J Korean Assoc Oral Maxillofac Surg.  2010 Oct;36(5):366-374.

The effect of silk fibroin and rhBMP-2 on bone regeneration in rat calvarial defect model

• Nam JH
• Noh KL
• Pang EO
• Yu WG
• Kang ES
• Kweon HY
• Kim SG
• Park YJ

Abstract

INTRODUCTION
This study evaluated the capability of silk fibroin (SF) and recombinant human bone morphogenetic protein-2 loaded SF (SF-BMP) as a bone defect replacement matrix when grafted in a calvarial bone defect of rats in vivo.
MATERIALS AND METHODS
A total 70 calvarial critical size defects (5.0 mm in diameter) made on 35 adult female Sprague-Dawley rats were used in this study. The defects were transplanted with (1) rhBMP-2 loaded silk fibroin graft (SF-BMP: 0.8+10 microg), (2) Silk fibroin (SF: 10 microg), and (3) no graft material (Raw). The samples were evaluated with soft x-rays, alkaline phosphatase activity, calcium/phosphate quantification, histological and histomorphometric analysis at postoperative 4 and 8 weeks.
RESULTS
The SF-BMP group (48.86+/-14.92%) had a significantly higher mean percentage bone area than the SF group (24.96+/-11.01%) at postoperative 4 weeks.(P<0.05) In addition, the SF-BMP group (40.01+/-12.43%) had a higher % bone area at postoperative 8 weeks than the SF group (33.26+/-5.15%). The mean ratio of gray scale levels to the host bone showed that the SF-BMP group (0.67+/-0.08) had a higher mean ratio level than the SF group (0.61+/-0.09) at postoperative 8 weeks. These differences were not statistically significant.(P=0.168 and P=0.243, respectively)
CONCLUSION
The rhBMP-2 loaded silk fibroin graft revealed fewer immunoreactions and inflammation as well as more new bone formation than the pure silk fibroin graft. Therefore, silk fibroin may be a candidate scaffold for tissue engineered bone regeneration.

Keyword

Silk; Fibroins; Recombinant human bone morphogenetic protein-2; Bone regeneration; Tissue scaffolds

MeSH Terms

Adult
Alkaline Phosphatase
Animals
Bone Morphogenetic Protein 2
Bone Regeneration
Female
Fibroins
Humans
Inflammation
Osteogenesis
Rats
Rats, Sprague-Dawley
Recombinant Proteins
Silk
Tissue Scaffolds
Transforming Growth Factor beta
Transplants
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Fibroins
Recombinant Proteins
Silk
Transforming Growth Factor beta

Figure

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  Fig. 1. Midsagittal reference line for histomorphometric analysis. A. Harvested calvaria, B. Soft X-ray image.

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  Fig. 2. % bone area of densitometric analysis. A. The SF-BMP group (48.86±14.92%) had a significant higher value than that of SF group (24.96±11.01%, *P<0.05) at postoperative 4 weeks. B. The SF-BMP group (40.01±12.43%) had a higher %bone area than that of SF group (33.26±5.15%) at postoperative 8 weeks. This difference was not, however, statistically significant.(P=0.168) (SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 3. Gray scale ratio to host bone. A. The SF-BMP group (0.70±0.07) and SF group (0.70±0.09) had a higher value than that of the Raw group (0.67±0.12) at postoperative 4 weeks. These differences were not, however, statistically significant.(P=0.418 and P=0.424, respectively) B. At postoperative 8 weeks, the SF-BMP group (0.67±0.08) had a higher value than that of the SF group (0.61±0.09), however, it was not statistically significant.(P=0.243) (SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 4. ALP activity. A. The SF-BMP group (23.71±8.60 U/L) had a significant higher value than that of the SF group (12.65± 6.47 U/L) at postoperative 4 weeks.(*P<0.05) B. The SF-BMP group (21.65±10.02 U/L) had a higher value than that of the SF group (16.72±7.35 U/L) at postoperative 8 weeks. However, there was not statistically significant difference.(P=0.263) (ALP: alkaline phosphatase, SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 5. Ca2+ quantification. A. The SF-BMP group (28.67±14.28 mg/dL) had a significant higher value than that of the SF group (12.53±5.01 mg/dL) at postoperative 4 weeks.(*P<0.05) B. The SF-BMP group (28.56±20.44 mg/dL) had a significant higher value than that of the SF group (2.83±1.38 mg/dL) at postoperative 8 weeks.(*P<0.05) (SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 6. PO42- quantification. A. The SF-BMP group (30.77±12.52 mg/dL) had a significant higher value than that of the SF group (12.28±11.87 mg/dL) at postoperative 4 weeks.(*P<0.05) B. The SF-BMP group(34.09±12.37 mg/dL) had a significant higher value than that of the SF group (4.25±2.34 mg/dL)at postoperative 8 weeks.(*P<0.05) (SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 7. A. Postoperative 4 weeks.(H&E staining, original magnification x100) B. Postoperative 8 weeks.(H&E staining, original magnification x100) C. Postoperative 4 weeks.(Masson's trichrome staining, original magnification x100) D. Postoperative 8 weeks.(Masson's trichrome staining, original magnification x100) E. Postoperative 4 weeks.(Osteocalcin immunoreactive staining, original magnification x100) F. Postoperative 8 weeks.(Osteocalcin immunoreactive staining, original magnification x100)(rhBMP-2: recombinant human bone morphogenetic protein-2, SF: silk fibroin, SF-BMP: recombinant human bone morphogenetic protein-2 loaded SF)

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  Fig. 8. A. Postoperative 4 weeks.(H&E staining, original magnification x100) B. Postoperative 8 weeks.(H&E staining, original magnification x100) C. Postoperative 4 weeks.(Masson's trichrome staining, original magnification x100) D. Postoperative 8 weeks.(Masson's trichrome staining, original magnification x100) E. Postoperative 4 weeks.(Osteocalcin immunoreactive staining, original magnification x100) F. Postoperative 8 weeks.(Osteocalcin immunoreactive staining, original magnification x100)(rhBMP-2: recombinant human bone morphogenetic protein-2, SF: silk fibroin)

Reference

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