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Int Chin J Dent 2007; 7: 87-91.

Non-vascularized bone isograft using GFP transgenic mice
Cheng Lin, DDS, PhD,a Bao-lin Liu, DDS,a Yan-pu Liu, DDS, PhD,a Xiao-hui Liu, DDS,PhD,b Xiao-guang Hu, DDS, MD,a Yan-liang Wang, DDS, PhD,a and De-lin Lei, DDS,MDa
aDepartment of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military MedicalbUniversity, Xi’an, and Department of Stomatology, General Hospital of Lanzhou Military District,Lan-Zhou, P. R. China.
Purpose: To further understand the viability of the non-vascularized bone graft and the origin of thebone-forming cells, we have developed a novel mouse femur model that permits direct-viewing analysis of bone
graft healing.
Materials and Methods: Critical size defects were prepared in one side of femur of 15 C57BL/6 mice andrepaired by non-vascularized femur with intact periosteum and parts of muscles from isogenous GFP transgenic
C57BL/6 mice. Sacrifice was performed 3 days, 1, 2, 3, and 4 weeks after surgery for histological andfluorescence microscopy analysis.
Results: Healing process of the transplants is similar to that of bone fracture. The osteoblasts and newly formedtrabeculae which were beneath the grafted periosteum showed green fluorescent protein (GFP) expression,
which was strongly expressed in the transplanted muscles.
Conclusion: These data demonstrate that part of non-vascularized grafted bone can keep viability and has thepotential of osteogenesis, indicating an important role for the grafted periosteum and bone in new boneformation. (Int Chin J Dent 2007; 7: 87-91.)
Key Words: GFP transgenic mice, isograft, non-vascularized bone graft.

Introduction
Non-vascularized bone graft, as an effective method for the repair of bone defects, is widely used in the world.
Conventional view of incorporation of non-vascularized bone graft to the recipient is called creeping
substitution.1 The grafts will finally be totally replaced by the host and all bone-forming cells originate from therecipients.2 However, some researchers hold a contrary opinion. Ray and Sabet3 used a radioactive tracer as abone label to perform bone transplantation between highly inbred mice. And Arora et al.4 identified sex
chromatin following sex-mismatched bone transplantation between highly inbred rabbits. They found that not
all the grafted bone suffered from creeping substitution, but a few parts of them could keep its viability.
Kakinoki et al.5 transferred non-vascularized tibia grafts from male to female rats, then extracted genomic DNA
and made Sry-specific PCR detection. Their results demonstrated that a small number of grafted bone survived
and proliferated over time.

However, these methods have low sensitivity, much complication; they haveprovided limited understanding of transplanted cells.
Green fluorescent protein (GFP) is a 27-KD protein, originated in jellyfish.

It provides a technicallyassessment of transgene activity and can be viewed as a real-time image in living tissues. Recently, Jiang et al.6
reported that GFP activity can be preserved in paraffin sections for standard fluorescence microscopy. Thus, it ishypothesized that GFP transgenic mouse as transplantation donors would be an easier way to determine theorigin of newly forming cells and comprehend clearly the behavior of grafted cells during the repair process ofbone defects.
In the present study, we transplanted non-vascularized femur grafts from GFP transgenic mice to its isogenous
mice. Fluorescence microscopic and histological assay were performed at 3 day, 1, 2, 3, and 4 weeks after
surgery, to identify the viability of the grafted bone and the origin of the bone-forming cells. This study may beof importance in the future development of clinical methods to reconstruct large mandible defects using

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non-vascularized bone grafts with dental implants at the same time.

Materials and Methods
The study procedure was approved by the animal center of Shan’xi province. All animal experiments wereperformed according to the guidelines of the animal committee of the Fourth Military Medical University.
Surgery procedure
Fifteen young adult male C57BL/6 mice (weighing 30-50 g) and fifteen 12-week male GFP transgenic
C57BL/6 mice (weighing 25-30 g) were used in this study.

All surgeries were performed under generalanesthesia with 1% sodium pentobarbital by intraperitoneal injection. A standardized metaphyseal bone defectof the femur (5 mm long, full thickness) was created in C57BL/6 mouse. Then a 5-mm-long-femur with intact
periosteum and parts of muscles was harvested from GFP transgenic C57BL/6 mouse. The graft was carefully
placed into the defect of the C57BL/6 mouse and fixed with sutures. Wound was closed in layers and fixed withbandage (Ex vivo time of the graft was less than 15 minutes). Animals were sacrificed at 3 days, 1, 2, 3, and4weeks after surgery respectively and the grafts were carefully harvested and fixed immediately into 10%
phosphate-buffered neutral formaldehyde for 24 hours.
Histology
For histology, the specimens were demineralized in 0.1 mol/L EDTA for 2 weeks, dehydrated in gradedethanol solutions, embedded in paraffin. Serial sections were cut in 4-µm-thick at different levels sufficiently farapart (about 200 µm) to avoid replicate sampling of a single surface event. Sections were then mounted on glass
slides and stored at -20˚C away from light. Finally, some sections were stained with hematoxylin-eosin (H-E)
and Masson trichrome, whereas others were left unstained for evaluation under fluorescence microscopy.
Fluorescence microscopy
All sections were observed under fluorescence microscopy. The average exposure time was 1-1.5 s, and theexcitation light was 488-530 nm blue light.

Results
All animals remained healthy during the healing period and healed uneventfully. Infection and immunologicalrejection were not observed.
HistologyFigs. 1-7 show histological findings (NB, new bone; GB, grafted bone; M, muscles; and OB, original bone).
Three days: There were lots of blood clots and inflammatory cells in the grafted areas. Transplanted bone waspartly absorbed and a thin layer of newly formed immature trabecular was on the surface of them.
One week: Blood clots had been replaced by vascularized granulation tissues which extended all the way to
the grafted bed.

The union between the grafted bone and recipient bone was full of mesenchymal cells

undergoing chondrocytic differentiation. Extensive bone absorption was observed at the periphery of the bone
graft indicated by the numbers of osteoclasts.
Two weeks: The grafted areas could be divided into five layers according to different tissues: grafted muscles,grafted periosteum, newly formed cartilage, newly formed trabeculae, and grafted bone.

Muscle fibers

interlacing with each other were in the outside of the grafted areas. Their cellular nuclei were on the margins of
the fibers. Connecting with muscle fibers, periosteum was intact and numbers of mesenchymal cells aggregated88Lin et al.

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in its germinal layer. Beneath the periosteum, there were lots of chondrocytes which seemed big and morekaryokinesis. Newly formed woven trabeculae occupied nearly two thirds of grafted areas, and numerous
osteoblasts lined around them. One third of the cortex of grafted bone had been absorbed and new boneformation occurred on the superficial of the remains. Grafted bone marrow was almost intact and within it lotsof lymphocyte and leukocytes got together.

Fig. 1.

Three days after surgery (H-E; Bar, 100 µm).

Fig. 3.

Two weeks after surgery.
(Masson trichormes)

Fig. 5.

Three weeks after surgery (H-E; Bar, 100 µm). Fig. 6.

Fig. 4.

Fig. 2.

One week after surgery (H-E, Bar, 100 µm).

Two weeks after surgery (H-E; Bar, 100 µm).

Four weeks after surgery (H-E, Bar, 100 µm).

Three weeks: The remained grafted bone began to integrate with newly formed mineralized trabeculae. New89

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medullary cavity occurred in the area initiallyoccupied by the bone grafts.
Four weeks: Newly formed bone incorporated to
the grafted bone and it’s difficult to distinguish them.
Fluorescence microscopy
Bright green light was found in transplanted
muscles all the time under fluorescent microscopy(Fig. 7). The osteoblasts and newly formed trabeculae,
which were beneath the grafted periosteum, also
showed GFP expression.
Fig. 7.

Two weeks after surgery (Bar, 100 µm).

Discussion
Although the theory of creeping substitution has been introduced for more than one hundred years, there arestill two questions not very clear: Does all the grafted bone be replaced by the newly formed bone? And where
are the osteoblasts or osteocytes in the new forming bone from?7,8 By now, large number of researches support
that parts of the grafted bone will survive and have the ability of osteogenesis.9-12 In this study, we investigatedthe viability of the non-vascularized grafted bone and its ability of new bone formation using isografting fromfluorescence specific GFP transgenic mice to its isogenous normal mice. Our results suggest that the transplants
can keep their viability and have the ability of osteogenesis. Although large part of the grafted bone wasabsorbed, the remains still kept its structure. Transplanted muscles and periosteum showed their viability bypositive expression of green lights. Green lights in the newly formed trabeculae and osteoblasts revealed thatbone forming cells were from the transplants. Thus our results indicate that the grafted bone and periosteum
play an important role in the process of new bone formation, and that the transplant can keep its viability during
the reparative process.
Owing to the early revascularization, transplanted bone, periosteum and muscles could keep their vitality andmedullary cavity of grafted bone could keep its structure, as the femur cortex of mouse is thin, which facilitates
blood vessels going through. Early revascularization was established by the blood-abundant recipient bed andthe transplanted soft tissues which can get blood easier than bone. Femur cortex of mouse is about 1 mm thick,through which blood vessels go easily. No fixation in medullary cavity could kept its structure and conduce torevascularization. Healing process of transplants is similar to the healing process of bone fracture.
In this study, we used GFP transgenic C57BL/6 mice as donor animals. These animals are very special withgreen muscles and bones in the sunlight and green eyes and ears under violet light. Histologically, muscles show
bright green light under fluorescence microscopy.

To bone, some researchers once worried that thedemineralization might destroy the expression of GFP. Recently, Jiang et al.6 has detected positive green
expression in demineralized bone sections. It is very interesting that positive expression only confine inosteoblasts and at the margin of the bone instead of the whole bone. Our study used 0.1 mol/L EDTA for 2
weeks’ demineralization, and the results indicated that GFP can be preserved in paraffin sections for standard
fluorescence microscopy. It is very helpful to use GFP transgenic mouse to be an animal model to study
non-vascularized bone graft.

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Acknowledgments
The authors would like to thank Mr. Ya-gang Wei for his valuable assistance in preparing the histological sections andworks in relation to the histomorphometry, professors Lian-jia Yang and Shao-zhong Dong for their professional help in
histological observations, and doctor Li-An Wu and professor Qin Ma for their kind help in English writing.

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Correspondence to:Dr. Bao-lin Liu
Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University
145 Changle Xi Road, Xi’an, 710032, P. R. China
Fax: +86-029-84772501 E-mail: kkzzzx@fmmu.edu.cn
Received October 19, 2007. Accepted December 4, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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