Alikhani M a,b, Alikhani M a, Sangsuwon C a, Oliveira SP a,c, Abdullah F a, Teixeira CC d
Figure 1: Schematic of model for application of static forces and periosteal stimulation. Calibrated springs used to produce static forces on the rat maxilla were fabricated from 0.016” stainless steel wires (3M Unitek, Monrovia, CA, USA) and secured to teeth by flowable resin. Photograph of spring installed in the rat maxilla (A). The springs were calibrated using a digital force gage to produce 100cN force when expanded from 4 to 6 mm (B). Periosteum stimulation was performed using 8 needles attached to a handle (C). The device was used to produce small perforations in the periosteum. Perforations were applied in the area of buccal cortical plate of second molars (D). (M1 = first molar, M2 = second molar, M3= third molar).
Figure 2: Static forces increase the number of osteoclasts in periosteum. In response to application of 100cN static forces osteoclasts number and activity increased significantly in the periosteum of the buccal surface of alveolar bone. Immunohistochemistry for TRAP was performed in paraffin sections to identify active osteoclasts in the area shown in this image (A). Light microphotographs show TRAP-positive osteoclasts in the surface of cortical bone at different time points osteoclasts are stained as dark red cells (arrowheads in B, magnification 20X). Mean numbers of osteoclasts at different time points (B), were defined as TRAP-positive cells on the surface of cortical bone of maxillary alveolar bone between the mesial border of the mesial roots of the first molars and distal border of the distal roots of the second molars as shown in the dashed box in A. Each value represents the mean ± SD of five animals (C). *Static group significantly different from sham, p<0.05.
Figure 3: Increase in osteoclasts in periosteum follows palatal width increase but precedes tooth movement. Palatal width and dental width of the maxilla were measured using micro CT 3D reconstructed images, and sections at the level of the mid-coronal plane of the maxillary second molar. Green line shows the width of palate (distance between the palatal walls at the level of intersection between palate and alveolar walls), and blue line shows the dental width (distance between height of contour of second molars) (A). The palatal widths were measured over time in both Static and Sham maxillae (B). Unilateral tooth movement was measured as described in Materials & Methods section at different time points (C). Data expressed as the mean ± SD of distances in mm. Each number represents the average of 5 samples. * Static group significantly different from sham, p<0.05. Histological section of periosteum in the area of second molar at day 7 shows osteoclasts activation in the periosteum ahead of tooth movement and before tooth reach the surface of cortical bone (D). Trap staining of the hemimaxilla in non-tooth bearing area midway between posterior teeth and anterior teeth demonstrates osteoclasts activation in periosteum and bone, in response to transverse forces applied to posterior teeth. Black arrows illustrate the direction of force (E).
Figure 4: Activation of osteoclasts is independent of changes in convexity of the cortical bone. 100 cN static force was applied perpendicular to hemimaxilla expanding the palate (increasing the convexity in buccal cortical plate), or constricting the palate (decreasing the convexity of buccal cortical plate). Histological sections were taken in area of buccal cortical plate of second molar. TRAP staining was performed on 14 days sections to identify osteoclasts. Application of static forces toward the cortical plate and periosteum (A) or in opposite direction (B), both stimulate osteoclasts activation in the periosteum. Black arrows show the direction of the force. Application of 100cN static force to posterior teeth stimulate TRAP-positive cells not only in periosteum but also in endosteum (C). Section includes bone in the area of second molar extending from suture to lingual cortical plate. TRAP staining shows day 14 of the static group. Osteoclasts activation was observed in suture, endosteum of palate and in the tension side of PDL.
Figure 5: Static forces increase inflammatory markers in periosteum. Mean protein concentration of IL-1α, TNF-β, CCL2, CCL5 in soft tissue (including periosteum) covering the buccal cortical plate was evaluated at different time points in periosteum by ELISA (A). Data expressed as the mean ± SD of concentration in picograms per 100 mg of tissue. Each number represents the average of 5 samples. * Static group significantly different from sham group at the same time point, p<0.05. TRAP staining of histological sections taken from buccal cortical plate of second molars in static and static plus anti-inflammatory medication after 7 days of application of static forces (B). TRAP staining of the first molar area after application of 100cN force in sagittal direction for 14 days (black arrow) (C).
Figure 6: Static forces activate osteoblasts in periosteum resulting in bone formation. 3D micro CT reconstructed images of coronal section of maxilla at the area of second molars were compared between sham and static groups. Static group demonstrate significant decrease in bone density in area of second molars at day 28. However, after 56 days, the animals that received static force showed significant reconstruction of buccal cortical plate (A). Change in expression of osteogenic markers TGFβ1,collagen 1, ALP, Osteopontin and Osteocalcin in soft tissue covering the cortical plate of posterior teeth at different time points was measured by RT-PCR. Data are expressed as mean ± SD “fold” change in expression in comparison to control. Each value represents the average of 5 samples. * Significantly different from control group, p<0.05 (B). Schematic showing the biomechanical analysis of the force applied to the crown of the maxillary molars (C). In response to this static force (F= force), a moment will appear in the system (M = F x d, where M = moment, and d = distance between force application and center of resistance of the tooth) that produces a high stress area in the alveolar crest of the buccal cortical plate where the higher magnitude of bone formation was observed (C).
Figure 7. Periosteal Stimulation Increases inflammation and the number of TRAP-positive cells. To study the effect of TRAP-positive cells on bone formation we increased the number of TRAP-positive cells in the periosteum by periosteal stimulation with small needles in the area of the second maxillary molar. Change in expression of CCL2, IL-1α, RANK, RANKL and Cathepsin K (Ctsk) in the soft tissue covering the cortical plate of posterior teeth was measured by RT- PCR at different time points. Data are expressed as mean ± SD “fold” change in expression in comparison to control. Each value represents the average of 5 samples. *Significantly different from static group at the same time point, p<0.05) (A and B). Immunohistochemistry for TRAP was performed in paraffin section of both “static” and “static + stim” groups to identify TRAP-positive cells in the periosteum of maxillary alveolar bone. Representative light microphotographs show TRAP-positive osteoclasts in surface of cortical bone at 3 and 7 days (C). Mean numbers of TRAP-positive cells at different time points, in the area between the mesial border of the mesial roots of the first molars and distal border of the distal roots of the second molars. Each value represents the mean ± SD of five animals. * Significantly different from control, p<0.05 (D).
Figure 8: Cortical bone formation was dependent on osteoclast activation in the periosteum. Osteoclasts activity was increased by bilateral application of periosteal stimulation in the area of the second molars and the effect on bone formation was compared by microCT images of coronal sections of maxilla in the area of second molars at day 56. Sections of Sham, Static and Static + Stim are shown 56 days after stimulation (A). Fluorescent microscopy images of coronal section of maxilla of Sham, Static, or Static + Stim at the area of second molars at day 56 are shown (B). Bone labeling was performed by Calcein green on day 0, 28 and 54. Micro CT images of mid-coronal section of maxilla at the area of second molars in animals that received static force in the maxilla and stimulation only in one side show asymmetrical bone formation (C). Fluorescent microscopy images of mid-coronal section of maxilla of animals that received static and unilateral periosteal stimulation in the area of second molars at day 56 (D). Bone labeling was performed by Calcein on days 0 (Green), and Xylenol Orange at day 26 and 54 (Orange). White arrows mark the change in the width of the cortical bone over the second molar area.
