SPINE Volume 35, Number 8, pp 881 886

2010, Lippincott Williams & Wilkins

Frequency of Lumbar Intervertebral Disc Calcification

and Angiogenesis, and Their Correlation With Clinical,
Surgical, and Magnetic Resonance Imaging Findings
Saeid Karamouzian, MD,* Hossein Eskandary, MD,* Mohcen Faramarzee, MD,
Mohammad Saba, MD, Hossein Safizade, MD, MPH,! Masoud Ghadipasha, MD,**
Afshar Reza Malekpoor, MD,* and Amin Ohadi, MD**

Study Design. This study measured the frequency of

lumbar intervertebral disc nucleus pulposus microscopic
calcification and angiogenesis in adult patients undergoing discectomy compared to normal cadavers.
Objective. The results were compared to determine
the relationship between disc microscopic calcification
with disc degeneration type, histopathological angiogenesis, patients age, gender, and duration of symptoms.
Summary of Background Data. True frequency of microscopic calcification in normal or degenerated lumbar
discs have not been fully defined nor linked to disc degeneration type and angiogenesis. Some studies demonstrated that angiogenesis and calcification are related to
each other in several another tissues.
Methods. The frequency of microscopic calcification in
specimens of disc nucleus pulpous obtained from 2
groups were measured: specimens were obtained during
surgery from 90 consecutive patients of 15 to 50 years old
suffering from disc herniation in single level of L4 L5 or
L5S1 between 2005 and 2006, 60 additional specimens of
lumbar disc nucleus pulposus were obtained from normal
cadavers of the same ages. Calcification was determined
microscopically by Von Kossa staining and angiogenesis
by H/E, and type of degeneration radiologically by Modic
classification.
Results. Frequency of microscopic calcification was
significantly higher in degenerated disc than normal cadaveric (54.4% vs. 6.7%) and was higher in Modic type III
than type I (III: 95.0%, II: 57.4%, I: 13.0%), also prevalence
of angiogenesis was significantly higher in patients than
cadaveric discs (41.0% vs. 6.7%) and in calcified than
noncalcified discs (59.2% vs. 19.5%) (P ! 0.001). There
was no relationship between disc calcification and patients gender and level of discectomy.
Conclusion. Disc nucleus pulposus microscopic calcification is a common event occurring in adult patients
suffering from lumbar disc herniation. Mechanisms that
From the *Neuroscience Research Center, Kerman, Iran; Department
of Neurosurgery; Kerman University of Medical Sciences, Kerman,
Iran; Afzal Research Institute (NGO), Kerman, Iran; Department of
Neurosurgery; Bahonar Hospital, Kerman University of Medical Sciences, Kerman, Iran; Department of Radiology, Kerman University of
Medical Sciences, Kerman, Iran; !Department of Community Medicine, Kerman Medical UniversityMedicine School, Kerman, Iran;
and **Legal Medicine Organization, Tehran, Iran.
Acknowledgment date: November 4, 2008. Revision date: May 31,
2009. Acceptance date: June 1, 2009.
The manuscript submitted does not contain information about medical
device(s)/drug(s).
No funds were received in support of this work. No benefits in any
form have been or will be received from a commercial party related
directly or indirectly to the subject of this manuscript.
Address correspondence and reprint requests to Hossein Eskandary,
MD, Afzal Research Institute, Samenol Hojaj, Khorshid St., Kerman,
Iran; E-mail: h_eskandary@yahoo.com

link disc degeneration, angiogenesis, and calcification remain a focus for further researches that may be useful in
future medical treatments before surgical treatment of
lumbar disc herniation.
Key words: herniated disc, pathology, angiogenesis,
degeneration, calcification, nucleus pulposus. Spine 2010;
35:881 886

Intervertebral disc herniation (HD) is one of the most

common causes of low back pain and/or sciatica which
results in disability among working age group in developed countries.1
The intervertebral disc, which consist of an outer fibrous anulus fibrosus (AF) surrounding an inner gelatinous nucleus pulposus (NP), is largely composed
of a cartilage component that consists of the proteoglycan aggrecan, and type II collagen.2 Herniated disc is
defined as a degenerated disc protruding into the spinal
canal or neural foramens with resultant compression of
nerve root.2
Histologically, herniated nucleus pulposus is surrounded by granulation tissues characterized by inflammatory cell infiltration and new formed vessels.3,4 It appears that both fibrotic and angiogenic reactions take
place in disc herniation.5 In several studies, radiographic
intradiscal calcification correlated significantly with disc
degeneration.6 Baron was the first to report a case of disc
calcification in a child in 1924.7 Von Lushka was the first to
describe intervertebral disc calcification in adults.8 Calcification in an intervertebral disc may occur in 3 places: anulus fibrosus, fibrocartilaginous plate, and centrally in the
nucleus pulposus. Calcification of nucleus pulposus consists of a deposit of amorphous calcium salts in degenerated
tissue and is usually seen in adults, more commonly in lumbar spine.9 In children, calcification within the intervertebral disc may resolve spontaneously. In adults, calcification
of anulus fibrosus is almost always asymptomatic and may
persist permanently.7,10,11 Disc calcification may have important implications. In one study, radiographic intradiscal calcification correlated significantly with the morphologic degree of degeneration.6 Other studies showed
that the calcium containing crystal deposits could disrupt
the discs extra cellular matrix,12 and these deposits were
likely to have dural adherence.13,14
The true prevalence of intervertebral disc calcification
is unknown.7 The reported prevalence of disc calcifica881

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Spine Volume 35 Number 8 2010

tion varies in different studies. It is reported as rare,7

3.1%,15 14.7% (microscopic in anulus),12 63% (microscopic),16 and 65% (radiologic).13 Previous studies
showed that factors such as TGF-, TNF-!, VEGF,
MMP-1, MMP-3 are associated with degeneration and
angiogenesis in disc tissue.2,5,17 On the other hand, other
studies showed that the same factors are associated with
ectopic calcification in several other tissues such as vessel, muscle,18 bone,19 and ligamentum flavum.20 The
purpose of this study was (1) to evaluate the prevalence
of microscopic calcification in normal cadaveric disc and
degenerated herniated disc, and to determine its relation
with the type of disc degeneration; and (2) to evaluate the
prevalence of disc angiogenesis and its probable relationship to disc calcification and the degeneration type.
Materials and Methods
Study Design
The study has an observational design with a case-control
component.

Study Participants
Cases were selected from consecutive patients who had an operation for lumbar disc herniation during the years 2005 and
2006 in the authors services. Subjects were included if they
were 15 to 50 years old, if they had only one level of disc
herniation (L4 L5 or L5S1), and if this was their first operation for disc herniation. The control group was obtained from
cadavers that were examined in the forensic medicine center of
Kerman. Cadavers were included if they belonged to a 15- to
50-year-old individual. They were excluded if there was a history of DH, lumbar canal stenosis, known causes of disc calcification, and systemic illness in the individuals. Only one disc
per patient or cadaver was examined.

Sampling
Samples of nucleus pulposus were obtained from either L4 L5
or L5S1 discs in the cases at the time of operation and from
L5S1 disc of the control cadavers. Care was taken to remove
all the granulation tissues and to sample only the nucleus pulposus. Specimens were placed in 10% neutral buffered formalin, then encoded and transported to laboratory for histologic
study.

Histopathologic Study
All samples were embedded in paraffin, sectioned by microtome, and stained with hematoxylin/eosin (H/E) for angiogenesis, and Von Kossa, for calcification. Two certified pathologists who were blinded to the study subjects examined the
samples and reported on any existing microscopic calcification
and microscopic angiogenesis.

Radiologic Study
All the cases underwent a lumbosacral MRI (GE 1.5 T unit,
General Electric Medical Systems, Signa, Exita) before the operation. A certified radiologist studied all the films on a blinded
fashion and reported on the type of disc degeneration. The
Modic classification21 was used to describe the degeneration
type.

Disc Consistency
All the specimens were examined by a single expert surgeon.
Disc consistency was classified as soft if disc material was

Table 1. Basic Characteristics of Cases and Controls

Case

Control

Number
90
60
Age (yr)
38.4 (SD, #8.59; range, 1750) 33.5 (SD, #9.17; range, 1950)
Male (n, %)
59 (65.6%)
41 (68.3%)
L4L5 HD
60 (66.6%)
20 (33.3%)
(n, %)
L5-S1 HD
30 (33.3%)
40 (66.6%)
(n, %)
Microscopic
49 (54.4%)
4 (6.7%)
calcification
Microscopic
37 (41%)
4 (6.7%)
angiogenesis

soft and easily removable, firm if the material was hard and
not easily removable, and soft to firm if the material had
intermediate consistency.

Disc Content
Disc content was defined as empty if the space did not contain significant nucleus pulposus, loaded if the space contained significant nucleus pulposus material that should be
evacuated by piece meal, and fragmented when the main
part of the nucleus pulposus detached itself from the rest of the
disc and was removed as a large fragment.

Statistical Analysis

SPSS version 11.5 was used to analyze the data. "2 test was used
to compare the differences in proportions and t test was used to
compare the means for continuous data. A P value less than
0.05 was considered statistically significant.

Results
About 90 cases and 40 controls were identified. Table 1
shows basic characteristics of the study subjects (Table 1).
Frequency of microscopic calcification in the disc
samples of the cases was significantly higher than controls, 54.4% versus 7.5% ("2 " 25.43, df " 1, P !
0.001). Microscopic angiogenesis was also more prevalent in cases than matched controls (40 cases), 41% versus 7.5% ("2 " 14.69, df " 1, P ! 0.001).
Table 2 shows the frequency of disc calcification and
angiogenesis in all the cases, categorized based on the disc
content and consistency. The presence of calcification was
significantly higher in samples with soft or firm consistency compared to soft to firm ("2 " 18.65, df " 2, P !
0.0001). The frequency of calcification was significantly
higher in the loaded discs compared to the other types of
disc content. ("2 " 7.05, df " 2, P ! 0.05). Neovascularization was also significantly more frequent in the samples
with soft or firm consistency compared to soft to firm
("2 " 18.65, df " 2, P ! 0.0001). It was more frequent in
the loaded discs compared to the other types of disc content
("2 " 7.05, df " 2, P ! 0.05).
The cases with disc calcification were significantly
older than cases without disc calcification (P " 0.002)
(Figure 1). Gender and level of degenerated disc was not
different between the cases with disc calcification and
those without.

Calcification of Degenerated Lumbar Disc Karamouzian et al 883

Table 2. Number of Disc Calcification and Angiogenesis in Different Categories of Disc Content, Disc Consistency,
and Modic Types

Total
Content
Loaded
Empty
Fragmented
P
Consistency
Soft
Soft to firm
Firm
P
Modic classification
Type 1
Type 2
Type 3
P

Cases
N

Calcification
N (%)

No. Calcification
N (%)

Angiogenesis
N (%)

No. Angiogenesis
N (%)

90

49 (54.4)

41 (45.6)

37 (41.1)

53 (58.9)

51
28
11

22 (43.1)
18 (64.3)
9 (81.8)

29 (56.9)
10 (35.7)
2 (18.2)

21 (41.1)
10 (35.7)
6 (54.5)

30 (58.9)
18 (64.3)
5 (45.6)

25
48
17

19 (76)
16 (33.3)
14 (82.4)

6 (24)
32 (66.7)
3 (17.6)

9 (36)
16 (33.3)
12 (70.6)

23
47
20

3 (13)
27 (57.4)
19 (95)

20 (87)
20 (42.6)
1 (5)

3 (13)
18 (38.3)
16 (80)

P ! 0.05

P ! 0.0001

P ! 0.0001

Figure 2 shows the number of the cases with and without disc calcification for each category of radiologic subtype. In the patients whose samples lacked calcification,
frequency of Modic type 1 was significantly higher ("2 "
20.13, df " 2, P ! 0.001). As stated above, soft to firm
consistency was more frequent in these cases ("2 "
18.65, df " 2, P ! 0.001). In the same cases, disc space
was loaded with disc material ("2 " 7.65, df " 2, P !
0.05).
In the cases whose disc samples did not show angiogenesis, frequency of Modic 1 was significantly higher than the
other patients ("2 " 20.13, df " 2, P ! 0.001). Soft to firm
consistency was found more frequent in this group of patients ("2 " 7.57, df " 2, P ! 0.05). There was no difference

Figure 1. Age difference in cases

with and without disc calcification.

P ! 0.05

P ! 0.0001

P ! 0.0001

16 (64)
32 (66.7)
5 (29.4)
20 (87)
29 (61.7)
4 (20)

in gender, level of herniated disc and disc content between

the cases with and without disc angiogenesis.
There was a significant association between microscopic
calcification and histopathological angiogenesis. Frequency
of microscopic angiogenesis in the disc samples of the patients with calcification was 59.2% (29/49) and in those
without calcification was 19.5% (8/41) ("2 " 25.51, df "
1, P ! 0.001).
Figure 3 and 4 show samples of microscopic disc calcification and disc angiogenesis.
Discussion
Our results demonstrate that microscopic calcification of
lumbar intervertebral disc is significantly more prevalent

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Spine Volume 35 Number 8 2010

Figure 2. Number of cases with

and without disc calcification for
categories of radiological subtype.

in degenerated herniated discs than normal discs (54.4%

vs. 7.5%), especially in higher type of degeneration
(13% in Modic type I, 57.4% in Modic type II, and 95%
in Modic type III). In addition, microscopic angiogenesis
of lumbar intervertebral disc is more prevalent in calcified herniated disc specimens (59.2% in calcified and
19.5% in noncalcified herniated disc specimens). The
prevalence of microscopic calcification of the herniated
lumbar disc was not different among men and women.
This prevalence was not different between the two different levels of HD (L4 L5 or L5S1).
Previous reports suggest that disc calcification in
adults occur mainly in anulus and is usually asymptomatic.7,9 Our study demonstrates that nucleus pulposus
calcification is in fact a common event occurring in adult
patients suffering from herniated disc.
Long-term changes related to intervertebral disc calcification may include scoliosis, loss of vertebral body
height, osteophyte formation and disc space narrowing.7

Calcium containing crystal deposits not only disrupt disc

extra cellular matrix but may also accelerate preexisting
degenerative changes via an elevation in matrix metalloproteinase.12 Calcified discs are Likely to have dural adherence and it should be noted that the majority of intradural penetrations occur in calcified discs.13,14
The etiology of disc calcification remains uncertain.10
Some recognized causes of calcification in intervertebral
discs include hyperparathyroidism, hemochromatosis,
ochronosis, pseudogout, gout, hypercalcemic states, and
degenerative diseases.9,10,11 The relationship between
crystals and disc degeneration merits further investigations.12 Feinberg et al showed that hydroxyapatite
(HAP) was generally associated with histologic signs
of disc degeneration.22 Whether degeneration initiates
crystal deposition or vice versa still remains a focus of
debate.23
At birth, the human disc has some vascular supply
within both the cartilage endplates and the anulus fibro-

Figure 3. Examples for microscopic calcification in disc samples.

Micrographs showing various calcification shapes in nucleus pulposus specimens under light.

Figure 4. Examples for microscopic angiogenesis in disc samples. Micrographs showing angiogenesis of disc nucleus pulposus
(H & E $ 400 and 1000).

Calcification of Degenerated Lumbar Disc Karamouzian et al 885

Figure 5. The linkage between disc degeneration, disc calcification and disc angiogenesis.

sus, but these vessels soon recede.8 With increasing age,

water is lost from the matrix, the proteoglycan content
changes and diminishes, the cartilage and endplates undergo thinning with altered cell density, formation of
fissures, and sclerosis of the subchondral bone.8,24 Our
findings show that calcification is more frequent in an
aging disc.
Starting in the second decade of life, blood supply to
the endplate diminishes, resulting in the tissue breakdown which begins in the nucleus pulposus.25 Histologically the granulation tissue of HD is characterized by
marked infiltration of macrophages.4 The interaction of
macrophages with disc tissue leads to the production of
tumor necrosis factor ! (TNF-!) resulting in the up regulation of expression of MMP-3 and MMP-7 as well as
vascular endothelial growth factor (VEGF) that may potentially induce new vessel formation.2,4 Therefore, interactions between VEGF and MMPs may tend to promote matrix degradation and neovascularization in
HD.2 Other factors such as fibroblast growth factor,26
transforming growth factor (TGF- )26 and pleiotrophin may play a role in neovascularization of damaged
disc tissue.5,26,27 Inflammatory cytokines, VEGF and
MMPs can promote ectopic calcification in several tissues such as ligamentum flavum, vessels, muscles, and
bones.18 In patients with rheumatic heart disease and
atherosclerosis, VEGF expression and calcification were
observed in the same areas.28 In another experimental
study, exogenous VEGF was capable of increasing adventitial angiogenesis, shifting macrophage infiltration
and osteopontin expression, and promoting ectopic calcification.29 In ossification of the supraspinous ligament,
possibly fibroblast or chondrocyte like cells respond to
some external stimulus, form fine fibrils, and produce
acid mucopolysaccharide. Undifferentiated mesenchymal cell then invade and transform into osteoblast, starting osteogenesis with progressive calcification.30 Angiogenesis may regulate ectopic calcification in several ways.
First, many angiogenic factors are now known to exert
both direct and indirect effects on bone and cartilage
formation. Second, cytokines released by endothelial
cells can induce the differentiation of osteoprogenitor
cells. Third, the new blood vessels provide oxygen and
nutrients to support calcification. Finally, the new blood
vessels can serve as a conduit for osteoprogenitor cells.18
We suggest 2 main mechanisms for disc calcification.
First, direct mechanism; in which inflammatory cytokines, VEGF and MMPs released in degenerated disc
tissue directly promote osteopontin expression, induce

the differentiation of osteoprogenitor cells and ultimately lead to ectopic calcification. Second, indirect
mechanism; in which these factors promote angiogenesis, and angiogenesis itself, promotes macrophage infiltration, provides more oxygen and nutrients, conducts
new osteoprogenitor cells, and ultimately causes progressive calcification. These mechanisms are summarized
in Figure 5 as a triangle. Further researches may elucidate
further interactions between the angles. Understanding
of these interactions may be useful in future medical
management of lumbar disc herniation. For example if
angiogenesis is a defense mechanism, drugs such as Statins may be useful to promote it and otherwise it should
be stopped by other mechanisms.
A better understanding of the underlying mechanisms
that link disc degeneration, angiogenesis and calcification
should provide a basis for the development of therapeutic
strategies to treat or arrest abnormal pathologic conditions.
One example is the role of nanobacteria that has been reported in several calcification- related diseases.31,32 If this is
a true association, that the disc calcification may be the
result of nanobacteria or self-replicating calcifying nanoparticles, it will support the authors hypothesis of disc
degeneration.33
Key Points

Disc nucleus pulposus microscopic calcification

and angiogenesis are significantly higher in degenerated disc than control group.
The frequency of angiogenesis is higher in calcified than noncalcified disc.
Calcification in Modic type III is more than type I.

Acknowledgments
The authors thank Dr. Ima Alimoradi and Ehsan MehrabiKermani for their careful reading of the manuscript.
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