| BackgroundPrimary stability is the one of the key factors for successful dental implant. During the healing phase, adequate primary stability prevent implant from excessive amount of micro-motion (100-150μm), result in osseointegration at the bone-implant interface by means of bone formation and remodeling around implant. It was suggested that choice of implant loading mainly depended on the primary stability. Distinct primary stability thresholds make sense since immediate or early loading implants were submitted to higher stresses and strains, therefore they should require a higher primary stability to withstand these biomechanical constraints. In clinical, the primary stability of an implant could be estimated usually by implant insertion torque value. By increasing the insertion torque it is possible to improve an implant’s primary stability. It has been suggested that implant could be expected to osseointegrate in immediate or early loading procedure, if implants were inserted with a torque of at least 30Ncm. However, high implant insertion torques produces compression and distortion on the peri-implant bone. This may induce deleterious effects on the local microcirculation, which may lead to bone necrosis. But it is disputed that higher insertion torque result in excessive bone absorption, even implant failure.Inflammation and trauma in the peri-implant bone induced by surgical drilling and compression from implant placement would lead to bone absorption and necrosis; therefore implant stability decreases in the early phase of healing. Subsequently, the implant stability in crease because of born regeneration and remodeling until ossintegration at bone-implant interface. It has been claimed that compression on the peri-implant bone would produced micro-fracture, further induced bone necrosis and disturbed osseointegration, when the implant was inserted with high insertion torque above 50-55Ncm. Duyck J et al. reported that high insertion torque (>50Ncm) caused significant marginal bone loss and overall peri-implant bone fraction around implants, and osseo-compression might have played a role in the marginal bone loss. Otherwise, some clinical reports and animal experiment showed that high implant insertion torque in dense cortical bone dose not induce bone necrosis or implant failure, but it dose increase the primary stability of implants, which is extremely important in immediate loading protocols.Installation of implants in the alveolar process elicits a sequence of healing events, including necrosis and subsequent absorption of traumatized bone around the titanium body concomitant with new bone formation. The first, osteoconduction, relies on the migration of differentiating osteogenic cells to the implant surface, through a temporary connective tissue scaffold. Osteoclasts are differentiated from osteogenic cells and migrate to bone surface, phagocytose necrosis bone tissue. Following the second phase, de novo bone formation, Osteoblast differentiated and maturated from osteoprogenitor, secrete un-mineralized collagen matrix (osteiod tissue). Alkaline phosphatase secreted from osteoblast, form alkaline the extracellular environment and result in a mineralized woven bone, being laid on the lacunae. At the bone remodeling phase, woven bone tissue will gradually replaced by lamellar bone. Osseointegration represents a dynamic process both during its establishment and its maintenance. In the establishment phase, there is a delicate interplay between bone resorption in contact regions between the titanium body and mineralized bone, and bone formation in ’contact-free’ areas by Wolf’s law.Osteoclasts (OC) and osteoblasts (OB) are the main participator for the bone modeling and remodeling process. Osteoclasts differentiate from bone marrow mesenchymal stem cells. In the bone regeneration and remodeling, OB is responsible for the synthesis, secretion and mineralization of bone matrix. After implant placement, because of stimulation by surgical trauma and compression, osteoclasts appearing on the surface of peri-implant bone lead to bone absorption and necrosis. Follow that, osteoblasts adhere to the necrotic bone’ surface, and secret and mineralize matrix resulting bone formation and regeneration. Recently, it is demonstrated that OPG/RANKL/RANK system is the major regulatory mechanisms of osteoclast differentiation and maturation. Osteoprotegerin (OPG)、 Receptor activitor of NF-κB (RANK) and Receptor activitor of NF-κB Ligand (RANKL) constitute OPG/RANKL/RANK cycle system, is an intercellular signal which induce formation and differentiation of osteoclasts. And so far, RANKL is the unique cytokine which directly induce osteoclast differentiation and regulate its function. OPG may link to RANK competitively, and block RANKL initiated signaling pathway by inhibiting the binding of RANKL and RANK. Therefore, ratio of RANK1/OPG expression determines the extent of bone absorption and necrosis. In addition, it was reported that the expression of RANKL and OPG in peri-implant bone would reach peak in 7d after implant installation, and then it would decrease gradually. OPG/RANKL system participates the formation of osteoclasts in the peri-implant bone, regulates bone absorption and necrosis, and affect on bone tissue metabolic microenvironment. The expression levels of OPG/RANKL/RNAK system would play a role to bone modeling and remodeling in peri-implant bone tissue.To study the effect of mechanical compression induced by implants inserted with low, medium and high insertion torque on peri-implant bone remodeling and osseointegration at bone-implant interface, and the effect of implant insertion torque on the expression of OPG/RANK/RANKL system and remodeling in the peri-implant bone during implant healing period, the animal experiments were designed. The study consists of two parts:experiment A, Effect of Insertion Torque on Implant Removal Torque; and experiment B, Effect of Insertion Torque on Expression of OPG/RANK/RANKL System in Peri-implant Bone Tissue.Experiment A:Effect of Insertion Torque on Implant Removal TorqueObjective To test the dental implants stability inserted with different insertion torque by removal torque test during a healing period, and to evaluate osseointegration on the implant-bone interface.Methods Nine healthy adult male Beagle dogs were selected, which were extracted of mandibular first to forth premolars to create edentulous regions,12 weeks later, titanium alloy dental implants with smooth surfaces (3.5×10mm)were inserted in the site of the extracted regions in each dog. Three dental implants were installed in the each side of the mandibular, the insertion torque were 10～30Ncm (low torque, group L),30～50Ncm (medium torque, group M), and >70Ncm (high torque, group H), respectively. The left side was considered as 1w group and the right side was considered as 4w group for three animals; the left side was considered as 4w group and the right side was considered as 8w group for another three animals; the left side was considered as 8w group and the right side was considered as 1w group for the rest of three animals. Clinical observation were taken at the corresponding time after the dental implant surgery, the animals were sacrificed and then were taken a reverse torque test(Rv) after a X-ray examination, the bone specimens were preserved for the subsequent experiment. Statistical analysis of the data was performed using ANOVA test by SPSS 18.0 software, statistically significant differences were accepted as P< 0.05.Results None of 9 experimental animals died, all of the 54 implants appeared no loosen and lost, the implant survival rate was 100%. All the implants were osseointegrated by X-ray examination. At post-operation 1w, the removal torque (Rv) were Rv= 57.83±10.00Ncm (group H), Rv=35.83±9.83Ncm(group M), Rv= 13.83±4.54Ncm(group L), respectively; a higher insertion torque revealed a higher reverse torque, there was a significant difference among the three groups(P<0.01). We found a decline tendency of implant stability in all the groups, the higher insertion torque showed a higher stability at the early time after the surgery. At post-operation 4w, the results were Rv= 58.33±8.29Ncm (group H), Rv= 35.00±9.25Ncm (group M), Rv=36.67±11.22Ncm (Group L), there was a significant difference among the three groups(P<0.01), there was no significant difference between the Group L and Group M (P> 0.05), however, there was a significant difference between Group L,M and Group H (P<0.01). Similar with Group M, the implant stability of Group L was significantly higher, but the implant stability is still lower than that of Group H. At post-operation 8w, the removal torque value were similar among the 3 groups, Rv=60.83±4.40 (Group H), Rv=59.83±7.94Ncm (Group M), Rv=60.67±3.61 (Group L), respectively, there was no significant difference among 3 groups in Rv(P>0.05). All implants have received good secondary stability and had ideal osseointegration at bone-implant interface.Conclusions Implanted with different insertion torque values, the stability declined at the early time after the surgery when compared with being implanted, but higher stability appears in high torque group. At the end of the 4w, the stability had obviously increased in low torque group, at the end of the 8w, all the implants have received good stability and osseointegration. Excessive bone tissue absorption around the implant didn’t be found with high insertion torque values. Implants will get enough stability during the bone healing if the insertion torque values can be controlled more than 30-50Ncm, also immediate or early load are accepted.Experiment B:Effect of Insertion Torque on Expression of OPG/RANK/ RANKL System in Peri-implant Bone TissueObjective The purpose of this experiment was to study the effect of mechanical compression induced by implants inserted with low, medium and high insertion torque respectively on the expression of OPG/RANK/RANKL system and the activation, differentiation and maturation of osteoclasts and osteoblasts in peri-implant bone tissue, to investigate the effect of implant insertion torque on the metabolic activity and bone regeneration and remodeling of the surrounding bone tissue from the perspective of molecular biology. Try to provide more theoretical basis and guidance for clinical treatment.Methods The experiment design and implant installation same as Experiment A. After the animals were sacrificed, the bone tissue specimens around the implant (5×5×10mm cuboid bone) were gotten by using electric reciprocating saw, and the upper half of specimens were cut out and saved in Carbon dioxide ice. Expression of OPG mRNA and RANKL mRNA were detected by fluorogenic quantitative PCR method. Statistical analysis (ANOVA) was performed with the SPSS statistical package (SPSS 18.0). A value<0.05 was considered statistical significant.Results Amplification curve inflection point clear, parallelism overall was well, and low concentration of the sample was significantly exponential amplification curve, implied specimens’ good amplification efficiency. Melting curve parallel overall. At post-operation 1w, the expression of OPG mRNA in 3 groups were at high level, group L= 6.65±0.34, group M=5.52±0.27, group H=6.53±0.30; there was a significant difference among the three groups(P<0.01). There was no significant difference between Group H and Groups L(P> 0.05); but there was significant difference between Group M and others groups(P<0.05). It implied bone regeneration activity of 3 groups were active, especially Group L and Group H were more obvious. At post-operation 4w, the expression of OPG mRNA were Group L=7.18±0.38, Group M=2.73±0.23 and Group H=5.16±0.36 respectively. There was significant difference between pairwise groups(P<0.01). Bone regeneration of Group M was to stabilization, and its of Group L and Group H were more active, especially Group L. Postoperative 8w, OPG mRNA expression values changed significantly, Group L=2.89±0.27, Group M=1.80±0.28, and Group H=2.21±0.27,there was a significant difference among the three groups(P<0.001). There was significant difference between Groups H and Group M(P<0.01); but there was no difference between Group H and Group M (P>0.05). OPG mRNA expression values of different groups were low, suggested that the peri-implant bone formation was to stabilization. At post-operation 1w, RANKL mRNA expression of 3 groups was at a high level, Group L=8.44±0.27, Group M=7.91±0.23, and Group H=6.09±0.25 respectively. There was significant difference between pairwise groups(P＜0.01). Osteoclast activity of 3 groups was active. Group L was the most, Group M followed, and H was the least. Postoperative 4w, RANKL mRNA expression was significantly decreased, Group L=5.51±0.51, Group M=4.63±0.38, and Group H=3.55±0.27. There was significant difference between pairwise groups (P<0.01). Osteoclast activity of 3 groups was stabilized. At post-operation 8w, RANKL mRNA expression were Group L=5.48±0.30, Group M=4.73±0.50, and Group H=4.84±0.42.there was a significant difference among the three groups (P<0.01). There was significant difference between Group M and Group L (P<0.05); and between Group H and Group L(P<0.05), but there was no difference between Group H and Group M. Osteoclast activity was similar to that of postoperative 4w. At post-operation 1w, the OPG/RANKL mRNA expression ratios were:Group L= 0.79±0.03, Group M=0.70±0.05, Group H=1.07±0.07. There was significant difference between pairwise groups (P<0.01). Bone remodeling of Group H was more active than the other two groups. At post-operation 4w, ratios were Group L=1.32±0.18, Group M=0.60±0.08, and Group H=1.46±0.11.there was a significant difference among the three groups (P<0.01). There were significant difference between Group M and other 2 groups (P<0.01); but there was no difference between Group L and Group H(P>0.05). Bone remodeling of Group H and Group L were active, but bone remodeling activity has stabilized. Postoperative 8w, the expression ratios were Group L=0.53±0.03, Group M=0.38±0.04, and Group H=0.46±0.07. there was a significant difference among the three groups (P＞0.01).There was no difference between Group L and Group H (P>0.05); but there were significant difference between Group M and the other 2 Groups (P<0.05). Because OPG/ RANKL mRNA expression ratios of each group were quiet low; and it indicated that bone remodeling activity of peri-implant bone tissue in 3 groups was stabilized.Conclusions:At post-operation 1w, OPG mRNA and RANKL mRNA expression were at a high level both, when implant were placed with different insertion torque, it implied bone regeneration and remodeling are active. To postoperative 8w, bone remodeling is to stabilization. OPG/RANKL mRNA ratio of Group H and Group L reach the peak at post-operation 4w, this may be related to the needs more new bone regeneration when implant is inserted with lower torque (<30Ncm) or higher torque (>70Ncm). Bone remodeling activity in Group M is stabilized at post-operation 4w means that peri-implant bone remodeling activity will be completion more evenly, when implant insertion torque is controlled between 30～50Ncm. The signal expression of OPG/RANKL/RANK system in the early stages of bone remodeling (7d postoperative) with high insertion torque (>70Ncm) should be study further. |
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