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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 μ g), (2) Silk fibroin (SF: 10 μ g), 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) The ratio of the calcium and phosphate contents of the SF-BMP (0.93±0.22) group was lower than that of the SF (1.90±1.42) group at postoperative 4 weeks. However, the SF-BMP group (0.75±0.31) had a higher Ca/PO4 ratio than the SF (0.68±0.04) at postoperative 8 weeks. These differences were not statistically significant.(P=0.126 and P=0.627, respectively) For the bone-specific alkaline phosphatase (ALP) activity, which is recognized as a reliable indicator of the osteoblast function, the SF-BMP (23.71±8.60 U/L) groups had a significantly higher value than the SF group (12.65± 6.47 U/L) at postoperative 4 weeks.(P<0.05) At postoperative 8 weeks, the SF-BMP (21.65±10.02 U/L) group had a lower bone-specific ALP activity than the SF group (16.72±7.35 U/L). This difference was not statistically significant.(P=0.263) For the histological evaluation, the SF-BMP group revealed less inflammation, lower foreign body reactions and higher bone healing than the SF group at postoperative 4 and 8 weeks. The SF group revealed more foreign body reactions at postoperative 4 weeks. However, this immunogenic reaction decreased and the remnant of grafted material was observed at postoperative 8 weeks. For histomorphometric analysis, the SF-BMP group had a significantly longer bone length to total length ratio than those of the SF group at postoperative 4 and 8 weeks.(P<0.05)
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Fig. 1.
Midsagittal reference line for histomorphometric analysis. A. Harvested calvaria, B. Soft X-ray image.
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)
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)
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)
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)
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)
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)
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)
Table 1.
Animal grouping
| Groups | N (sites/animals) | Composition | Graft | |
|---|---|---|---|---|
| 4 weeks | 8 weeks | |||
| SF-BMP | 12 (6) | 10 (6) | rhBMP-2 loaded silk fibroin | 0.8+10 μ g |
| SF | 12 (6) | 12 (6) | Silk fibroin | 10 μ g |
| Raw | 12 (6) | 12 (5) | Raw defect | None |
Table 2.
Dry weight
| Groups | 4 weeks (mg) | 8 weeks (mg) |
|---|---|---|
| SF-BMP | 635.83±21.09 | 32.17±23.05 |
| SF | 611.75±4.03 | 7.00±2.83 |
| Raw | 616.83±13.35 | 28.17±13.47 |
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