Fig 1.
Schematic diagram of the experimental design for PB or BM-based bone engineering with rhBMP-2, and the preliminary study to determine the suboptimal-dose of rhBMP2 on bone formation.
Typical histological appearance in specimens at 2 weeks following transplantation. (A) The 6 fractions (1, PB; 2, BM; 3, PPP; 4, PRP; 5, BM-PPP; 6, BMC) were mixed with the alloplastic material (β-TCP granules) containing a suboptimal dose (0.5 μg) of rhBMP2. (B) When 20mg β-TCP granules after adsorbed 0, 0,5 1 or 2 μg of rhBMP-2 were transplanted subcutaneously (as a model of ectopic bone formation), 1 μg rhBMP-2 could induce the minimal bone formation (arrows). Scale bars represent 200 μm. (C) When 20mg β-TCP granules after adsorbed 0, 0,5 or 1, 5 μg of rhBMP-2 were transplanted onto the cranium (as a model of vertical bone augmentation), 0.5 μg rhBMP-2 could induce the minimal bone formation. Scale bars represent 200 μm. The yellow dotted line indicates the boundary between the host bone and the specimen. Blue-arrows indicated the augmented bones.
Table 1.
Control- and experimental-groups for transplantation.
Table 2.
Numbers of mononuclear cells (MNCs) and platelets (PLTs) in each fraction.
Fig 2.
Typical histological appearance in specimens at 2 and 4 weeks following subcutaneous transplantation to the dorsal skin.
For each group, sections were stained with hematoxylin and eosin (H&E), and the scale bars represent 200 μm. The left and right panels show the typical appearance at 2 and 4 weeks, respectively, following transplantation. In the (A) PB, (B) PPP, (C) PRP, (D) BM, and (E) BM-PPP groups, minimal ectopic bone was detectable at 2 weeks but promoted bone formation (blue-arrows) was observed at 4 weeks. In the (F) BMC group, abundant ectopic bone (blue-arrows) was observed at 2 and 4 weeks. (G) The average area (%) of ectopic bone per whole area was measured in each group. At 2 weeks, the BMC group exhibited significantly bone formation (9.3±1.4%) compared to the other groups (approximately 1–2%) (p<0.05). At 4 weeks, the BMC and PRP groups showed abundant ectopic bone (10.2±3.3% and 10.7±3.8%), however, there were no significant differences among the groups. Values are the means ± standard deviation of five sections from each of the five specimens per group. The asterisk represents statistical significance (*p <0.05) between the BMC group and other groups.
Fig 3.
Typical histological appearance in specimens at 2 weeks following transplantation onto the cranium.
Coronal plane sections were stained with hematoxylin and eosin (H&E). For each group, the left panel shows the whole area of specimens (scale bars represent 1 mm), and the right panel shows the magnified image of the white box in the left panel (scale bars represent 200 μm). A small amount of new bone was detected along the host bone in the (A) PB, (B) PPP, (C) PRP, and (E) BM-PPP groups. The (D) BM and (F) BMC groups showed obvious bone formation along the host bone. (G) New bone area surrounded the β-TCP particles and connected with the host bone [box area in (F)]. (H) Masson’s trichrome staining showed the immature (blue) and mature (red) bone in the new bone area [box area in (F)]. (I) Human vimentin immuno-staining showed a few positive cells at the surface of newly formed bone adjacent to the β-TCP particles. The yellow dotted line indicates the boundary between the host bone and the specimen.
Fig 4.
Typical histological appearance in specimens at 4 weeks following transplantation onto the cranium.
Coronal plane sections were stained with hematoxylin and eosin (H&E). For each group, the left panel shows the whole area of specimens (scale bars represent 1 mm), and the right panel shows the magnified image of the white box in the left panel (scale bars represent 200 μm). New bone area was obviously promoted along the host bone and β-TCP particles in the (A) PB, (B) PPP, (C) PRP, (D) BM, (E) BM-PPP, and (F) BMC groups, and replacement bone tissue was clearly observed at the surface of the β-TCP particles in the magnified areas. (G, H) The surface of β-TCP particles was being resorbed and replaced with new bone at the far site from the host bone, and newly formed bone was observed to be more mature (red stained by Masson’s trichrome) [box area in (F)]. (I, J) The newly formed bone was sufficiently integrated with the host bone, and appeared mature (red stained by Masson’s trichrome) [box area in (F)]. The yellow dotted line indicates the boundary between the host bone and the specimen.
Fig 5.
Average area (%) of newly formed bone in the whole area (A) and at the surface of β-TCP particles (B). (A) At 2 weeks, BMC and BM groups showed considerable new bone formation, and the amount of new bone in the BM group was significantly increased compared to the Control, PB, PPP, PRP, and BM-PPP groups (p <0.05). At 4 weeks, bone formation was promoted in all groups. In particular, new bone area was obviously augmented in the BMC, BM, and PRP groups. (B) When the area of replaced bone tissue at the surface of β-TCP particles was assessed, BMC and BM groups showed significantly increased area compared to the Control group at 2 weeks (p <0.05). Then, the BMC group showed more prominent bone formation at 4 weeks. In particular, a significant difference was found between BMC and PRP groups. Values are the means ± standard deviations of five sections from each of the five specimens per group. The asterisk represents statistical significance (*p <0.05) among experimental groups, and the triangle-mark represents statistical significance (▲p <0.05) between the Control and other groups.
Fig 6.
Synergistic effect of BMC and BMP-2 on bone formation.
(A) Bone formation at 2 weeks post-transplantation. Scale bars represent 200 μm. The newly formed bone was integrated with the host bone. rhBMP-2 at 0.5 μg induced a limited amount of new bone formation while 1 μg rhBMP-2 and BMC (BMC represents the BMC group) promoted bone formation to surround β-TCP particles and connect to the host bone. Black asterisk: β-TCP particles, blue arrow: newly formed bone, and yellow dotted line: boundary between the host bone and specimen. (B) Comparison of the area (%) of newly formed bone in each group. PRP represents the PRP group. Values are the means ± standard deviations of five sections from each of the five specimens per group. The asterisk represents statistical significance (*p <0.05) among groups.