J Korean Assoc Oral Maxillofac Surg.  2017 Dec;43(6):373-387. 10.5125/jkaoms.2017.43.6.373.

Stepwise verification of bone regeneration using recombinant human bone morphogenetic protein-2 in rat fibula model

• Nam JW
• Kim HJ

• KMID: 2398992
• DOI: http://doi.org/10.5125/jkaoms.2017.43.6.373

Abstract

OBJECTIVES
The purpose of this study was to introduce our three experiments on bone morphogenetic protein (BMP) and its carriers performed using the critical sized segmental defect (CSD) model in rat fibula and to investigate development of animal models and carriers for more effective bone regeneration.
MATERIALS AND METHODS
For the experiments, 14, 16, and 24 rats with CSDs on both fibulae were used in Experiments 1, 2, and 3, respectively. BMP-2 with absorbable collagen sponge (ACS) (Experiments 1 and 2), autoclaved autogenous bone (AAB) and fibrin glue (FG) (Experiment 3), and xenogenic bone (Experiment 2) were used in the experimental groups. Radiographic and histomorphological evaluations were performed during the follow-up period of each experiment.
RESULTS
Significant new bone formation was commonly observed in all experimental groups using BMP-2 compared to control and xenograft (porcine bone) groups. Although there was some difference based on BMP carrier, regenerated bone volume was typically reduced by remodeling after initially forming excessive bone.
CONCLUSION
BMP-2 demonstrates excellent ability for bone regeneration because of its osteoinductivity, but efficacy can be significantly different depending on its delivery system. ACS and FG showed relatively good bone regeneration capacity, satisfying the essential conditions of localization and release-control when used as BMP carriers. AAB could not provide release-control as a BMP carrier, but its space-maintenance role was remarkable. Carriers and scaffolds that can provide sufficient support to the BMP/carrier complex are necessary for large bone defects, and AAB is thought to be able to act as an effective scaffold. The CSD model of rat fibula is simple and useful for initial estimate of bone regeneration by agents including BMPs.

Keyword

Bone regeneration; Bone morphogenetic proteins; Carrier; Autoclaved autogenous bone; Rat fibula

MeSH Terms

Animals
Bone Morphogenetic Proteins
Bone Regeneration*
Collagen
Fibrin Tissue Adhesive
Fibula*
Follow-Up Studies
Heterografts
Humans*
Models, Animal
Osteogenesis
Porifera
Rats*
Bone Morphogenetic Proteins
Collagen
Fibrin Tissue Adhesive

Figure

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  Fig. 1 A critical sized segmental defect of 6 mm was formed in rat fibula model.

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  Fig. 2 Experimental sites of rat fibulae in Experiments 1–3. A. Absorbable collagen sponge with/without recombinant human bone morphogenetic protein-2 (rhBMP-2) in Experiments 1 and 2. B. Xenogenic bone (CollaOss; Bioland, Korea) in Experiment 2. C. Autoclaved autogenous bone (arrow, inset) with/without rhBMP-2 in Group A of Experiment 3. D. Fibrin glue block (arrow, inset) with/without rhBMP-2 in Group B of Experiment 3.

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  Fig. 3 Gross findings in Experiment 3 (A–D: Group A, E–H: Group B). A, C, D. The 2-, 4-, 8-week experimental groups with rhBMP-2/AAB. Complete bony unions were observed in all groups. The 2-week group showed an ectopic irregular and very excessive generated bone, but the amount of newly formed bone decreased over time through remodeling. E, G, H. The 2-, 4-, 8-week experimental groups with rhBMP-2/FG. Complete bony unions were observed in all groups as Group A. However, the new bone of the 2-week group was formed within relatively limited area comparing with Group A. In the 4-week group, similar or more newly formed bone was observed than in the 2-week group, and ectopic bony growth was also seen. In the 8-week group, a complete union having significantly diminishing the amount of regenerated bone than in the 4-week group was seen. B, F. The 2-week control groups with AAB or FG only. B. Non-union was observed between AAB and the residual fibula ends. F. Segmental bony defect remained on the fibula. (rhBMP-2: recombinant human bone morphogenetic protein-2, AAB: autoclaved autogenous bone, FG: fibrin glue)

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  Fig. 4 Microtomography (micro-CT) findings in the experimental groups of Group A in Experiment 3. Comparison of the bone density between the tibia and the newly formed fibula using Micro-CT images. A. The 2-week group; A high-density bi-cortical bone and a low-density regular bone marrow was observed on the adjacent tibia, in contrast with the newly formed fibula bone, which showed an irregular pattern. A high-density bi-cortical bone was also observed on grafted autoclaved autogenous bone (AAB) area. B. The 4-week group; The newly formed fibula bone showed a similar bone density pattern as the adjacent tibia with lower intensity of its cortical portion. Both also showed a low-dense and regular bone marrow. C. The 8-week group; Compared with the 4-week group, the distribution of the bone density was similar to normal bone. The formation of a regular and low-dense cancellous bone was noticeable between the two peaks traced by the cortex of the newly formed bone.

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  Fig. 5 Microtomography (micro-CT) findings in the experimental groups of Group B in Experiment 3. Comparison of the bone density between the tibia and the newly formed fibula using Micro-CT images. A. The 2-week group; In the adjacent tibia, a high dense cortical bone was seen as well as a regular and less dense inner cancellous bone. In the fibula site, an over-regenerated bone with an irregular bone density was observed. B. The 4-week group; Same findings as the 2-week experimental group with no remarkable change in bone density. C. The 8-week group; The density pattern of the newly formed fibula, composed of the cortical bone and bone marrow, was similar to that of the adjacent tibia.

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  Fig. 6 Radiographic and microtomography findings in the xenograft groups in Experiment 2. A. The 4-week group; An irregular density of grafted material was observed (arrow). B. The 8-week group; Reduced grafted material with similar density pattern to the 4-week groups was observed (arrow).

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  Fig. 7 Histomorphologic findings in Group A of Experiment 3 (H&E staining, A–D: ×10, inset: ×100). A. The 2-week experimental group; The formation of immature woven bone and simultaneous bone remodeling, surrounding the transplanted autoclaved autogenous bone (AAB) were observed (arrowhead). B. The 4-week experimental group; A continuous connecting cortical bone was noticeable between remaining fibula bone and newly formed bone and the inner part was gradually replaced by bone marrow. C. The 8-week experimental group; A complete union between the regenerated bone and the pre-existing fibula was observed with a continuous and homogeneous connection of the bone marrow and cortical bone. D. The 8-week control group; A complete union between the newly formed bone and the remaining fibula was observed. The new bone formation and ossification progress were seen surrounding the grafted AAB (arrowhead).

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  Fig. 8 Histomorphologic findings in Group B of Experiment 3 (H&E staining, A–D: ×10, inset: ×100). A. The 2-week experimental group; The newly formed bone was observed including endochondral ossification in the both borders near the existing fibula. B. The 4-week experimental group; The cortical layer of the newly formed bone showed a similar continuity as the remaining fibula. The inner part was gradually replaced by bone marrow. C, D. The 8-week experimental and control groups; Both experimental and control groups showed complete unions of the fibula defects.

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