Osteoclasts: The Biological Knife In Sutural Responses To Mechanical Stimulation

Mani Alikhani, Sarah Alansari, Mohammed M Al Jearah , Niraj Gadhavi, Mohammad A Hamidaddin, Fadwa A Shembesh, Chinapa Sangsuwon, Jeanne M Nervina, Cristina C Teixeira

Figure 1: Calibrated springs used to produce transverse forces in the rat maxilla. Springs were fabricated from 0.016” stainless steel wires (3M Unitek, Monrovia, CA, USA), with two vertical loops used to tie the spring to the maxillary first molars using 0.008” stainless steel ligature ties and composite. (A) Photograph of springs installed in the rat maxilla. (B) The springs were calibrated using a digital force gage to produce 100cN force when expanded from 4mm (left) to 6mm (right) (VL = vertical loop, SST = stainless steel tie, M1 = first molar, M2 = second molar, M3= third molar).

Figure 2: Changes in sutural, palatal and dental widths over time. (A) Palatal width, dental width and mid-palatal suture widths were measured using µCT 3D reconstructed images and sections at the level of the mid-coronal plane of the maxillary first molar. Red line shows the width of the mid-palatal suture (distance between the borders of the suture), Green line shows the width of palate (distance between the palatal walls at the level of intersection between the palate and alveolar walls), and Blue line shows the dental width (distance between height of contour of first molars). (B) Sutural, palatal and dental widths were measured over time in the Experimental and Sham maxillae. Data expressed as the mean ± SD of distances in mm. Each number represents the average of 5 samples. (* Experimental width significantly different from Sham, p<0.05). (C) Coronal (through the mid-coronal sections of the maxillary first molars) and occlusal (from oral cavity) µCT images of the mid-palatal suture opening at different time points.

Figure 3: Increase in sutural width in response to transverse forces occurs in both tensile and compressive sutures. (A) 3D µCT reconstructed images of rat heads showing surrounding sutures that connect the maxilla to adjacent bones. Arrows point to the location of the zygomaticomaxillary, zygomaticotemporal, transverse and frontomaxillary sutures. (B) 3D µCT reconstructed images of the sutures at day 7 for both Sham and Experimental group maxillae show increased width for all the sutures (red box detail). (C) Widths of sutures were measured over time in both Experimental and Sham maxillae. Data expressed as the mean ± SD of distances in mm. Each number represents the average of 5 samples. (* Experimental width significantly different from Sham, p<0.05).

Figure 4: Osteoclast number and activity increase significantly in the mid-palatal suture in response to tensile forces. (A) Immunohistochemistry for TRAP was performed in paraffin section of both Experimental and Sham maxillae to identify active osteoclasts in the area. Light microphotographs show TRAP-positive osteoclasts in the mid-palatal suture and surrounding bone at different time points. All images were collected at the suture area between the first and second molars. Osteoclasts are stained as multi-nucleated red cells (black arrows, magnification 10X). (B) Mean numbers of osteoclasts at different time points, in the area of the mid-palatal suture and adjacent bone. Each value represents the mean ± SD of five animals. (* Significantly different from Sham, p<0.05). C) Change in expression of osteoclast markers (RANKL, RANK, OPG and CtsK) in the mid-palatal suture of the Experimental and Sham groups at different time points during application of transverse forces was measured by RT-PCR. Data presented as mean “fold” change in expression in comparison to Sham expression. Each number represents the average of 5 samples. (* Significantly different from Sham at the same time point, p<0.05). (D) Bone density was quantified using µCT data in the area of the mid-palatal suture and surrounding palatal bone. Data shown as mean ± SD of BV/TV percentage (bone volume/ tissue volume). (* Significantly different from Sham at same time point, p<0.05).

Figure 5: Increased and sustained expression of inflammatory markers in both the mid-palatal suture and PDL correlate with osteoclast activity. Expression of chemokines (A), cytokines (B), and their receptors (C) in the mid-palatal suture and PDL of the Sham and Experimental groups was measure by RT-PCR 24 hours after force application. Data are expressed as the Experimental mean “fold” change ± SD in gene expression compared to the Sham group. Each number represents the average of 5 samples. All 29 genes were significantly different between Experimental and Sham at both locations. (* Significantly different between suture and PDL in experimental group, p <0.05). (D) Mean concentration of IL-1, TNF-, CCL2, CCL5 in mid-palatal suture and PDL at different time points was evaluated by ELISA. Data expressed as the mean ± SD of concentration in picograms per 100 mg of tissue. Each number represents the average of 5 samples. (* Significantly different from Sham; # significantly different between Experimental suture and Experimental PDL, p<0.05). (E) Mean concentration of RANKL in the mid-palatal suture and PDL of Sham and Experimental groups at different time points was evaluated by ELISA. Data expressed as the mean ± SD of concentration in picograms per 100 mg of tissue. Each number represents the average of 5 samples. (*Significantly different from Sham, p<0.05).

Figure 6: Anti-inflammatory medication blocks the catabolic effect of transverse forces. (A) µCT images of representative maxillae from Sham, Experimental, and Experimental + NSAIDs groups after 14 days of force application. (B) H&E and TRAP staining of histological sections taken from the mid-palatal suture in the area of contact between the first and second molars in Sham, Experimental and Experimental + NSAIDs groups after 7 and 14 days of transverse force application. Osteoclasts are red multinucleated cells (black arrows). Change in expression of (C) RANKL, Ctsk and (D) IL-1 and CCL2 in the mid-palatal suture in the presence and absence of NSAID was measured by RT- PCR at Days 3 and 7. Data are expressed as mean ± SD “fold-change” in expression in comparison to Sham. Each value represents the average of 5 samples. (* Significantly different from Sham group, p<0.05).

Table 1: Morphological changes in the maxillary palatal, dental and sutural widths in response to application of transverse forces in the absence (Exp) and presence (Exp + NSAID) of anti-inflammatory medication. Data shown as mean + SEM of 5 samples (* Significantly different from Sham; # Significantly different from Exp +NSAID)

Figure 7: Osteoclasts activity is a pre-requisite for osteoblast activity in the mid-palatal suture in response to transverse forces. (A) Change in expression of osteogenic markers (collagen I, ALP, Osteopontin and osteocalcin) in the mid-palatal suture at different time points by RT-PCR. Data are expressed as mean ± SD “fold-change” in expression in comparison to Sham. Each value represents the average of 5 samples. (* Significantly different from Sham group, p<0.05). (B) Change in expression of ALP, Osteopontin and Osteocalcin in the mid-palatal suture in the presence or absence of anti-inflammatory medication was measure by RT-PCR at Day 14. Data are expressed as the mean ± SD “fold-change” in expression compared to Sham. Each value represents the average of 5 samples. (* Significantly different from Experimental + NSAID group and from the Sham group, p <0.05). (C) Mean activity of ALP during 28 days was measure spectrophotometrically. Transverse forces were able to increase ALP in Experimental group. Each value represents the average of 5 samples. (* Significantly different from Sham group, p<0.05). (D) Bone density was quantified using µCT data in the inter-radicular area of the first maxillary molar at Day 28. Data shown as mean± SD of BV/TV percentage (bone volume/ tissue volume) (* Significantly different from Sham group, p<0.05). (E) Fluorescence microscopy images of mid-palatal suture after 28 days of expansion. Bone labeling was performed by Calcein on days 0 (Green), Xylenol Orange at day 12 (Orange) and Demoxicycline at day 26 (Yellow). Experimental group maxilla demonstrates significant bone formation activity around the suture evidenced by increase fluorescence, which was reduced in animals that received anti-inflammatory medication (Experimental +NSAIDs).

Figure 8: Expression of transitional molecules activated during sutural response to transverse forces. Gene expression of BMP-6, TGF- and Wnt10b in the mid-palatal suture was measured by RT-PCR from Day 3 to Day 28. Data expressed as the mean ± SD “fold” change in comparison to Sham group. Each value represents the average of 5 samples (* significantly different from Sham group, p<0.05).

Figure 9: Model of events in sutures in response to orthopedic transverse tensile forces. As an initial response to tensile forces, a robust and sustained inflammatory response (markers: IL1, TNF-, CCL-2, CCL-5) recruits osteoclasts precursors into the area of mid-palatal suture and adjacent bone. As a result of osteoclast activity (markers: RANK and RANKL, CtsK, TRAP) and bone resorption, there is a significant reduction in the bone density in the area and the suture width is visibly increased. After this initial stage of overt catabolism in both tensile and compressive sutures, there is a transition marked by stimulation of bone formation via coupling factors including diffusible factors, membrane bound molecules and factors embedded in bone matrix (markers: TGF-, BMP6, WNT10b and SP1). During this period and only after the catabolic stage, does the palatal and dental widths significantly increase, and physical expansion finally occurs. This transition period results in osteoblast stimulation and is followed by a strong anabolic phase (markers: Coll I, ALP, OC, OP) that ensures that the resorbed bone is replaced with new bone, re-establishing the integrity of the suture in the framework of the new skeletal dimensions.