Cell and Tissue Banking 5: 231–238, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands. 231

-1

Experimental and clinical study of the demineralized bone allografts

manufactured in the tissue bank of CITO

M.V. Lekishvili1,*, A.I. Snetkov1, M.G. Vasiliev1, V.K. Il’ina1,

N.I. Tarasov2, E.D. Gorbunova3, A.S. Pankratov4, O. Yu. Barakina3,
N.S. Gavryushenko1 and S. Yu. Batrakov1
1
Central Institute of Traumatology and Orthopedics (CITO), Moscow, Russia; 2Municipal Pediatric Clinics
(MMPC) 13, Moscow, Russia; 3Municipal Pediatric Clinics (MMPC) 1, Moscow, Russia; 4Russian State
Medical University Moscow, Russia; *Author for correspondence(e-mail: leki@mail.ru; maxox@mail.ru;
phone: +7-095-4509981; fax: +7-095-1543139)

Received 8 March 2004; accepted in revised form 6 July 2004

Key words: Demineralized bone allografts, Irradiated allograft, Lyophilized, Tissue bank

Abstract

The technology for producing demineralized bone allografts (DBA) with definable degree of demineral-
ization and sterilization by high energy electron bean was developed in the tissue bank of Central Institute
of Traumatology and Orthopaedics (CITO). The authors consider the technology to be one of the ways of
producing demineralized bones. The results of the experiments show that time of demineralization process
as well as the absorption dose of radioactive high energy electron beam change substantially mechanical
toughness and osteoinductive properties of DBA. Mechanical properties of DBA were tested by the uni-
versal testing machine ‘Zwick 1464’. Quantitative assessment of DBA osteoinductive properties resulted
from the investigation of DBA samples in the culture of stromal precursor bone marrow cells and in the
culture of human skin fibroblasts. Cloning efficiency of fibroblasts was considered as indecies of prolifer-
ative potential of stromal bone marrow cells, i.e. osteogenic precursor cells. The growth of the cell mass
after definite time as well as the index of 3H-timidin marked cells within biological inductor were considered
as indecies of proliferative potential of skin fibroblasts. The obtained results showed, that inductive
properties of allografts improve when the degree of their demineralization increases whereas the dose of
high energy electron radiation decreases. Mechanical toughness of DBA deteriorates when both degree of
their demineralization and radiation doses increase. This emphasizes the importance of optimizing tech-
nological stages in DBA producing. Since 1998 DBA have been used in Russian clinics for bone plasty in
traumatology and orthopaedics, maxilla-facial surgery, ophthalmology, and neurosurgery. The resulting
analysis is based on case histories of 257 patients operated from March 1998 to July 2002. The majority of
patients were children and teenagers of 3–18 years old with prime tumors, tumor-like and systemic
inherited diseases of skeleton, post-traumatic complications. Observation periods were from 1.5 to 5 years
and 10 months. Good and satisfactory results were obtained in 93.4% cases.

Abbreviations: DBA – demineralized bone allografts; CITO – Central Institute of Traumatology and
Orthopaedics, Moscow, Russia
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Introduction
It is well known that development and creation of
universal bioactive materials is urgent now and
cause a lot of problems connected with processing,
preserving, sterilizing, and storing allografts. De-
spite the impressive achievements of tissue engi-
neering there remain many unsolved problems.
The most important of them are problems con-
nected with ensuring safety and preserving plastic
potential of the material, as well as creating
diversity of material forms and mechanical char-
acteristics (Von Versen 1993, p. 4).
A principal technology for manufacturing DBA
has been developed and used in the CITO tissue
bank on the basis of accumulated worldwide
experience. The main stages of this technology are
the following: the demineralization of bone tissue
at +18–20 with hydrochloric acid of various
concentrations under varying exposure time; the
lyophilization of the frozen material ( 35); and
radioactive sterilization of the end product by a
high-velocity electron beam of 20–25 kGy
absorption dose. As we were the first to use a high-
energy electron beam for sterilizing biological
materials the main aim was to study some prop-
erties of our materials through experiments and
assess the results of bone plasty surgeries in which
CITO produced DBA were used.
Materials and methods
To carry out biomechanical experiments some
sample plates were produced from human shin-
bone cortical layer with dimensions of 65 ± 5 mm
length, 20 ± 1 mm width and thickness from
2 ± 0.2 mm. Donor material sources were chosen
at random irrespectively of age, sex and other
donor characteristics.
Changes of mechanical properties of deminer-
alized allografts after sterilization by high-velocity
electron beam of different doses (20, 30 and
50 kGy) were assessed in the first part of the
experiment. The samples were demineralized with
0.6 H solution of hydrochloric acid within 12 h,
frozen at 35 C, lyophilized in sublimation unit
‘LZ 9.2’ within 48 h and sterilized by a high-
velocity electron beam. The experimental samples
differed in absorbed radiation doses mentioned
above, non-radiated demineralized bone tissue
samples being the control of the experiment. The
technological stages for producing a group of
experimental samples of 20 kGy radiation dose
were alike our technology of producing DBA.
Before testing mechanical toughness of the sam-
ples they were soaked in physiological solution for
25–30 min at room temperature.
The second part of the experiment dealt with the
samples which were used for assessment of
mechanical property dependence on the deminer-
alization degree. The samples were divided into
four groups, each of them being demineralized
within 15, 30, 45, and 60 h, respectively. The
demineralization was carried out in the solution of
1.2 H hydrochloric acid with 1:10–15 ratio of the
material weight to the acid volume at room tem-
perature. Then the material was stored for 10–
14 days at 35 C, lyophilized within 48 h and
sterilized by a high-velocity electron beam. The
radiation dose was 25 kGy. The control group
contained not processed samples of cortical layer
native bone. These samples were taken from the
same parts of donor shin-bone. Before mechanical
testing lyophilized samples were soaked in physi-
ological solution for 25–30 min at room tempera-
ture.
The manufactured sample variants were tested
for 3-point bend by the universal testing machine
‘Zwick 1464’ (Germany). The velocity of loading
was 15 mm/min and 1 kH force indicator with
±1% accuracy was used. The applied force and
sample deformation were automatically recorded
in ‘force-elongation’ coordinates. The deformation
of the samples was measured with analogue indi-
cator. The scale of the record was 0.01 mm per
1 mm of the diagram. Specific toughness was de-
fined through dividing the break force by the
sample cross section.
The investigation of safety and degree of
inductive properties of allograft components were
carried out in the culture of human stromal pre-
cursor bone marrow cells. The degree of inductive
properties of bio-components under the study de-
pended directly on the cloning efficiency of stromal
osteogenic bone marrow cells.
Demineralized bone allograft samples of differ-
ent demineralization degree and different radiation
dose having the dimensions of 1 · 1 cm were ad-
ded piece by piece to culture containers simulta-
neously with explantation of 5 · 105 human
stromal bone marrow cells. The cells were culti-
 233

vated in the medium 199 with 10% embryonal calf bone-plasty operations complicated with major
serum for 14 days until cell colonies were formed. defects of bone tissue in low extremities. In such
The cultures were fixed, the colonies were counted cases in CITO besides DBA frozen cortical allo-
and characterized: multiplayer, friable big, and grafts were used in combination with supporting
friable little. metallo-osteosynthesis.
Properties of stromal bone marrow cells as Ophthalmologically related were 19 children
precursors of osteo- and chondrogenesis as well as and teenagers (5–16 years old) hospitalized ur-
their similarity with skin fibroblasts allowed to use gently mainly with trauma of orbit walls accom-
the information about both types of connective panied by serious vision decay. Moreover, five
tissue cells (skin fibroblasts and stromal bone patients had major defects of frontal and parietal
marrow cells) as the assessment criteria for the bones due to car accidents and mine-missile
influence of biological inductors on proliferative wounds. Before 2000 surface-demineralized DBA
cell potential. plates from long bone cortical layer were used for
Cell mass growth after the definite period of filling bone defects in three cases of orbit wall
time in the presence of DBA samples and auto- trauma at the Moscow Children Municipal Clinic
radiographically defined index of 3H-timidin No.1. However, rather high mechanical proper-
marked cells were considered the main indices of ties and limited size of the material gave no
cell culture proliferative potential of skin fibro- possibility to use it for vast and irregularly
blasts. shaped defects of face and sinciput. The disad-
The index of marked cells was defined after 24-h vantages of these allografts enabled us to develop
incubation of cells with 3H-timidin in 1 lj Ku/ml and produce plastic material from donor sinciput
concentration by introducing isotope into con- bones. The place of raw material extraction was
tainers with DBA samples 24 h after sowing the
cells. The obtained preparations with autograph
Table 1. Bone system diseases treated with DBA of different
were colored in Gimza solution. The index of demineralization degree.
marked cells was defined in percent as the ratio of
the marked cell number to the total number of Pathology Patients
analyzed cells (minimum 500).
Benign tumors
Since March 1998 DBA with different demin- Osteoma 5
eralization degree and form (plates, chips, crumb) Osteoid-osteoma 2
have been used in Moscow specialized clinics for Chondroma 3
restoring bone tissue of patients with different Osteochondroma 5
Chondroblastoma 9
bone pathologies. These clinics specialize in trau-
Gigant-cell tumors 9
matology and orthopedics, maxilla-facial surgery,
Other tumors
ophthalmology, and neurosurgery. Since that time
Adamantinoma 1
up to July 2002 several hundred reconstructive
bone plastic operations had been made. We could Tumor-like diseases
Bone cysts (aneurismal and solitarian) 86
analyze distant results of bone plasty with DBA Metaphysal bone defect 15
for 257 patients who had been operated within Eosinophilic granuloma 1
that period. The post-operation supervision lasted Fibroid dysplasia 37
from 1.5 to 5 years 10 months. The main methods Other diseases
for control and assessment of allograft recon- Imperfecta osteogenesis 2
struction dynamics in post-operation period were Development anomalies 2
clinical observation, X-ray and computer tomog- Exostosous chondrodysplasia 3
Perthes disease 2
raphy. Dyschondroplasia 2
The majority of operated patients – 212 out of Initial chronic osteomyelitis 1
223 (95%) were children and teenagers of 3– Larcen syndrome 1
18 years old who related to traumatology and Inbred false joint 3
orthopedics. Nosology of bone pathology in which Ollier’s disease 5
Vitamin-D resistant rachitis 3
DBA were used involved wide range of diseases Post-traumatic deformations and false joints 26
(Table 1). Additional difficulties were caused by
234

chosen taking into account common embryonal

origin of bones from upper and middle face zones
having particular structure. This production
technology was the same as that of DBA from
long bone cortical layer. From October 2000 to
June 2002 this material was applied to 16 trau-
matized children including five complex trauma
of orbit walls with defects of sinciput zone cov-
ering 25 cm2. Trauma diagnosis and following
dynamic control over the allograft rebuilding
were carried out through computer. Control
examinations were made every other 4–6 months
after the operation.
Among the observed patients there were 15
adults with dystrophic changes of upper jaw bone
tissue and posttraumatic false joint of lower jaw
resulting from missile wounds and post-traumatic
osteomyelitis. Not large DBA plates were applied
to 9 patients in maxilla-facial surgery for enlarging
bone mass of degeneratively changed jaws. The
contents of transplanted material, that was appl-
pied to 5 patients for substituting missile and
osteomyelitic defects of lower jaw, varied. The
plasty was made through DBA plates, lyophilized
and demineralized bone chips and crumb, autot-
issues taken from patient’s iliac and ultrahigh
dispersion hydroxyapatite ‘Ostim’ which were in
different proportions (Figure 1). The advantage of
‘Ostim’ as compared to other modifications of
synthetic hydroxyapatite lies in the fact that it
contains crystals analogous in size and form to
natural bone tissue hydroxyapatite. One patient
experienced the plasty of frontal bone defect ob-
tained from heavy downward blow at a construc-
tion site. A DBA plate was used in combination
with autotissue taken from sinciput bones
(Figure 2).

Results

The experiments showed that after 3 h deminer-

alization with 1.2 H solution of hydrochloric acid
the amount of hydroxyapatite in the samples falls
on an average by 33.9%, after 6 h it falls by 47%
and after 12 h – by 58.6%.
Assessing the dependence of allograft mechani-
cal properties on different radiation doses of high
energy electron absorption we revealed that mini-
mum change of allograft toughness occurs when
the doses range 20–30 kGy. Further decay in
Figure 1. Patient X, aged 37. Osteomyelitis of lower jaw.
Roentgenograms: (a) before the operation; (b) 2 months after
the operation; (c) 6 months after.
toughness of the material was more vivid when the
doses grew from 30 to 50 kGy (Diagram 1).
While assessing the dependence of mechanical
toughness of the samples on demineralization time
we obtained the expected results: the more time the

Figure 2. Patient A, aged 28. Reconstructive computer tomo-
gram of front bone defect: (a) before the operation; (b) imme-
diately after the bone plasty.
demineralization process took, the worse the
allograft characteristics were (Diagram 2).
All samples of demineralized bone allografts in-
duced forming colonies in the culture of stromal
precursor bone marrow cells. According to the
statistics the number and the sizes of the colonies
grew up. The DBA samples with demineralization
degree of 47 and 58% influenced bone marrow cells
Diagram 1. Dynamics of sample bending strength variations
depending on the time of demineralization.
235
Diagram 2. Dynamics of sample bending strength depending
on radiation dose.
to form even multiplayer colonies (Diagram 3).
The obtained results showed that inductive prop-
erties of allografts depend on the degree of their
demineralization: the higher the degree of tested
bone samples is, the more vivid their properties
enhancing proliferative potential of culture cells
are. However, while sterilizing demineralized bone
allografts the increased absorption dose of high
velocity electron beam resulted in worse inductive
properties of the allografts tested.
The skin fibroblast culture enabled all the
demineralized allograft samples to increase pro-
liferation of culture cells which was more explicit
near the samples.
Two hundred and ten patients with traumato-
logic and orthopedic pathologies had positive
results after DBA treatment (94.2%). Nineteen
children and teenagers with trauma of orbit lower
wall and defects of sinciput bones as well as 9
adults with degenerative bone changes had 100%
positive result. It is necessary to add that 3 of 5
Diagram 3. Colony-forming efficiency of osteogenic cells in
marrow stromal cell culture with the DBA of different demin-
eralization extent.
236

children with major defects of sinciput have

uncompleted allograft regeneration. However,
regeneration control shows formation of natural
tissue both on the border with uninjured bone
and in the center of the allografts (Figure 3).
DBA plasty was made to patients with degener-
atively changed upper jaw bones. After 6 months
a new bone formed where dental allografts were
implanted. Worse results were obtained in treat-
ment of patients with firearm and osteomyelitic
defects of lower jaw having vast soft tissue
damage: 2 patients of 5 (40%) had the return of
osteomyelitis. One patient with frontal bone
plasty left Russia and there is no information
about him.
In traumatology and orthopedy only those
operations were considered to be successful after
which patients had no complaints and difficulties
in using the operated extremity. In some cases
X-ray showed complete regeneration of the allo-
graft and formation of a new bone. In other cases
DBA adhered firmly to each other and to the
mother bone but their outlines were visible and
their regeneration was not complete. At the same
time there were no signs of the return of the dis-
ease. The time of native bone formation in the
operated zone depended on the volume of this
defect, nosonomy and the course of the disease,
the quantity and quality of bone plastic material,
the character of surgical manipulations and a
number of other concomitant factors (Snetkov
et al. 2003, p. 74).
Consolidation periods for fractures in case of
posttraumatic false joints were 4–10 months (Fig-
ure 4), whereas organotypical regeneration of
plastic material took 12–24 months. Average bone
regeneration period was 12 months (Figure 5).
After massive DBA plasty of fibroid dysplasia
allograft reconstruction lasted 15 month and
longer. When frozen cortical allografts and
metallic constructions were used the period of
bone tissue regeneration was 24–30 months and
longer (Figure 6).
On the whole positive results of bone plasty
operations using DBA amounts to 93.4% (241 out
of 257 operated patients). However, 16 cases were
considered to be not satisfactory. The main rea- Figure 3. Patient G, aged 5. Computer tomogram of post-
sons of that were the following: the returns of bone traumatic defect on orbir upper wall and sinciput: (a) before the
operation; (b) 1 year after; (c) 2 year after the operation.
cysts obtained by children (aneurismal – 5 cases
and solitarian – 1); the return of fibroid dysplasia –
3 cases; suppuration on the background of osteo-

Figure 4. Patient Sh, aged 9. Treatment of the false joint: (a)
before the operation; (b) bone plasty combined with osteosyn-
thesis; (c) the state 14 months after the plasty.
237
Figure 5. Patient Sh, aged 16. Computer test 15 months after
DBA alloplasty for aneurysmic cyst.
Figure 6. Patient M, aged 16. Fibroid dysplasia of the right
femur. Roentgenograms: (a) before the operation; (b) vast edge
resection and DBA plasty in combination with frozen cortical
allografts fixed with L-shaped plate; (c) 3 years later before
extracting the plate; (d) 5 years after the operation.
238
myelitis – 6 cases and 1 case of not clear result
from front bone plasty when DBA with autografts
were used. The surgeons did not managed to
substitute the whole defect, especially the defect of
the orbit upper wall.
Discussion
The results of experimental study and clinical
application of DBA confirm that the material
developed and manufactured at CITO tissue bank
possesses all the qualities of demineralized bone.
However, it is necessary to remember that DBA
properties depend on many concomitant factors:
the quality of donor material, methods of its
conservation, sterilization, storage, etc. (Dziec-
Goclawska 2000, p. 57). So while manufacturing
DBA it is possible to add some property, let us say
osteoinductivity, to the material being manufac-
tured and vice versa to obtain allografts with
properties of biologically inert material. We pro-
duced the material which has osteoinductive
properties together with high safety, and this was
confirmed by DBA application in clinics (Mironov
et al. 2002, p. 89).
Our study enabled us to assess properties of
materials with different degree of demineraliza-
tion and sterilized by high energy electro-beam.
The absorption dose of 20–25 kGy does not af-
fect noticeably mechanical and osteoinductive
DBA properties. Thus we can state that DBA
with high degree of demineralization have poor
mechanical and osteoinductive characteristics.
These DBA properties were taken into account in
manufacturing plastic material from sinciput
bones for the plasty of facial and sinciput defects.
Mechanical properties of DBA with low degree of
demineralization can be used for operations of
supporting skeleton parts. Combination of allo-
grafts with different degree of demineralization
should be used for replacement of low extremity
defects. In these cases one part of DBA performs
mechanical functions the other stimulates osteo-
genesis.
Failures in surgical treatment with DBA showed
that the main reasons lay in inadequate complex
treatment of patients with bone cysts and osteo-
myelitis rather than in disadvantages of DBA.
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