J Bone Metab.  2017 Feb;24(1):23-30. 10.11005/jbm.2017.24.1.23.

Effects of Escherichia Coli-derived Recombinant Human Bone Morphogenetic Protein-2 Loaded Porous Hydroxyaptite-based Ceramics on Calvarial Defect in Rabbits

• Kim SY
• Lee Y
• Seo SJ
• Lim JH
• Kim YG

• KMID: 2371829
• DOI: http://doi.org/10.11005/jbm.2017.24.1.23

Abstract

BACKGROUND
Recombinant human bone morphogenetic proteins (rhBMPs) have been widely used in regenerative therapies to promote bone formation. The production of rhBMPs using bacterial systems such as Escherichia coli (E. coli) is estimated to facilitate clinical applications by lowering the cost without compromising biological activity. In clinical practice, rhBMP-2 and osteoconductive carriers (e.g., hydroxyapatite [HA] and bovine bone xenograft) are used together. This study examined the effect of E. coli-derived rhBMP-2 combined with porous HA-based ceramics on calvarial defect in rabbits.
METHODS
Six adult male New Zealand white rabbits were used in this study. The experimental groups were divided into the following 4 groups: untreated (NC), bovine bone graft (BO), porous HA (HA) and porous HA with rhBMP-2 (HA-BMP). Four transosseous defects of 8 mm in diameter were prepared using stainless steel trephine bur in the frontal and parietal bones. Histological and histomorphometric analyses at 4 weeks after surgery revealed significant new bone formation by porous HA alone.
RESULTS
HA-BMP showed significantly higher degree of bone formation compared with BO and HA group (P<0.05). The average new bone formation % (new bone area per total defect area) of NC, BO, HA, and HA-BMP at 4-week after surgery were 12.65±5.89%, 29.63±6.99%, 28.86±6.17% and 49.56±8.23%, respectively. However, there was no statistical difference in the bone formation between HA and BO groups.
CONCLUSIONS
HA-BMP promoted more bone formation than NC, BO and HA alone. Thus, using E. coli-derived rhBMP-2 combined with porous HA-based ceramics can promote new bone formation.

Keyword

Bone morphogenetic proteins; Eschericia coli; Hydroxyapatites; Osteogenesis

MeSH Terms

Adult
Bone Morphogenetic Proteins
Ceramics*
Durapatite
Escherichia coli
Escherichia*
Humans*
Hydroxyapatites
Male
Osteogenesis
Parietal Bone
Rabbits*
Stainless Steel
Transplants
Bone Morphogenetic Proteins
Durapatite
Hydroxyapatites
Stainless Steel

Figure

• 

  Fig. 1 Clinical photograph of the surgery. (A) Sagittal incisions were made along the midline of the calvaria in six rabbits under anesthesia. Four transosseous defects of 8 mm in diameter were prepared using stainless steel trephine bur in the frontal and parietal bones. (B) Defects were filled with porous hydroxyapatite (HA), porous HA with recombinant human bone morphogenetic protein-2, bovine bone xenograft, or left untreated for control.

• 

  Fig. 2 Surface structures of porous hydroxyapatite (HA) and bovine bone xenograft (BO). Graft materials were coated with platinum and were subjected to the scanning electron microscopy. Microscopic structures of porous HA (A) and bovine bone xenograft (B) were shown in ×100 magnification. Scale bars indicate 500 µm.

• 

  Fig. 3 Histological evaluation of bone regeneration. Rabbit calvarial defects were either untreated (negative control [NC]), filled with bovine bone xenograft (BO; positive control), filled with porous hydroxyapatite (HA), or filled with porous HA plus Escherichia coli-derived recombinant human bone morphogenetic protein-2 (rhBMP-2). At 4 weeks after surgery, calvariae were removed, fixed, decalcified, and embedded in paraffin. Tissue sections of 3 m thickness were prepared using a microtome. (A) The tissue sections were subjected to hematoxylin and eosin staining. Microscopic images with along the coronal plane of the calvarial defects were obtained (×12.5 magnification). (B) Higher magnification (×40) images of the boxed areas in (A) area shown. Scale bars indicate 500 µm. Representative images from 6 rabbits are presented.

• 

  Fig. 4 Histomorphometric evaluation of bone regeneration. Quantitative analysis of new bone formation was performed by calculating new bone area per total defect area using the I-solution software (IMT i-Solution Inc., Vancouver, BC, Canada) as described in materials and methods. Data are mean±standard error of the mean. *P<0.05 vs. untreated control. **P<0.05 vs. porous hydroxyapatite (HA) alone or bovine bone xenograft. rhBMP-2, recombinant human bone morphogenetic protein-2.

• 

  Fig. 5 The effect of Escherichia coli-derived recombinant human bone morphogenetic protein-2 in bone regeneration in vivo. Calvariae were subjected to micro-computer tomography analysis. Representative data from per group are shown (newly formed bone; pink: hydroxyapatite (HA) group, blue: HA-bone morphogenetic protein group, green: bovine bone xenograft group, and yellow: negative control group). Although the HA particles are not fully resorbed after 4 weeks after surgery, but are discernible from newly formed bones in defect area.

• 

  Fig. 6 Histologic analysis for the adipogenesis induced by Escherichia coli-derived recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo. Among the 6 samples per group, adipogenesis was only observed in 1 slide in porous hydroxyapatite plus rhBMP-2 group (hematoxylin and eosin [H & E], ×100 magnification). Scale bar indicates 500 µm. The arrowhead indicates adipocytes.

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