeneration of bone (Orsini et al. 2004),

stimulating angiogenesis in rabbits and

aiding the formation of bone by acting as

an osteoconductive matrix for the ingrowth

of blood vessels and associated fibrogenic

and osteogenic cells (Strocchi et al. 2002).

CaS is susceptible to resorption by os-

teoclasts in vitro (Sidqui et al. 1995) and

in vivo resorption may occur due to a

combination of cellular activity and disso-

lution in body fluids. Animal studies have

shown that the dissolution of CaS cement

is accompanied by the precipitation of a

calcium phosphate (CaP) layer around the

particles, to which osteoblasts attach (Ricci

et al. 2000; Orsini et al. 2004) and in a

study in rabbits concentric rings of bone

were seen to have developed around the

dissolving particles (Orsini et al. 2004). At

the molecular level, CaS has been shown to

promote higher levels of signalling mole-

cules such as transforming growth factor-b(TGF-b) and bone morphogenetic proteins,

which may be responsible for the ingress of

osteoprogenitor cells and their differentiation

into mature osteoblasts (Walsh et al. 2003).

Recently, CaS has been reported to modu-

late gene expression in MC3T3-E1 mouse

pre-osteoblastic cells, and specifically to

augment expression of genes involved in

fracture healing including alkaline phospha-

tase, type II collagen and fibronectin 1, in

comparison with polymethylmethacrylate

(Lazary et al. 2007).

Although CaS has been used extensively

as a bone graft substitute material in hu-

mans the mechanism by which it resorbs is

still not clear. To investigate this, it is

necessary to examine healing bone at an

early or intermediate stage when some of

the original graft material is still present.

At the University of Gothenburg, histolo-

gical examination of samples of bone re-

trieved from patients 4 months after sinus

lift augmentation procedures, at the time of

second surgery for placement of dental

implants, showed that they still contained

some residual graft material. The aim of

this study was to use a combination of

back-scattered electron (BSE) imaging and

elemental microanalysis to investigate the

composition of this residual material in

order to shed light on the healing process.

The material used in this study consists

of a mixture of a and b forms of CaS, from

which arsenic, bismuth and strontium

have been removed to achieve a high degree

of purity. This product has been previously

used in humans as a graft material in

extraction sockets (Guarnieri et al. 2004),

for maxillary sinus augmentation (Scarano

et al. 2006) and for peri-implant bone

regeneration (Scarano et al. 2007).

Materials and methods

Surgery and bone retrieval

Specimens from a previous clinical and

histological investigation were used for

further analyses in the present study. In

brief, 10 patients attended the department

of Oral and Maxillofacial Surgery, Gavle

Hospital in Gavle Sweden for maxillary

sinus augmentation because of the lack of

sufficient bone tissue for the placement of

endosseous implants. The inclusion criter-

ion for maxillary sinus augmentation was

o5 mm alveolar bone remaining in the

floor of the sinus as determined by conven-

tional tomographic radiography. The pro-

tocol to harvest bone samples was approved

by the local ethics committee and informed

written consent for research was obtained

from all patients.

The maxillary sinus lift procedure was

performed under local anesthesia. A crestal

incision and a vertical releasing incision

were performed, and a mucoperiosteal

flap was elevated and reflected laterally to

expose the lateral wall of the sinus. Using a

round bur under sterile saline solution

irrigation, a 20-mm-wide and 10-mm-

high window was outlined and a ‘trap

door’ was made in the lateral sinus wall.

The door was rotated inward and upward

with a top hinge to a horizontal position.

The Schneiderian membrane was elevated

without laceration and CaS granules (Sur-

giplaster, Ghimas, Bologna, Italy) were

a

b

Fig. 1. Section of retrieved bone stained with toluidine blue from the maxillary sinus of a patient showing

newly formed bone and residual graft material (stained black) 4 months after augmentation with calcium

sulphate. (b). Back-scattered electron image of an area containing bone (left) and graft material (right) from the

other half of the same specimen block.

Slater et al . Calcium sulphate resorption in human maxillary sinus

c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard 815 | Clin. Oral Impl. Res. 19, 2008 / 814–822

mixed following the manufacturer’s guide-

lines and packed into the sinus cavity.

After a healing period of 4 months, the

second-stage surgery was performed to

place dental implants and at the same

time harvest bone biopsies for analysis.

Seven bone samples were retrieved from

the lateral wall using a 4 mm � 10 mm

diameter trephine under sterile saline solu-

tion irrigation. The samples were fixed in

4% formalin, dehydrated with ethanol and

embedded in hexamethylmethacrylate re-

sin. Resin blocks containing the bone sam-

ples were then sectioned under water

irrigation with a band saw to expose the

layer of bone fragment. Sections from half

of each block were processed for histology

and stained with toluidine blue to show

bone and graft material. Blocks were

selected from seven patients in which

residual bone graft substitute had been

identified by histological examination,

as, for example, in Fig. 1. The unused

half of each block was processed for scan-

ning electron microscopy (SEM) and

elemental analysis of the tissues as

described below.

Electron microscopy andelemental analysis

Microscopy and analysis were carried out at

the Centre for Electron Microscopy, Univer-

sity of Birmingham, UK. The surface of each

resin block containing the bone fragment

was polished using a manual grinder

with 800-grit silicone carbide paper. The

blocks were then mounted on an aluminium

stub and carbon coated (Polaron sputter

coater, Quorum Technologies, Ringmer,

UK).

Samples were examined using a field

emission environmental scanning electron

microscope (Philips XL 30 FEG ESEM,

FEI, Eindhoven, The Netherlands) operat-

ing in high vacuum mode at a working

distance of 10 mm and an accelerating vol-

tage of 15 kV. BSE imaging was used to

provide contrast between resin, bone and

biomaterial: generally the resin appeared

black, bone white and biomaterial various

shades of grey. Energy dispersive X-ray spec-

troscopy (EDS) was used to identify and

evaluate the relative concentrations of all

the chemical elements present in the tissues

and was carried out using Oxford INCAt

EDS system (High Wycombe, UK), using

point analysis, line-scan and mapping facil-

ities. Preliminary EDS analysis of Surgipla-

ster graft material confirmed that it

consisted of pure CaS with no impurities

[Ca : S ratio (atomic %)¼1.0].

A total of more than 200-point analyses

were carried out on the seven sections.

These included at least three sites within

bone in areas with no visible adjacent

biomaterial in each section, to determine

the elemental composition of the natural

bone as a base-line for comparison. Data

were then collected from deliberately tar-

geted sites of interest within the biomater-

ial, close to and distal from bone and at the

bone–implant interface if present (an aver-

age of approximately 25 points/section,

depending on the biomaterial content). Ad-

ditional information was obtained from

line scans and elemental maps.

Results

Figure 1a shows an overall view of a section

of one of the seven samples of retrieved bone

stained with toluidine blue. This appearance

was typical of all the samples with some

variations in the amount of biomaterial pre-

sent. The graft material was concentrated in

specific areas surrounded by newly formed

bone and consisted of irregularly shaped

particles up to 1 mm in length or granules

and appeared blue–black, in contrast to the

uniformly blue-stained trabecular bone with

visible osteocyte lacunae and lamellar struc-

ture. A section from a similar area of the

other half of the same block is shown as

observed by SEM, using BSE imaging. Frag-

ments of biomaterial were identified in these

images by their generally grey colour, irre-

1

5

2 4

6

7

3

500µm

Fig. 2. Back-scattered electron image of a resin-embedded bone section containing bone and residual

biomaterial. EDS spectra were collected at the points indicated by the numbered crosses.

Table 1. Elemental composition (atomic %) at each of the points shown in Fig. 2. In thisexample Mg was identified at just one of the points identified within residual biomaterialbut at other locations trace amounts of Na, Mg and occasionally S were detected in bone.

Spectrum C O Na Mg P S Ca Material

1 47.9 37.9 0.4 5.4 8.5 Bone2 73.3 16.1 2.1 2.8 5.8 Biomaterial3 76.3 11.1 0.9 5.2 6.5 Biomaterial4 62.6 25.1 0.2 3.7 1.7 6.8 Biomaterial5 62 23.8 0.2 3.2 2.8 8.0 Biomaterial6 30.3 37.4 0.5 0.2 11.6 20.1 Biomaterial7 59.5 27.7 4.6 0.9 7.3 Bone


816 | Clin. Oral Impl. Res. 19, 2008 / 814–822 c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard

gular shape, or granular appearance, in

contrast to the bone which had a trabecular

structure with well-defined margins, osteo-

cyte lacunae, cement lines, occasional

blood vessel canals and generally a brighter,

whiter and denser appearance in comparison

with the graft biomaterial (Fig. 1b). Full

details of the histology will be reported

elsewhere. From the BSE electron micro-

scopy images, the trabecular bone appeared

to be well-formed and contained regions of

mature bone with spindle-shaped osteocyte

lacunae and aligned collagen fibres and areas

of woven bone, characterized by larger

rounder osteocyte lacunae and non-aligned

collagen.

Elemental point analysis

EDS point analysis was used to identify and

determine the relative concentrations of

all elements present at 27 separate points

within bone and 187 points within

biomaterial in the seven sections. A typical

site of interest and the results of

point analysis are shown in Fig. 2 and

corresponding Table 1. Ca, P, C and O,

were always detected in bone, together

with trace amounts of Na, Mg and S

although these were not always detected.

Na and Mg detected in ‘biomaterial’ could

be attributed to signal from underlying

or adjacent bone in the section or to

substitution with these ions. The average

Ca : P ratio in bone was 1.77� 0.47

(atomic %; n¼27).

EDS analysis of the residual graft material

particles in the retrieved tissue revealed CaS

and CaP in every variable relative proportions.

The material was classified into three differ-

ent categories (A, B and C) on the basis of the

relative amounts of Ca, S and P (Fig. 3) from

the point analysis data. Material A most

closely resembled the original graft material

with approximately equal amounts of Ca and

S and low amounts of P (atomic %

S/P � 2.41, or no P detected). Material B

had higher but very variable relative amounts

of S and P (S/P¼0.11–2.4); Material C con-

tained large amounts of Ca and P and very

little S (S/P�0.1). Material C was of a more

uniform consistency and appeared denser

than A or B. The average ratios of the three

elements, Ca, S and P are shown in Table 2

together with the standard deviation of the

means. The more consistent composition of

type C CaP material (Fig. 3 and Table 2)

suggests that this could represent the final

stage of the replacement process, where

dissolution/precipitation has reached a steady

state. This material had an average Ca/P

ratio of 1.63� 0.21 (Table 2), consistent

with calcium-deficient hydroxyapatite

(stoichiometric hydroxyapatite has a Ca/P

ratio of 1.67).

b

Ato

mic

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Sample No.

0

5

10

15

20

25P S Ca

0

5

10

15

20

25

30

35

40

45

Ato

mic

%

P S Ca

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

a

Ato

mic

%

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Sample No.

P S Cac

Fig. 3. Atomic percentages of Ca, P and S within residual graft material at different points of analysis in

different samples, illustrating the difference between biomaterial classified as A, B or C. (a) Biomaterial A: high

Ca and S, low P; (b) Biomaterial B: intermediate levels of all three elements; (c) Biomaterial C: high Ca and P,

low S. For clarity data from only 20 samples in each group are shown. Total numbers in each category and

average atomic percentage ratios of each pair of elements are shown in Table 2.

Table 2. Comparison of the relative amounts of Ca, P and S in Biomaterial classified as A, Band C from point analysis results shown in Fig. 3

Biomaterial BSE appearance Ca/P Ca/S S/P N

A Dense grey 8.5 � 5.5 1.4 � 0.6 6.8 � 6 32B Granular grey 2 � 1.4 5.4 � 5.4 0.7 � 0.5 79C Dense white 1.6 � 0.2 39.1 � 15.5 0.06 � 0.11 76Total 187

Figures indicate the average (atomic %) ratio of each pair of elements � the standard deviation.

N is the number of points analysed. Highest levels of sulphur were detected in A and least in

C which appeared denser and whiter than A or B but was still distinguishable from bone by the

absence of lacunae.

BSE, back-scattered electron.



Hydroxyapatite is the most stable form

of CaP above pH 4 and the most similar

to the mineral phase of bone, which con-

sists of carbonate-substituted hydroxyapa-

tite. These results are consistent with

a gradual resorption of the CaS graft

material and its replacement by CaP,

as occurs in animals (Ricci et al. 2000;

Orsini et al. 2004).

Line scans

To investigate the distribution of CaS and

CaP in selected areas and individual bio-

material particles, line scans were carried

out, as shown in an example in Fig. 4. The

analysis indicated that this individual par-

ticle contained both CaS and CaP with

CaS more concentrated at one end and CaP

at the other with a mixture of the two in

the central region. Line scans were per-

formed on particles in other sections and

in every case high levels of P occurred in

regions with low levels of S (and vice

versa), consistent with the replacement of

CaS with precipitated CaP as it gradually

dissolves.

Elemental maps

Elemental mapping, as illustrated in Figs 5

and 6, showed the infiltration of CaP into

regions of resorbing CaS. Figure 5 shows an

area of graft material consisting of a grey

granular area with an adjacent whiter and

denser ‘crust-like’ area. The smaller grains

of darker grey appearance were CaS while

the denser whiter material consisted

mainly of CaP. Figure 6 shows the analysis

of granular material consisting of areas of

large roughly spherical particles and smal-

ler granules: CaP was concentrated in the

larger particles and the smaller granules

were CaS. There were also a few very small

fragments of apparently pure CaS, indi-

cated by arrows in Fig. 6.

Discussion

CaS has been used very successfully for

augmentation of bone in the maxillary

sinus in preparation for insertion of dental

root implants. Radiographic and histologi-

cal evidence suggests that the material

gradually resorbs completely as it is slowly

replaced by bone, by a process of creeping

substitution. In animals, this process has

been shown to involve the formation of a

CaP layer, which forms on the surface of

the dissolving material. The purpose of this

investigation was to use EDS microanaly-

sis to observe whether the same phenom-

enon occurs in human bone. In humans, it

is clearly not possible to obtain samples of

bone at different time points during heal-

ing; it is only ethical to retrieve bone at the

time of second surgery to place the im-

plants. However, because resorption is a

gradual process, if implant placement and

bone retrieval is done early enough it may

be possible to observe and analyse the

bone-substitute graft material at different

stages of resorption. Previous studies on

human bone augmented with CaS 6

months after initial surgery indicated that

no CaS remained in the tissues at this time

Fig. 4. Line-scan showing relative concentrations of Ca, P and S along a line passing through bone (1) and a

graft biomaterial particle (areas 2 and 3): Ca (green); P (blue); S (red). For clarity the scan result is also shown

separately in the lower figure. Bone is distinguishable from graft material buy the presence of lacunae, whiter

appearance and the low level of sulphur. Area 2 of the biomaterial contains CaS and CaP. Area 3 of the same

particle consists mainly of CaP.

Fig. 5. Elemental maps showing relative concentra-

tions of Ca, P and S within resorbing graft material.

The upper bse image shows the material in relation

to surrounding bone (B). The lower figures show

corresponding elemental maps: Ca (green); P (blue);

S (red). Most of the material in this area is CaP,

consisting of an outer denser brighter "crust-like"

layer (below, right) and a grey more granular region

(above). S was detected throughout the area in low

concentrations, with a higher level along a "seam"

(arrowed) between the granular and denser CaP

regions.



although there were small fragments

of biomaterial present, as shown by

polarized light and fluorescence micro-

scopy (Orsini et al. 2004; Traini et al.

2008). The present study was a retrospec-

tive investigation of bone biopsies

from patients retrieved 4 months after in-

itial surgery, in which residual biomaterial

had been previously identified by light

microscopy.

The results of this study indicated that

the residual biomaterial consisted of a mix-

ture of CaS and CaP. Because it had been

confirmed that the graft material consisted

only of CaS, any phosphate present in the

biomaterial must have originated from host

bone or body fluids. The results obtained

are consistent with particle resorption from

the outside inwards (Ricci et al. 2000;

Orsini et al. 2004; Atilgan et al. 2007)

with the formation of a CaP layer at the

surface of dissolving CaS, as has been

shown to occur in animals and can be

demonstrated to occur in vitro in simulated

body fluid (Ricci et al. 2000; Mamidwar

et al. 2007). The formation of a CaP layer

also occurs on other calcium salts used as

graft materials, for example calcium

carbonate (Damien et al. 1994). CaP was

also present as small granules, which may

Fig. 6. Elemental map showing the distribution of Ca, P and S in a granular area of resorbing graft material. The upper bse image shows the material in relation to adjacent bone

(B); the lower figures show a magnified bse image of the area within the box (size bar ¼ 100 mm) and corresponding elemental maps: Ca (green); P (blue); S (red). S was mainly

detected in the smaller granules or very small, apparently pure CaS fragments, indicated by arrow. The larger, brighter, roughly spherical particles consisted of CaP.



have originated from disintegrating larger

particles or a surface layer or be formed by

spontaneous precipitation from saturated

body fluids. Biomaterial C would appear

to be the final stage of the replacement

process, as suggested by its more consistent

composition and its Ca/P ratio, which was

consistent with calcium-deficient hydro-

xyapatite, possibly substituted with

sodium, carbonate and magnesium ions.

The formation of a CaP layer on the

surface of CaS may be the key to its

biocompatibility because the mechanism

of bone-bonding to CaP graft materials

such as hydroxyapatite and other bioactive

materials is believed to involve precipita-

tion of calcium and phosphate ions at the

bone-biomaterial interface with the forma-

tion of carbonate apatite, forming a bridge

to the host bone (Ducheyne et al. 1992;

Kokubo et al. 1992). In addition, high

levels of Ca2þ may stimulate osteogenesis

by their effects on osteoblast gene expres-

sion (Lazary et al. 2007).

The fate of the newly formed CaP parti-

cles is not known although all the histolo-

gical evidence suggests that they are

resorbed or incorporated into bone where

they may undergo further remodelling. As

mentioned above, histological studies of

bone retrieved from human patients under-

going sinus lift procedures, 6 months after

initial surgery revealed residual biomaterial

particles adjacent to woven or mature bone

with no signs of inflammatory infiltrate

or soft tissue at the interface (Traini et al.

2008). In that publication the residual

biomaterial was referred to as CaS;

however, subsequent EDS analysis of

one of same specimens has shown that a

particle identified on the basis of polarized

light microscopy as biomaterial, was

actually CaP. The continued presence of

CaP particles after the CaS has disappeared

may be advantageous because they

maintain space and may encourage angio-

genesis.

The presence of relatively large amounts

of biomaterial in these samples after 4

months is perhaps surprising since previous

studies both in animals and humans have

reported faster resorption rates and almost

complete disappearance of the biomaterial

at earlier or comparable times. However,

many reports are based on radiographic

evidence which may not reveal residual

granules especially if they are disintegrat-

ing into smaller particles and have trans-

formed to CaP and are thus very similar in

composition to bone. Histological findings

may reveal residual particles not seen in

radiographs, as in a study of CaS resorption

in a medullary defect in canines after 13

weeks (Turner et al. 2003). Variation in

individual rates of resorption can be seen

even with a small number of patients, as in

this study, in which histological examina-

tion of specimens from three of the 10

patients did not show any residual parti-

cles. Further studies are necessary to in-

vestigate what factors may be responsible

for the variation. These could include

location of particles within the graft site

and the degree of compaction, individual

patient sinus anatomy and healing

capacity.

In conclusion, this study has clearly

shown the presence of CaP as well as

CaS in human bone tissue augmented by

CaS in the maxillary sinus and the

results suggest that as CaS dissolves it is

replaced by precipitated CaP, as has been

previously shown to occur in animals.

The formation of a CaP layer may

be an essential intermediate step in bone

bonding and contribute to the excellent

biocompatibility of this bone graft substi-

tute material.

Acknowledgements: We thank Paul

Stanley of the University of

Birmingham Centre for Electron

Microscopy for assistance with SEM and

elemental analysis.

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