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The experimental study of cryopreserved osteoblasts combined withbioactive glass repairing mandibular defects in rabbits
Wanye Tan, MD,a Fengcai Wei, MD,a Shanzhen Sun, MD,b and Mingxia Sun, MDbaDepartment of Stomatology, Qi-Lu Hospital, Shandong University, and bSchool of
Dentistry, Shandong University, Jinan, P. R. China

Purpose: The purpose of this study was to observe the biological character of cultured periosteal-derived
osteoblasts (POBs) preserved in liquid nitrogen in vitro, and to have a preliminary study on theosteogenetic capability of bioactive glass ceramics (BGC) combined with POBs.
Materials and Methods: The POBs were acquired from New Zealand big white rabbit and after several
generations culture some of them were kept in liquid nitrogen. The cryopreserved cells for six months were
cultured in DMEM medium and examined by morphological and histological observation. Then thosegrowing well in vitro were seeded into the porous BGC materials. A week later, the combined materials
were implanted into the bone defects of rabbits’ mandible, the control groups were implanted into thesingle BGC (no cells). After two, four, eight, and 12 weeks of operation, the specimens were respectively
excised and examined by X-ray and histological chemistry observation.
Results: The cryopreserved POBs grew well in vitro and also had the typical characters of mature
osteoblasts. Cultured with BGC materials, the cells could attach grow and proliferate well on the surface ofmost endopores. After four weeks of operation the transplanted osteoblasts began to form new osteoid orbone-like tissue in most pores of implanted BGC, and the bone defects were repaired better and earlier.
Conclusion: It was practical to use the cryopreserved osteoblasts as seeded cell for further study on bone
tissue engineering. It suggested that the “living” bone substitute would get more application and play amore important role in bone restoration and reconstruction. (Int Chin J Dent 2003; 3: 91-99.)
Clinical Significance: The results of this study demonstrate that the porous bioactive glass ceramics
combined with cryopreserved osteoblasts can repair the bone loss of rabbits’ mandible, and what we havedone will do good to future clinical application.
Key Words: bioactive glass ceramics, bone tissue engineering, bone defcents, cell culture, cryopreserved
osteoblast.

Introduction
With the rapid development of cell biology and material biology in the past 10 years, great progress has
been made on bone tissue engineering. It was an exciting direction to restore and reconstruct bone defects
by using correlative techniques of tissue engineering. Many scholars1-7 had been being engaged in such91Tan et al.

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studies as the osteoblasts culture and amplification, cell transplantation, mixed-cultivation between the cells
and some bioactive materials. Most of the osteoblasts they used as the seeded cell were freshly acquired
from the host body, and a repeated process of the cells’ segregation, purification, amplification andidentification would be done in every experiment.

Then it became very important to establish an“osteoblast bank” and use the crypopreserved cells to do the studies on bone tissue engineering. In ourstudy, we chose the cryopreserved periosteal-derived osteoblasts (POBs) as the seeded cell, the bioactive
glass ceramics (BGC) as the cell carrier, and after having been mixed- cultured in vitro for one week, thecombined BGC was transplanted into the mandibular defects of rabbit. Through some examinations wewould observe the osteogenesis of the transplanted osteoblasts and discuss the possibility of cryopreserved
osteoblasts transplantation.

Materials and Methods
Cell Isolation, Culture, and Cryopreservation
The POBs were obtained from newborn rabbits of the New Zealand strain. The tibia periosteum was
excised aseptically, cleaned of other soft tissues, sheared into pieces, and digested in 0.25% trypsin for 30
minutes, 1% collagenase for two hours. The released cells were collected in plastic culture flasks, and theculture medium was Dulbecco modified eagle medium (DMEM) containing 20% fetal bovine serum (FBS),
the culture condition was in a humidified atmosphere consisting of 5% CO2 at 37˚C, and 100% relative
humidity. When the cells in primary culture grew to approach confluence, they were digested by 0.25%
trypsin and subcultured in new flasks. The cultured cells of fourth to sixth generations growing well werechosen to reduce the temperature step by step to -100˚C and then kept in liquid nitrogen (the freezing
medium included 10% dimethyl sulfoxide, 40% FBS, 50% DMEM).
Cell Anabiosis and Identification
The cells preserved in liquid nitrogen for six months were revived and continued to subculture until to getenough cells. Some cells were examined by alkaline phosphatase (ALP) stain (using the method ofmodified Gomori’s). Some were cultured in conditioned medium (concluding 10% FBS, 10 mmol/Lß-sodium glycerophosphate, 50 mg/L vitamin C) for 30 days, and then stained in situ with alizarin red S toobserve the bone-like nodes forming. Other cells were used in the followed experiment.
BGC Preparation and Mixed-cultivation with Osteoblasts in Vitro
All the porous BGC materials were prefabricated nubbly shapes (10x8x3 mm), cleaned by ultrasound for
30 minutes, washed by distilled water three times, then sterilized for later use.
The anabiotic osteoblasts were diluted by DMEM concluding 20% FBS until a density of (4-5)x106/mL,
and each 0.1 mL cell suspension was seeded into the porous BGC which had been put in the 12-well plastic
culture plate. Then they were cultured in a humidified atmosphere consisting of 95% air and 5% CO2 at
37˚C, and 100% relative humidity. And 30 minutes later more DMEM was added to the wells until on theupper surface of BGC. The medium was changed every second day.
After mixed-cultured in vitro one week, some combined BGC species combining many osteoblasts were92

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examined by scanning electron microscope, others were used in the followed experiments.
Animal Experiment
A total of 12 mature New Zealand big white rabbits were chose and randomly divided into four groups,
and each group had three rabbits. Bone defects were made in all the rabbits’ bilateral mandible, and one
side was implanted the combined BGC (as the experimental group), the other side was implanted the single
BGC (as the control group).
The operation procedure as follows: Rabbits were anaesthetized by intravenous injection of 3% sodium
pentobarbital (30 mg/kg weight). Under sterile conditions the skin on the mandibular infraborder before
the masseter muscle attachment was incised, and the mandibular body was exposed. With the help ofdentistry drill, bone defect was made the similar size as BGC implant (10x8x3 mm). The combined BGC
implant were then fixed in the position of bone loss, and the host periosteum was well protected andsutured. The skin and other soft tissues were closed demixingly. The wound was open for air. The
operation of control groups was as the above. All the experimental rabbits were given cidomycin for threedays after operation, and feed routine diet.
At two, four, six, and eight weeks after operation, three rabbits of one group were killed and the wholemandibles were harvested. The specimens including the implant and some surrounding bone tissue were
carefully excised, and first of all examined by X-ray under same conditions with the dental digital X-ray
imaging system, fixed in formalin, decalcified in 5% azotic acid for four days, and then embedded inparaffin. Sections of 5-7 µm in thickness were deparaffinized for hematoxylin and eosin staining, and thenwere observed under general light microscope.

Results
The primary POBs at the firstly beginning were round monocyte. One day later, they began to stick onto
the floor of the culture flasks, then they extended and disfigured as the polygonal appearance. After
cultured three to four days, the adherent cells began to grow and proliferate quickly, which exhibitedmorphologies ranging from the spindle-shape typical of fobroblasts to polygonal-shape. At eight to ninedays, the adherent cells reached confluency and mainly shaped in spindle. The anabiotic cells had thesimilar morphologies as subcultured cells, and after growing in vitro for three days, they also expressedsignificantly high ALP activity, which showed mass gray or black deposit in the cytoplasm of mostcultured cells. After cultured in conditioned medium for 30 days, the anabiotic cells formed mineralized
nodes which took on the red, round shapes by stain of alizarin red S (Figs. 1-3).
When mixed-cultured in vitro with porous BGC, the osteoblasts could stick to the surface of mostendopores of the material, and continue to grow and proliferate, even excrete extra celluar matrix (ECM)(Fig. 4).
The X-ray results and histological results of combined BGC repairing mandibular defects of rabbits indifferent time were showed in Tables 1 and 2, and Figs. 5 and 6.

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1

2

Fig. 1. After 24 hours cultured in vitro, the anabiotic osteoblasts were mainly spindle-shaped or
polygonal-shaped (Inverted phase-contrast microscope, x200).
Fig. 2. After three days cultured in vitro, the anabiotic osteoblasts were stained black or brown incytoplasm (Modified Gomori’s method, x400).

3

4

Fig. 3. After 30 days cultured in conditioned medium in vitro, the osteoblasts formed roundminerlization nodes stained red (Red alizarin stain, x100).
Fig. 4. After one week of the cultured cells combined with BGC in vitro, many cells grew andproliferated on the surface of most endopores (SEM examination, x500).

5

6Fig. 5. After eight weeks of the combined materials implanted into the bone defects, thetransplanted osteoblasts formed osteoid tissue (HE stain, x100).
Fig. 6. After 12 weeks of the combined materials implanted into the bone defects, thetransplanted osteoblasts formed new bone tissue (HE stain, x100).

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Table 1. X-ray results of combined BGC repairing bone defects in different time.
Time(Post-operation)

Combined BGC(Experimental group)

Single BGC(Control group)

2 weeks

Apparent border between BGC andhost bone, local density significantly
lower than surrounding bone tissue.

Similar as the experimentalgroup.

4 weeks

The border was clouding, synostosis
began to form, local density was nearto that of surrounding host bone.

The border was also distinct,
less synotosis formed, lower
density than host bone.

8 weeks

The border was unclear, synostosis
completely formed, local density wasas high as surrounding bone.

The border became clouding,
local density was near tohost bone tissue.

12 weeks

The border disappeared, BGC implantcompletely repaired the bone defects.

Nearly the same as theexperimental group.

Table 2. Histological results of combined BGC repairing bone defects in different time.
Time(Post-operation)

Combined BGC(Experimental group)

Single BGC(Control group)

2 weeks

Fibrous tissue grew into surrounding
pores, new blood vessel could be seen;
the adherent cells in most endoporesgrew well ,excreted much ECM.

Fibrous tissue grew into thethe surrounding pores of
BGC; no cells could be seen
in most endopores.

4 weeks

Osteoid formed on the border; newbone-like tissue could be seen in mostendopores, a few of lymphocytes andacidophil granulation cells were seenaround the new tissue.

Bone-like tissue could be seen
only on the border, no new
tissue formed in endopores,
some lymphocytes andgigantocells could be seen.

8 weeks

Many fresh bone tissue formed inmost endopores; synotosis began
to form on the border.

Osteogenesis could also beseen in the pores of the border,
no bone formed in endopores.

12 weeks

In most endopores many lamellar-likebone tissue formed, vasculogenesis
was seen much.

Synotosis formed between the
BGC and host bone,no newbone was found in endopores.

Discussion
The Choice of the Seeded Cell
The seeded cell of bone tissue engineering was the osteoblast, which could be acquired by many ways invivo.

Such as the bone marrow stromal cells (BMSCs) from bone marrow, the periosteal-derived

osteoblasts (POBs) from periosteum, the osteoblasts from bone tissue and the heterotopic mesenchymal
stem cells from other tissues. At present the BMSCs and the POBs were applied much more than others on

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the experimental studies. We chose the young rabbit’ POBs as the seeded cell, and found that they not only
grew and proliferated well in vitro but also could be induced to differentiate into mature osteoblasts. We
didn’t see the aging change in all the passages (in our experiment the cultured cells were observed by 20th
generation). These results were also verificated by other scholars.
The periosteum concluded such cells as osteoblasts, fibroblasts, blood cells, blood vessel endothelial
cells and so on. After digested by pancreatin and collagenase, the periosteum were dissected into theabove-mentioned cells, which were mixed in the primary cell culture. Blood cells were suspended cells,
which could not stick to the bottle floor in vitro and were cleaned gradually in the process of the changing
of culture medium. Blood vessel endothelial cells were much less than osteoblasts and couldn’t grow for along time without the stimulation of some definite growth factor.

The fibroblasts were similar as

osteoblasts in appearance but were less in quantities, and under some definite conditions they could be
induced to the cells having some characters of osteoblasts. Thus through unceasing cell culture andpassages, the periosteum cells were gradually converted into the more pure cell (osteoblasts, orosteoblast-like cell) line.
The Superiority of Using the Cryopreserved Osteoblasts
Many studies8 had confirmed that the cultured cells in vitro could be kept in a low temperature
circumstance for a long time, and the anabiotic cells also had the similar principal functions as before. Our
experiment showed that the cryopreserved osteoblasts had not only a high ability of growth andproliferation but also the typical functions of mature osteoblasts such as the synthesis of ALP and bone-liketissue. This suggested that it was practical to establish an “osteoblast bank” preserving cultured cells anduse the cryopreserved cells to work as the seeded cell of bone tissue engineering. We knew that thecultured cells grew quickly in vitro, and if we didn’t take some measures to preserve them, there would be
too many cells to use, which would result in much meaningless consumption. At the same time too many
passages might make the cultured cells becoming aging, old, or even dead. Thus it was essential to choose
the cultured cells growing well in vitro to preserve in liquid nitrogen for future use.

The quickdevelopment of modern low temperature medicine could also make this being possible.
There were many advantages for us to use the anabiotic osteoblasts to carry on studies on the bone tissue
engineering: (1) Timing: Whenever we had the demand for osteoblasts we could resuscitate thecryopreserved cells to culture and proliferate. (2) Saving: It needed only the liquid nitrogen jar for keeping
the cells, which avoided much expense of the primary and secondary cell culture. (3) Repeating: Theanabiotic osteoblasts from one same bottle had the similar growing behavior and biological function. And
after several generations’ amplification we could acquire enough cells and use them for the contrast
experimental research.
Choice of the Cell Carrier
Cell carrier3-4 was some material that could provide an enough space for cells’ growth and transplantion.
If the cultured osteoblasts were only injected into the body in the manner of liquid, new bone-like tissue
would not form or form little, which was mainly because the transplanted cells couldn’t keep the effective

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concentration in the local region. While the cell carrier had a porous structure, and the cultured osteoblasts
could attach and grow onto the surface of the most endopores of the material. Then after transplanted into
the body, the carrier could make the adherent cells form new bone-like tissue in a relatively steadysurrounding. This suggested that it was significant for the bone tissue engineering to select an appropriate
cell carrier.
The bioactive glass ceramic9-12 was a sort of degradable material, which had been widespreadly used as
the substitution of bone to restore and reconstruct the bone defects, especially the cranofacial bone loss.
Recently many scholars4-7 had made use of the porous bioglass as the cell carrier. Our experiment showedthat the BGC material had good bio-compatibility, less inflammatory reaction and foreign body rejection.
It could composite large quantities of cultured osteoblasts in vitro which continued to proliferate in vivo
and then formed new bone-like tissue. The degradation ratio of BGC was very low, and at the 12th week
after transplantation we only found a little portion of material being in resorption. But some scholars
considered that as long as the attaching osteoblasts could grow well and form new osteoid tissue, the cellcarrier materials would have good clinical value even if they were absorbed so slowly.
The Osteogenesis Principles of the Combined BGC
After the combined BGC material was transplanted into the location of the mandibular defects of rabbits,
the adherent osteoblasts could also maintain the ability of growth and proliferation with the host offerednutrien, which put up a satisfactory osteogenesis potential.

The combined BGC was a “living”

tissue-engineered bone substitution material, which combined many osteoblasts and could repair the rabbit’
mandibular bone loss better and earlier. The principles were discussed as followed.
The BGC Material had Good Bone Conductibility and Bio-compatibility
When the porous BGC was transplanted into the body, the blood, bone marrow and ECM around theoperation region began to aggregate and contact with the material, and then an ion exchange occurred on
the surface of the implant. As a result a sort of biochemistry couple formed between the BGC and thesurrounding bone tissue. Simultaneously a layer of hydroxyapatite (HA) containing some bicarbonate
compound came into being on the surface of the BGC. The HA layer, which had the similar structure as
the inorganic component of common bone tissue, could be discerned and accepted by the host body. Someof the osteoblasts and collagen fibers around the operation region could deposit and grow on the surface ofthe HA layer. With time went on, some new bone-like tissue gradually formed in the HA layer. All theseabove mentioned were the rudiment of the synostosis forming between the implant and the surrounding
bone tissues. Our experiment also confirmed that there would be much satisfactory synostosis formed at
the edge of the implant no matter what the BGC combined the cultured osteoblasts. The X-ray andhistological examination results gave the detailed interpretation.
The Role of the Transplanted Osteoblasts Combined into Most Endopores of the BGC
After cultured in vitro with the porous BGC for one week, there would be large quantities of osteoblasts
attaching onto the surface of most endopores. Some experiments4-7 in vitro suggested that the combined
osteoblasts not only grew well and excreted abundant ECM but also kept the fundamental biological97

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characters of mature osteoblasts. At the beginning of the combined BGC implanted into the body, theattached cells began to grow by sucking some nutrien component of the surrounding micro-environment.
And some time later, blood capillary began to grow into the endopores of the porous BGC. Then an
exchange of the nutrien matters and metabolism products between the transplanted cells and the host bodyestabilished. Thus in a new circumstance these transplanted osteoblasts could continue to grow andproliferate, synthesize and excrete ECM, even form bone tissue. In our study we observed that the densityof two groups at fourth week was both lower than the surrounding bone tissue (because of the porous
structure). At eighth week their density all became higher than before, but that of the experimental group,
which was as high as the host bone tissue, increased much more than the control group. This was mostly
because the transplanted cells could form new bone in the bosom of the implant, which made the implanttake on a higher density by the X-ray examination, while the osteogenesis of the control group only limitedon the border of the material. The histological results also showed in the experimental group many newbone-like tissue formed within most endopores, while there was no new tissue formed in the center of thecontrol group at all time segments. In addition, we found that the synostosis time (as displayed in X-ray) of
the experimental group was earlier (at eighth week) than the control group (at 12th week). We discussed
the possible causes was as followed: (1) The synostosis of the experimental group was due to the
osteogenesis of both the host osteoblasts and the transplanted cells, while the control group was only due tothe host osteoblasts. (2) The transplanted cells were some mature osteoblasts and could excrete somebioactive factors, which might stimulate the differentiation of the host stromal cells and augment theosteogenesis ability. But the concrete principles should be confirmed by further studies.

Conclusion
In our experiment, we used the porous BGC as the cell carrier and the cryopreserved osteoblasts as theseeded cell to replace the bone loss of rabbits’ mandible, and acquired successful results were obtained. It
suggested that the bone tissue engineering would become a best choice to restore human bone defects infuture.

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Reprint request to:Dr. Wanye Tan
Department of Stomatology, Qi-Lu Hospital, Shandong University
107, Cultural Western Road, Jinan 250012, P. R. China
Fax: +86-0531-6927544 Email: tanwanye@hotmail.com
Received on March 28, 2003. Revised on July 26, 2003. Accepted on August 12, 2003.
Copyright ©2003 by the Editorial Council of the International Chinese Journal of Dentistry.

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