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Morphology of the osteonal cement line in human bone.

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THE ANATOMICAL RECORD 217:223-228 (1987)
Morphology of the Osteonal Cement Line
in Human Bone
MITCHELL B. SCHAFFLER, DAVID B. BURR, AND RICHARD G . FREDERICKSON
Department of Anatomy, (M.B.S., D.B.B., R. G.F) and Orthopedic Research Laboratory
(M.B.S., D.B.B.), West Virginia University Medical Center, Morgantown, WV 26506
ABSTRACT
While current consensus suggests the absence of collagen in osteonal cement lines, the extent of cement line mineralization and the nature of the
ground substance within the cement line are unclear. Samples of human radius
were examined by using scanning electron microscopy, electron microprobe, and
histochemical techniques. X-ray intensities were used to compare the amount of
calcium, phosphorus, and sulfur in cement lines with amounts in surrounding
lamellar bone. The results indicate that cement lines contain significantly less
calcium and phosphorus, but significantly more sulfur, than surrounding bone
matrix. The Ca/P ratio of cement lines was significantly greater than that of
lamellar bone, suggesting that the mineral in cement lines may not be in the form
of mature hydroxyapatite. No selective staining of the cement lines could be demonstrated by using periodic acid-Schiff, Sudan black B, or alcian blue critical electrolyte concentration techniques.
Numerous functional and biomechanical theories and
observations attach considerable functional importance
to the cement lines which separate secondary osteons,
or Haversian systems, from the surrounding bone matrix. They are variously implicated in fracture processes,
energy absorption, viscous damping and elastic function, and fatigue processes in compact bone (Piekarski,
1970; Carter and Hayes, 1977; Lakes and Saha, 1979;
Gottesman and Hashin, 1980; Katz, 1981; Martin and
Burr, 1982; Burr et al., 1985). In short, there is no scarcity of ideas regarding cement line function. There are,
however, inadequate morphological bases to either explain experimental observations of cement line action or
to allow the evaluation of various theories of cement
line function.
While it has been suggested that cement lines are
optical artefacts resulting from the juncture of the differently oriented components of osteonal and extraosteonal
bone (Schmidt, 19591, these thin refractile structures are
generally recognized as morphologically distinct entities
since they were so described by von Ebner (1875; also
see Sokoloff, 1973). However, the compositional correlates of this morphological distinctiveness, compared to
the organic and mineral components of surrounding bone
matrix, are unclear. Light microscopic methods, such as
collagen specific stains and silver impregnation techniques, indicate that cement lines are collagen deficient
(Weidenreich, 1930; Sokoloff, 1973); scanning electron
microscopic study finds similarly (Frasca, 1981). Alternatively, collagen X-ray diffraction patterns have been
shown in cement lines (Philipson, 1965). Investigations
into, the nature of noncollagenous organic matrix, or
ground substance, in cement lines yield no consensus as
to which possible organic components are contained
within (Sokoloff, 1973; Frasca, 1981). Similarly, it is
unclear whether cement lines are more calcified than
surrounding bone (Philipson, 1965; Frasca, 198l), less
0 1987 ALAN R. LISS, INC.
calcified than surrounding bone (Fawns and Landells,
1953), or not different from surrounding bone in this
regard (Mellors, 1964). It is also not known whether the
mineral in cement lines differs qualitatively from the
mineral contained in lamellar bone (i.e.,hydroxyapatite).
The purpose of this investigation is to examine the
osteon cement line by using scanning electron microscopy, X-ray microprobe, and histochemistry in order to
address two principal areas of morphological ambiguity:
1)the mineralization of the cement line relative to surrounding bone matrix and 2) the presence and characteristics of cement line noncollagenous organic matrix
components.
MATERIALS AND METHODS
Diaphyseal blocks were cut from six fresh human radii
obtained at autopsy. Four bones were from males (ages
38, 57, 58, and 81 years) and the others were from
females (ages 62 and 81 years); no history of metabolic
or bone disease was noted. Undecalcified cross sections
(150-200 pm thick) were cut with an Isomet metallurgical saw (Buehler Instruments, Evanston, E)and hand
ground and polished, and each section was then cut into
segeral smaller pieces. Specimens were then dried at
60 C, mounted on copper trays by using double-sided
tape, sputter coated with a thin layer of carbon, and
viewed in a JEOL 1OOCX TEMSCAN electron microscope. Specimens were imaged topographically by using
the secondary electron image (SEMI and compositionally by using the back-scattered electron image @SE).
Received July 28, 1986; accepted October 22,1986.
M.B. Schaffler’s current address is the Division of Radiobiology,
University of Utah School of Medicine, Salt Lake City, UT 84112.
Richard G. Frederickson’s current address is the Veterans Administration Hospital, Seattle, WA 98108.
224
M.B. SCHAFFLER, D.B. BURR, AND R.G. FREDERICKSON
X-ray microanalysis was undertaken on the same sections employed for electron microscopy; the analyses
were performed by using a Tracor-Northern X-ray spectrophotometer with a 30-mm2energy-dispersive detector
(model TN-2000, Middleton, WI) installed on the JEOL
lOOCX unit. Continuum suppression was achieved by
digital filtering through the analytic system. Sampling
for the microprobe study consisted of three sites for each
of 32 randomly selected complete secondary osteons; the
sample sites were the cement line and areas of interstitial and osteonal lamellae 25 pm on either side of the
cement line sample site. The electron beam was positioned over a sample by viewing the BSE image at
~ 4 , 0 0 0magnification. A 1-minute sampling period was
used to obtain X-ray spectra; the accelerating voltage
was 60 kV. Sampling was confined to either the cement
line or the sample lamellae, as these structures are a t
least 1 pm or greater in width. Relative amounts of
calcium and phosphorus among three sample sites were
compared from X-ray intensities by using correlated ttests. Quantification of these intensities was achieved
by using a variation of the internal standard ratio technique (Dorge et al., 1978) as a ZAF correction was not
available for data reduction. Characteristic X-ray intensities obtained for calcium and phosphorus a t cement
lines and osteonal lamellae were compared directly to
the intensities for interstitial bone, defined as the internal standard for a sample group, from the corresponding
sample area. The internal standard method of normalization assumes that interstitial bone is fully mineralized, mature hydroxyapatite [3Ca3 (PO& * Ca(OH)2];
this assumption is well supported in the literature (Amprino and Engstrom, 1952, Wergedal and Baylink, 1974;
Landis and Glimcher, 1978; Glimcher, 1984). Comparison of Ca/P molar ratios was performed by using data
normalized by the internal standard; correlated t-tests
were used.
Specimen portions not used for electron microscopyKray omicroprobe analysis were frozen and stored at
-80 C . Specimens were subsequently thawed and fixed
in formol-cetylpyridinium chloride (4-8 hours). Undecalcified specimens were cut as described earlier. Another
set of fixed specimens was completely decalcified in 5%
EDTA and paraffin embedded. Sudan black B was used
to determine whether lipid is present in cement lines
(Pearse, 1980). Periodic acid-Schiff reaction was used to
assess the presence of glycoproteins and covalently
bound sugars in cement lines (Pearse, 1980). The alcian
blue critical electrolyte concentration (CEC) technique,
which uses different molar concentrations of MgC12 to
differentially stain glycosaminoglycans, was employed
to examine cement lines as well (Scott et al., 1964; Scott
and Dorling, 1965).
RESULTS
Scanning Electron Microscopy (SEM)
a dark band between osteonal and interstitial lamellae
(Fig. 2). Because backscattered electrons reflect the mass
density of a region, the dark-band appearance of cement
lines indicates that they have less mass and are compositionally different from surrounding bone. This difference may indicate that cement lines contain less calcified
material than the surrounding bone matrix, that the
materials contained within are composed of elements of
lower atomic number, or some combination of both
possibilities.
Electron Microprobe Analysis
Relative intensities for calcium and phosphorus (Fig.
3) indicate that cement lines are less mineralized than
surrounding bone matrix. Cement line calcium and
phosphorus intensities were significantly lower than
those in either interstitial lamellae (mean differences:
Ca= 10%, P= 18%; P < .01) or osteonal lamellae (mean
differences Ca = 6%,P= 12%;P < .01).While the amount
of sulfur in bone matrix is typically small and the current data support this, elevated sulfur peaks (12-15%
greater intensity than surrounding bone) were found in
cement lines. It should be noted that sulfur, as examined
in cells with the electron microprobe, tends to evaporate
or burn off rapidly (Andrews et al., 1983). As no attempt
was made to prevent this, such as using a cooled specimen stage, the actual sulfur differences observed may
underestimate systematically the amount of sulfur present. Because no standard was used for sulfur, higher
sulfur intensities at cement lines can only be interpreted
qualitatively relative to surrounding bone at a sample
site to indicate that sulfur-containing moieties are contained within cement lines.
Osteonal lamellae were found to be less highly mineralized than extraosteonal bone, which is consistent with
results of previous studies (Amprino and Engstrom,
1952; Bonucci et al., 1970). Osteonal and interstitial
bone, however, did not differ with regard to sulfur.
Quantitative comparison is based on molar proportions of calcium and phosphorus in hydroxyapatite;
mean normalized number of moles for each of these
elements at cement lines and osteonal and interstitial
lamellae are shown in Table 1. Calcium-phosphorus molar ratios (CdP), calculated from these normalized data,
are shown in Figure 4. Ca/P molar ratio in cement lines
(mean= 1.84) is significantly greater than in interstitial
or osteonal lamellae (P<.OOl). Ca/P molar ratios in osteonal and interstitial bone do not differ significantly
(means= 1.69 vs. 1.67, respectively).
Histochemistry
Examination of bone sections with periodic acid-Schiff
(PAS) reaction did not reveal any staining in cement
lines, which suggests that little, if any, glycoprotein is
contained within cement lines. Similarly, Sudan black
B did not reveal any lipid in cement lines. Studies of
cement lines with the alcian blue CEC technique did
not show selective intensification of staining at cement
lines, although some diffuse staining of the cement lines
and the entire bone matrix was observed in the chondroitin sulfate and keratan sulfate ranges.
SEM indicated that cement lines are topographically
indistinct from surrounding bone (Fig. 1).These topographic views reveal no separation of osteons from the
surrounding bone matrix, a potential artefact of drying
the specimens for electron microscopic examination.
Images generated by using BSE, which are produced
DISCUSSION
by interactions of incident electrons with the various
elemental masses within the specimen, revealed a dis- When intracortical bone resorption and refilling occur,
tinct morphology for cement lines; these interfwes of the resulting secondary osteon, or Haversian system, is
osteons with surrounding bone appeared consistently as separated from the the surrounding bone matrix by an
OSTEONAL CEMENT LINE COMPOSITION
225
Fig. 1. The secondary electron image revealed that the cement line (CL) is topographically
indistinct from surrounding lamellar bone. X 3,000 original magnification.
interface structure known as the “cement line.” While
standard histological descriptions of osteonal bone indicate that cement lines are characteristic features of this
type of tissue, the question of whether they are optical
artefacts resulting from the juncture of osteonal and
extraosteonal lamellae has been raised (Schmidt, 1959).
Nevertheless, the prevailing opinion in the literature is
that these thin, refractile bands, several micrometers
wide, are real and unique structural entitites (von Ebner, 1875; Weidenreich, 1930;Fawns and Landells, 1953;
Philipson, 1965; Sokoloff, 1973; Frasca, 1981). However,
the nature of cement line composition is controversial.
Bone is composed principally of collagen fibers, calcium phosphate salts primarily in the form of hydroxyapatite, and small amounts of glycoprotein and
proteoglycan collectively termed ground substance and
water. The extent to which each of these components
appears in lamellar bone is well documented (Howland
et al., 1926; Easthoe and Easthoe, 1954; Woodward, 1964;
Robinson, 1975). The extent to which these components
appear in cement lines is unclear. Weidenreich (1930)
and Sokoloff (1973) indicate that stains for collagen fail
to reveal its presence in cement lines; silver impregnation techniques, which are specificfor collagen and reticular fibers, are selectively rejected by cement lines but
not by the surrounding bone matrix. Scanning electron
microscopy studies of mechanically disrupted cement
lines similarly do not show collagen fibers in cement
lines (Frasca, 1981). One study stands in contradistinction to these other studies; Philipson (1965) used X-ray
diffraction t o study orangutan and whale bone samples
and obtained characteristic collagen diffraction patterns
in cement lines. However, inherent methodologicalfaults
and inconsistencies within that study (see Sokoloff, 1973
for discussion) have led to the belief that Philipson’s
data for cement line collagen are incorrect. Consequently, consensus is that cement lines are collagen
deficient.
The current histochemical studies reveal no PAS
staining at cement lines, suggesting that little or no
glycoprotein is contained within. Because glycoproteins
in connective tissue are typically intimately associated
with collagen, the lack of PAS staining also implies that
cement lines are collagen deficient. In addition, no lipid
was found in cement lines. These data are consistent
with earlier characterizations of cement line constituents (Weidenreich, 1930; Sokoloff, 1973; Frasca, 1981).
Previous studies have not examined whether glycosaminoglycans are present in cement lines. The current
studies with alcian blue failed to reveal any differential
staining between the cement lines and the bone matrix,
suggesting that sulfated glycosaminoglycans are no
226
M.B. SCHAFFLER, D.B. BURR, AND R.G. FREDERICKSON
Fig. 2. The backscattered electron image indicated that the cement line (CL) is a region of
low mass density which is compositionally distinct from adjacent lamellar bone. x 1,000 original
magnification.
I_
Sullur
C a l ciuiii
400.000
inlerstitial
Lamellae
1,600
70.000
1.400
65,000
Lo
m
r
c
*
=
13
L
350.001
60,000
55.000
300.001
50.00
Fig. 3.The relative X-ray intensities for calcium and phosphorus indicate that cement lines
contain 510% less calcium and 10-16% less phosphorus than adjacent bone matrix ( P < .001).
Cement lines contain about 20% more sulfur than adjacent bone ( P < .001).
227
OSTEONAL CEMENT LINE COMPOSITION
more prevalent in cement lines than in the lamellar
bone matrix. However, the microprobe data consistently
indicated that higher sulfur levels are present in cement
lines, and the most likely location for large amounts of
sulfur in bone is in sulfated glycosaminoglycan. Baylink
et al. (1972), using electron microprobe techniques, demonstrated high sulfur levels in osteoid and also showed
that the amount of sulfur present varied directly with
the amount of chondroitin sulfate present in the tissue.
Accordingly, while the histochemical analyses were unable to demonstrate glycosaminoglycans, the sulfur
found in cement lines suggests that these complexes
may be present.
Most equivocal is whether cement lines are more, less,
or similarly mineralized than surrounding bone. Fawns
and Landells (1953)were unable to demonstrate calcific
materials in cement lines with the aid of either alizarin
red or von Kossa stain. Philipson (1965) used X-ray absorption and found that mineralization of cement lines
was comparable to heavily mineralized osteons. Similarly, Frasca (1981) indicated that cement lines are more
highly mineralized than surrounding bone. Mellors
(1964) found that mineralization of cement lines does
not differ from surrounding bone. Cement lines are characteristically radiolucent when seen in microradiographs of cortical bone; this has been generally
a..
a
:.:.:.:
I .90
Interstitial lamellae
(Internal Standard)
Cement Line
Osteonal
Lamellae
I .8
Ca/P
I .7
I .6
Fig. 4. The C a R ratio in cement lines was significantly greater than
that in surrounding lamellar bone (P < .001),suggesting that cement
lines do not contain mature hydroxyapatite.
interpreted as indicating a lower relative mineralization
for cement lines. However, Sokoloff (1973) pointed out
that such radiolucency could also be a n artefact due to
specimen shrinkage resulting in cement line separation
during the preparation of undecalcified bone sections. In
the current study, SEM indicated that shrinkage separation a t cement lines did not occur. BSE images, like
typical microradiographs, showed that cement lines are
regions of lower mass density than either osteonal or
interstitial bone. The X-ray microprobe data indicated
that this relative lower mass density corresponds to both
less calcium and less phosphorus in cement lines than
in surrounding bone.
From the empirical formula for hydroxyapatite, the
Ca/P molar ratio can be calculated. Experimentally derived Ca/P molar ratios for bone approximate this theoretical value well. Strandh (1960) used microchemical
analyses and found a Ca/P molar ratio of 1.71for human
bone. Mellors (1964) determined Ca/P molar ratio in
human cortical bone by using electron microprobe; his
data show a mean ratio of 1.70. Wergedal and Baylink
(1974) employed electron microprobe techniques to examine mineralization in developing chick bones; a t the
completion of mineralization Ca/P molar ratio closely
approximated the expected values for hydroxyapatite.
Accordingly, the high degree of concordance between
theoretically and experimentally determined calciumphosphorus molar ratios supports the use of the internal
standard (interstitial bone) normalization used here.
Because interstitial bone was assumed for this study
to have a C a / p molar ratio of 1.67, comparison among
interstitial bone sample sites cannot be performed. However, comparison among interstitial bone, osteonal bone,
and cement line ratios are possible. The mean Ca/p
molar ratio of osteonal lamellae in this study was
1.69+0.02, which is essentially identical to that expected for hydroxyapatite and to previous determinations in mature bone (Strandh, 1960; Woodward, 1964;
Mellors, 1964; Wergedal and Baylink, 1974; Glimcher,
1984).
In the current study, cement line Ca/P molar ratio was
significantly higher (1.84 Ifr 0.03) than in any surrounding area of bone. This higher ratio primarily represents
the influence of the relatively decreased amounts of
phosphorus observed in cement lines. Phosphorus emits
“soft”or low-energy X-rays under X-ray excitation; these
X-rays are likely to be attenuated by the matrix through
which they must pass. It is possible that the different
composition of the cement line matrix, evidenced by the
relatively increased presence of sulfur, absorbs more
TABLE 1. Elemental analysis of cement line composition in bone
Cement line
Ca
P
Intensity
(cpm)
Relative
intensity
361,048
(+20,532)
90.2
(k0.8)
52,431
(+3,411)
80.2
(k1.7)
Osteonal lamellae
Ca
P
405,896
(+20,413)
95.2
(kl.1)
59,217
(+3,763)
95.0
(k1.7)
Interstitial lamellae
Ca
P
416,264
(f21,505)
100
62,851
(+3,738)
100
(-1
(-1
(%)
Relative no.
moles
CafP molar
ratio
9.02
(k0.08)
4.81
(kO.10)
1.84
(k0.03)
9.52
(kO.11)
5.70
(+0.10)
1.69
(k0.02)
10
6
(-1
(-1
1.67
(-1
228
M.B. SCHAFFLER. D.B. BURR, AND R.G. FREDERICKSON
phosphorus-emitted X-rays than the surrounding bone,
and consequently the intensity of phosphorus peaks detected for cement lines would appear relatively decreased.
Alternatively, the relatively increased CaiP molar ratio in cement lines may suggest a real difference in their
mineralization relative to lamellar bone. Pellegrino and
Biltz (1968) showed a linear relationship between increasing Ca/P molar ratio and increasing amounts of
carbonate in bone mineral. Hohling et al. (1970), using
electron microprobe, demonstrated higher CaiP molar
ratios than expected for hydroxyapatite in initial mineralization deposits in embryonic chick bones. In a similar developmental model, an inverse relationship was
shown between acid phosphate and carbonate concentrations (Pellegrino and Biltz, 1972). The presence of carbonate rather than phosphate in bone mineral would
result in an increased calcium-phosphorus molar ratio
and is a likely explanation for the high Ca/P molar ratio
seen in cement lines. This cannot be assessed, however,
within the current detection limits of electron microprobe analysis. Similarly, in order to determine the influence of cement line matrix absorption on X-rays
emitted from phosphorus, a ZAF correction would have
to be used.
Data from the current investigation indicate that cement lines are compositionally distinct from lamellar
bone. They are both less highly mineralized and differently mineralized than the surrounding bone matrix.
Taken together with the higher sulfur levels present in
cement lines, these data suggest that cement lines may
represent a residuum of mineralized “ground substance” from the initial reversal phase of formation of a
secondary osteon. These data also suggest that cement
lines may be more mechanically compliant than surrounding bone and may also help explain some of the
biomechanical functions attributed to cement lines.
ACKNOWLEDGMENTS
This work was supported by NM Biomedical Research
Grant SO7 RR0443-18 and NM grant AM27127. The
authors wish to thank Dr. Gary Kirk for his helpful
discussion, comments, and criticism on the manuscript,
and Professor John Scott for supplying the alcian blue.
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