Effects of mechanical repetitive load on bone quality around implants in rat maxillae

Abstract

Greater understanding and acceptance of the new concept "bone quality", which was proposed by the National Institutes of Health and is based on bone cells and collagen fibers, are required. The novel protein Semaphorin3A (Sema3A) is associated with osteoprotection by regulating bone cells. The aims of this study were to investigate the effects of mechanical loads on Sema3A production and bone quality based on bone cells and collagen fibers around implants in rat maxillae. Grade IV-titanium threaded implants were placed at 4 weeks post-extraction in maxillary first molars. Implants received mechanical loads (10 N, 3 Hz for 1800 cycles, 2 days/week) for 5 weeks from 3 weeks post-implant placement to minimize the effects of wound healing processes by implant placement. Bone structures, bone mineral density (BMD), Sema3A production and bone quality based on bone cells and collagen fibers were analyzed using microcomputed tomography, histomorphometry, immunohistomorphometry, polarized light microscopy and birefringence measurement system inside of the first and second thread (designated as thread A and B, respectively), as mechanical stresses are concentrated and differently distributed on the first two threads from the implant neck. Mechanical load significantly increased BMD, but not bone volume around implants. Inside thread B, but not thread A, mechanical load significantly accelerated Sema3A production with increased number of osteoblasts and osteocytes, and enhanced production of both type I and III collagen. Moreover, mechanical load also significantly induced preferential alignment of collagen fibers in the lower flank of thread B. These data demonstrate that mechanical load has different effects on Sema3A production and bone quality based on bone cells and collagen fibers between the inside threads of A and B. Mechanical load-induced Sema3A production may be differentially regulated by the type of bone structure or distinct stress distribution, resulting in control of bone quality around implants in jaw bones.

Fig 1. Experimental design.

(A) Scheme of titanium implant. Length and diameter of implants, and pitch, height and angle of thread were presented. (B) Implant placement was performed after extraction of both maxillary first molars (M2 and M3 indicate second and third molar, respectively). (C) Experimental schedule. Seven rats were used. A randomly selected implant per rat received mechanical loads (n = 7 sites). Remaining implants received no mechanical loads (n = 7 sites, control). (D) Mechanical repetitive load (10N, 3Hz, and 1800 cycles) was applied twice a week for 5 weeks at 3 weeks post-implant placement. (E) Region of interest (ROI; aqua area) for micro computed tomography (microCT) analysis. ROI did not include the area from the implant surface to 50μm away from implants to avoid titanium metal artifacts. (F) Areas of interest (AOIs; yellowish area, aqua area and yellow-green area) for histological analyses. Analyses were performed on the inside and outside areas of the first thread (designated as thread A) and the second thread (designated as thread B).

Fig 2. Effects of mechanical repetitive load on bone around implants.

(A) Representative transverse microCT images at the neck, center and top of the implant. (B) Bone volume/tissue volume (BV/TV) around implants did not change between groups (Mesial root: mesial root of maxillary second molars). (C) Trabecular number (Tb.N) was significantly larger in the loading vs. control groups. (D) Trabecular thickness (Tb.Th) was the same between groups. (E) Trabecular separation (Tb.Sp) was similar between groups. (F) Bone mineral density (BMD) around implants increased significantly in the loading vs. control groups. (n = 7, *p<0.05).

Fig 3

Effects of mechanical repetitive load on bone inside threads A and B (A) Representative images of H-E-stained sections (Bar: 100 μm). (B) Osteocyte density was the same inside thread A in the loading vs. control groups. (C) Representative images of H-E-stained sections (Bar: 100 μm). (D) Osteocyte density was significantly higher inside thread B in the loading vs. control groups. (E) Representative images of Runx2 stained-sections (Bar: 100μm; Arrowheads indicate Runx2⁺ osteoblasts). (F) Osteoblast number (N.Ob/BS) was the same inside thread A in the loading vs. control groups. (G) Representative images of Runx2-stained sections (Bar: 100μm; arrowheads indicate Runx2⁺ osteoblasts). (H) N.Ob/BS was significantly increased inside thread B in the loading vs. control group. (I) Representative images of TRAP-stained sections (Bar: 100 μm; Arrowheads indicate TRAP⁺ osteoclasts). (J) Osteoclast number (N.Oc/T.A) did not change inside thread A between groups. (K) Representative images of TRAP-stained sections (Bar: 100 μm; arrowheads indicate TRAP⁺ osteoclasts). (L) N.Oc/T.A was the same between groups. (n = 7 each, *p<0.05).

Fig 4

Production of Sema3A inside threads A and B (A) Representative images of Sema3A-stained sections of the inside area of thread A (Bar: 100 μm). (B) Sema3A levels were similar between groups. (C) Representative images of Sema3A-stained sections of the inside area of thread B (Bar: 100μm). (D) Sema3A levels were significantly higher in the loading vs. control group. (arrowhead: Sema3A, n = 7, *p<0.05).

Fig 5

Effects of mechanical repetitive load on collagen fibers inside and outside area of threads A and B (A) Representative images of picrosirius red-stained sections with polarized microscopy and emphasized images in thread A (Bar: 100 μm; green and yellow-orange indicate type III and type I collagen, respectively). (B) Type I collagen area fraction was significantly smaller inside thread A vs. outside thread A at 200–400 μm away from implants under non-loaded conditions. (C) Type III collagen area fraction was similar, irrespective of analyzed areas under non-loaded conditions. (D) Type I collagen area fraction was higher in the loading vs. control group, but the difference was not significant. (E) Type III collagen area fraction was significantly higher in the loading vs. control groups. (F) Representative images of picrosirius red-stained sections with polarized microscopy and emphasized image in thread B. (Bar: 100 μm; green and yellow-orange represent type III and type I collagen fibers, respectively) (G) Type I collagen area fraction was smaller inside thread B vs. outside thread B at 200–400 μm away from implants under non-loaded conditions. (H) Type III collagen area fraction was smaller inside thread B vs. outside thread B at 0–200 μm and 200–400 μm away from implants under non-loaded conditions. (I) Type I collagen area fraction was significantly higher in the loading vs. control group. (J) Type III collagen area fraction was significantly higher in the loading vs. control groups. (K) Angle differences of collagen alignment to the long axis of implants were similar among groups under non-loaded conditions. (L) Representative images of the preferential alignment of collagen fibers in thread A (yellow represents collagen fibers and double arrowheads indicate the preferential alignment of collagen fibers). (M) Angle difference of collagen alignment in the upper flank of thread A was similar between groups. (N) Angle difference of collagen alignment in the lower flank of thread A was similar between groups. (O) The angle difference of collagen alignment to the long axis of implants inside thread B was bigger than that outside thread B, but the difference was not statistically significant. (P) Representative images of the preferential alignment of collagen fibers in thread B. (Q) Angle difference of collagen alignment in the upper flank angle of thread B was the same between groups. (R) Angle difference of collagen alignment in the upper flank angle of thread B was significantly smaller in the loading vs. control groups. (Bar: 100μm) (n = 7 each, *p<0.05, **p<0.01).