Randomised Controlled Trial

Dental Implants

Int. J. Oral Maxillofac. Surg. 2018; 47: 651–657https://doi.org/10.1016/j.ijom.2017.11.001, available online at https://www.sciencedirect.com

The impact of different torquesfor the insertion of immediatelyloaded implants on the peri-implant levels of angiogenesis-and bone-related markersA. Verrastro Neto, R. Andrade, M. G. Correa, R. C. V. Casarin, M. Z. Casati, S. P.Pimentel, F. V. Ribeiro, F. R. Cirano: The impact of different torques for the insertionof immediately loaded implants on the peri-implant levels of angiogenesis- and bone-related markers. Int. J. Oral Maxillofac. Surg. 2018; 47: 651–657. ã 2017International Association of Oral and Maxillofacial Surgeons. Published by ElsevierLtd. All rights reserved.

Abstract. The aim of this split-mouth, randomized, double-blind, controlled clinicaltrial was to evaluate the influence of different insertion torque values for dentalimplants on bone- and angiogenesis-related marker profiles. Eighteen edentulouspatients received dental implants and fixed complete-arch mandibular prostheses.The implants (n = 36) were assigned randomly to two groups: reduced torque(n = 18), with insertion torque <30 N cm; and conventional torque (n = 18), withinsertion torque �30 N cm. Levels of vascular endothelial growth factor (VEGF),placental growth factor, bone morphogenetic protein 9 (BMP-9), periostin,osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) in the peri-implant fluid were quantified at 7, 14, 30, and 120 days after implant placement.Inter-group comparisons showed that VEGF and OPG levels were higher in the low-level torque group than in the conventional torque group on days 7 and 30,respectively (P < 0.05). BMP-9 and periostin levels were higher in the conventionalgroup than in the low-level torque group on day 120, and TRAP was up-regulatedaround implants inserted with conventional torque when compared to those insertedwith lower-level torque at all time points evaluated (P < 0.05). In conclusion, theuse of different levels of torque for implantation of immediately loaded implantssignificantly influenced the levels of bone- and angiogenesis-related markers duringearly peri-implant repair.

0901-5027/050651 + 07 ã 2017 International Association of Oral and Maxillofacial Surge

A. Verrastro Neto, R. Andrade,M. G. Correa, R. C. V. Casarin,M. Z. Casati, S. P. Pimentel,F. V. Ribeiro, F. R. CiranoDental Research Division, School of Dentistry,Paulista University, Sao Paulo, Brazil

Key words: torque; immediate dental implantloading; bone; biological markers; protein array.

Accepted for publication 3 November 2017Available online 23 November 2017

ons. Published by Elsevier Ltd. All rights reserved.

652 Verrastro Neto et al.

Treatment with dental implants is anestablished and predictable technique forthe rehabilitation of partially and totallyedentulous patients1,2. The use of imme-diate- and early-loading implants has in-creased over the years, accelerating thefunction of the implants3,4. Primary stabil-ity, defined as the absence of implantmicromovements and directly influencedby the mechanical connection between theimplant surface and the surrounding bone,has been described as essential for thesuccess of dental implants, and is impor-tant to support early functional loading5–8.Experimental and clinical studies have

attempted to identify a minimum insertiontorque value to obtain adequate stabilityfor immediate loading. It has been pro-posed that implants need to be insertedwith a torque of at least 30 N cm for animmediate-loading protocol9. In a clinicalstudy on the immediate loading of single-tooth implants, Ottoni et al. establishedthat immediate loading should only beproposed when the insertion torque ishigher than 32 N cm10. In line with this,Neugebauer et al. concluded that implantsplaced with an average insertion torquehigher than 35 N cm were the most suc-cessful in minipigs11.However, there are conflicting reports

in the literature regarding the conse-quences of using different levels of inser-tion torque during implant placement1,9,12.From a biological perspective, some stud-ies suggest that a higher insertion torqueresults in bone resorption before integra-tion through bone formation13–15. On theother hand, other studies have reportedthat lower insertion torques allow implantmicromovement and connective tissueformation, leading to an absence ofosseointegration5,15. Furthermore, it hasbeen reported that the use of a reducedinsertion torque may result in emptyspaces between the implant and the osteot-omy site, which may lead to the formationof a blood clot immediately after implantplacement and rapid replacement withwoven bone, without prior bone resorp-tion13. Considering the results of theseprevious studies, it could be hypothesizedthat implants inserted with a reduced tor-que may promote a more promising pat-tern of bone repair when compared tothose inserted with higher insertion tor-ques, although this goes against therecommendations for insertion torque pro-posed in some studies9–11.Information in the literature regarding

the peri-implant bone healing profile un-der different types of insertion torque isscarce, particularly for immediately load-ed implants. In light of this, the objective

of this study was to evaluate whether theuse of different levels of insertion torquefor immediately loaded dental implantscould interfere with the levels of angio-genesis mediators and osteoblastogenesis-and osteoclastogenesis-related factors inthe peri-implant crevicular fluid duringearly peri-implant repair. These moleculesare key markers of the host’s response andare thought to be critical mediators of bonerepair.Due to the emerging clinical demand for

immediate implant loading, new evidenceregarding the peri-implant angiogenesis-and bone-related biomarker profiles asso-ciated with immediately loaded implantsinserted with different levels of torquewould help to establish better surgicaltechniques for implant placement, therebyimproving peri-implant repair. This in turncould contribute to improving the successof immediately loaded implants.It was hypothesized that the use of

different levels of torque for the insertionof immediately loaded dental implantscould modulate the local pattern of boneand vascular mediators during early bonehealing around implants. Benefits in termsof the release of at least some bone andvascular mediators were expected usingthe reduced torque approach, i.e. the up-regulation of angiogenic factors (vascularendothelial growth factor (VEGF) andplacental growth factor (PLGF)) andosteoblastogenic factors (bone morphoge-netic protein 9 (BMP-9) and osteoprote-gerin (OPG)), and the down-regulation ofa marker of osteoclastogenesis (tartrate-resistant acid phosphatase (TRAP)).

Materials and methods

Patient population

The population of this prospective, split-mouth, randomized, double-blind, con-trolled clinical trial was recruited frompatients referred to Paulista Universitybetween August 2014 and February2016. The clinical procedures and evalua-tions were performed between October2014 and March 2016. Data entry andstatistical analyses were conducted bythe end of October 2016. Eighteen patientswere selected. Seven were male and 11were female, and they ranged in age from39 to 65 years. This study was approved bythe university ethics committee.

Inclusion and exclusion criteria

The inclusion criteria for this study wereas follows: patients with an edentulousmandibular arch indicated for rehabilita-

tion with implants; any extractions per-formed at least 4 months before treatment;and age between 18 and 65 years.The following exclusion criteria were

applied: presence of systemic diseases thatmay interfere with bone repair (includingdiabetes, arthritis, hypothyroidism, hyper-parathyroidism, and osteoporosis); the useof medications that would contraindicatethe placement of implants or that areknown to alter implant osseointegration(including anti-inflammatories andbisphosphonates) up to 6 months beforesurgery; bone grafts or a history of previ-ous regenerative procedures in the area forimplantation; insufficient bone for implantinsertion; pregnant or breastfeeding wom-en; and smokers or ex-smokers.All eligible patients were provided with

detailed information on the nature of thestudy and the potential risks and benefitsof their participation, and they each signedan informed consent document.

Experimental groups

The selected patients received five imme-diately loaded implants between the men-tal foramina (single-stage dental implants)and a fixed implant-supported completearch prosthesis. Using a computer-gener-ated list (managed by F.R.C), two of theimplants were assigned randomly to thefollowing groups: reduced torque ap-proach (n = 18), in which the implantswere inserted with an insertion torque of<30 N cm; or conventional torque ap-proach (n = 18), in which the implantswere inserted with an insertion torque of�30 N cm (see Fig. 1 for the positions ofthe implants)9.

Implant therapy

The surgeries and all postoperative fol-low-up appointments were performed atthe dental clinic of Paulista University,and all patients received five cylindricaldental implants (diameter 4.1 mm) withexternal hexagon connections (Implacil deBortoli, Sao Paulo, Brazil) in the mandi-ble. One surgeon (A.V.N) with clinicalexperience in implant dentistry performedall surgical procedures.Briefly, the surgical area was anaesthe-

tized, following which a crestal incisionwas made and a mucoperiosteal flap wasraised. For the two experimental implants,careful site preparation was performed inorder to obtain primary stability accordingto the insertion torque determined previ-ously for each implant (<30 N cm or�30 N cm). The torque level was recordedby the drilling device (Chiropro; Bien-Air

Insertion torque and angiogenesis-bone markers 653

Fig. 1. Schematic illustration of the experimental design. Arrows indicate the implants includedin the study groups.

Fig. 2. Flowchart of the study showing the patients enrolled in the pre-study phase and theselection of individuals for the study phase.

Dental SA, Bienne, Switzerland). For theimplants inserted with the conventionaltorque approach, the surgical sequencefollowed the drilling protocol describedby the implant company (single-stage den-tal implants). For the reduced torque ap-proach implants, the surgical procedurewas followed as described by the implantcompany, but over-preparation was per-formed (3.7 mm final twist drill). Allpatients were blinded to the implant groupallocation.Interrupted absorbable polyglactin 910

sutures were placed (Vicryl; Ethicon,Somerville, NJ, USA). Amoxicillin (2 gadministered 1 h before the procedure),dipyrone (500 mg every 6 h for 2 dayspostoperative), and 0.12% chlorhexidinemouthwash (every 12 h for 7 days) wereprescribed.All patients received Branemark full-

arch prostheses within 24 h of implantplacement.

Evaluation of the angiogenesis- and

bone-related marker profile by multiplex

bead immunoassay (Luminex)

An examiner who was blinded to theimplant allocation (R.A.) collected theperi-implant crevicular fluid from theimplants in each group using filter paperstrips (PerioPaper; Oraflow, Hewlett, NY,USA) at 7, 14, 30, and 120 days afterimplant insertion, as described previous-ly16. Briefly, the site was dried and isolat-ed, following which an absorbent paperstrip was inserted in the peri-implant sul-cus until resistance was met. The strip wasthen left in place for 30 seconds. Thisprocedure was performed at four sitesper implant. The strips were then placedin separate tubes containing phosphate-buffered saline (PBS)/Tween. The fluidvolume was measured using a calibrateddevice (Periotron 8000; Oraflow) and the

peri-implant fluid samples were stored at�80 �C.The levels of angiogenesis mediators

(VEGF and PLGF), osteoblastogenesis-re-lated factors (BMP-9, periostin, and OPG),and an osteoclastogenesis-related marker(TRAP) were determined using specific kits(HBNMAG–51 K, HRNKLMAG-31K-01,and HAGP1MAG–12 K; Millipore Corpo-ration, Billerica, MA, USA) and a multipleximmunoassay instrument (MAGpix; Mir-aiBio, Alameda, CA, USA). The sampleswere analyzed individually, and the levelswere estimated using a five-parameter poly-nomial curve (xPONENT software; Milli-pore Corporation). All results were adjustedfor the volume of peri-implant crevicularfluid collected for each implant, and valueswere expressed in units of pg/ml.

Data analysis

The number of patients included in thisstudy was based on previous investigationsthat have found significant differences inperi-implant and gingival crevicular fluidlevels of various bone-related and immu-noinflammatory markers16,17.The data were initially analyzed for

homogeneity using the Shapiro–Wilk test.Between-group comparisons of the datawere then performed using analysis ofvariance (ANOVA) with the Tukeypost-hoc test (bone/vascular markers) orthe paired Student t-test (insertion torque).All analyses were conducted using SAS9.1 (SAS Institute Inc., Cary, NC, USA),considering each patient as an experimen-tal unit and a = 5%.

Results

A total of 121 patients were initially se-lected, but 103 were excluded as they didnot meet the inclusion criteria. Thus, 18patients were included in the study; all ofthese patients completed the study andthere were no drop-outs (Fig. 2). Of the18 patients, 11 were female (61.1%) andseven were male (38.9%); their mean agewas 58.06 � 7.42 years (range 39–65years). Measurements of insertion torquewere averaged for both groups and areillustrated in Fig. 3. The average insertiontorque in the low-level torque group was19.18 � 3.56 N cm and in the convention-al torque group was 46.04 � 7.48 N cm. A

654 Verrastro Neto et al.

Fig. 3. Insertion torque for the dental implants in each group; mean � standard deviationvalues. *Statistically significant difference between the groups; paired Student t-test, P < 0.05.

statistically significant difference wasobserved between the groups, with higherinsertion torque values in the conventionaltorque group (P < 0.05).

Levels of angiogenesis- and bone-related

markers

The inter-group analysis showed higherVEGF and OPG levels in the low-leveltorque group when compared to the con-ventional torque group on days 7 and 30,respectively (both P < 0.05) (Fig. 4A andB, respectively). In addition, the inter-group analysis showed significantly re-duced levels of TRAP for implants withlow-level insertion torque at all timepoints evaluated (7, 14, 30, and 120 days)when compared to those of the conven-tional insertion torque group (all P < 0.05;Fig. 4C). Conversely, regarding BMP-9and periostin levels, both markers wereincreased in the conventional torque groupcompared to the low-level torque group atday 120 (both P < 0.05; Fig. 4D and E,respectively). Intra-group comparisonrevealed higher levels of PLGF at120 days when compared to days 7 and30 in the group with conventional inser-tion torque (both P < 0.05; Fig. 4F).

Discussion

The insertion torque is related to primarystability, which may interfere with theosseointegration process and consequentlyaffect the success of immediately loadedimplants. Although a minimum torque of30 N cm has been proposed for an imme-diate-loading implant9–11, other studieshave suggested that a greater insertiontorque results in bone resorption beforebiological integration through bone for-mation, whereas lower torque could pro-mote a more promising pattern of bonerepair compared to higher insertiontorques13–15,18. It appears that no clinical

study to date has evaluated the impact ofdifferent levels of insertion torque on thepattern of peri-implant bone repair forimmediately loaded implants. The presentstudy investigated the levels of certainmarkers of osteoclastogenesis, osteoblas-togenesis, and angiogenesis in the peri-implant fluid surrounding immediatelyloaded implants that had been insertedat different torques. In general, the resultsindicate that the torque applied affectedthe bone- and angiogenesis-related med-iators released during early peri-implantrepair.Interestingly, the results revealed ele-

vated concentrations of VEGF and OPG inthe peri-implant fluid of implants insertedwith a lower torque when compared tothose inserted with conventional torque ondays 7 and 30, respectively (P < 0.05).While VEGF is an important growth factorin the process of angiogenesis, related tochondrocyte and osteoblastdifferentiation19–21, OPG inhibits the dif-ferentiation and function of osteoclasts22,and these factors act to protect againstbone loss.In this trial, higher VEGF levels were

observed at the beginning of bone repair (7days) under the low-level insertion torqueprotocol when compared to the conven-tional torque protocol (P < 0.05; Fig. 4A).This outcome supports a probable benefi-cial effect of using a reduced torque duringthe surgical procedure for implant inser-tion, as this favours angiogenesis and themicrocirculation. In agreement, animalstudies have suggested that damage tothe bone tissue and vessel obstructionleading to bone necrosis are related tothe elevated pressure generated throughthe use of a high level of torque for implantinsertion14,15,23.Some in vitro investigations have dem-

onstrated osteoblast differentiation byVEGF in a dose-dependent manner24,and additional evidence has confirmed

the chemoattractive effect of VEGF tohuman osteoblasts and mesenchymal pro-genitor cells25,26. Interestingly, consider-ing the essential role of this angiogenesismarker in bone formation mechanisms,the increased level of VEGF observed inthe initial stage (7 days) of repair inimplants inserted with low-level torquein the present study may have positivelyinfluenced the subsequent up-regulation ofOPG observed at 30 days when comparedto the implants inserted with conventionaltorque (P < 0.05; Fig. 4B). This indicatesthe cooperation of angiogenic and osteo-genic signalling to benefit peri-implantbone formation. OPG is an osteoblasto-genesis-related marker that is importantfor preventing bone resorption. OPG bindsto the receptor activator of nuclear factorkappa-B ligand (RANKL) to inhibit bind-ing to its membrane receptor (RANK).Considering the favourable impact ofVEGF and OPG during the bone remodel-ling process, the presence of higher levelsof these mediators in the peri-implant fluidof implants inserted with low torque at thebeginning of bone repair suggests a prom-ising impact of this surgical technique inthe modulation of key markers involvedwith osseous healing around implants.Another outcome from the current in-

vestigation relates to the osteoclast-specif-ic enzyme TRAP and suggests a positiveimpact of low insertion torque on the localhost response in the area surrounding theimplants. The data showed significantlyreduced levels of TRAP around implantsinserted under low torque when comparedto those inserted with conventional torqueat all time points evaluated (7, 14, 30, and120 days) (P < 0.05, Fig. 4C). TRAP haslong been established as a marker of oste-oclast function27, and its activity is elevat-ed in the serum of patients with bonedisorders and harmful osseous repair28.Thus, the down-regulation of this markerin the peri-implant fluid of implantsinserted with low torque indicates that itlikely plays a role in the molecular eventsthat occur during peri-implant healing.Conversely, the current trial revealed thateven conventional torque values (average46.04 N cm; Fig. 3) promoted the elevatedrelease of TRAP throughout the study.This finding is in agreement with previousdata demonstrating that the use of aninsertion torque higher than 40–45 N cmmay disturb the local microcirculation,leading to necrosis of the osteocytes andconsequently to bone resorption29.However, the current investigation

revealed that implants inserted with con-ventional insertion torque showed favour-able modulation of some

Insertion torque and angiogenesis-bone markers 655

Fig. 4. Levels of vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), tartrate-resistant acid phosphatase (TRAP), bonemorphogenetic protein 9 (BMP-9), periostin, and placental growth factor (PLGF) in the peri-implant fluid of implants inserted with the reducedtorque approach and the conventional torque approach (pg/ml); two-way ANOVA with post-hoc Tukey test. (A) VEGF: *statistically significantdifference between the groups, P < 0.05; no statistically significant difference in the intra-group analysis (P > 0.05). (B) OPG: *statisticallysignificant difference between the groups, P < 0.05; no statistically significant difference in the intra-group analysis (P > 0.05). (C) TRAP:*statistically significant difference between the groups, P < 0.05; no statistically significant difference in the intra-group analysis (P > 0.05). (D)BMP-9: *statistically significant difference between the groups, P < 0.05; no statistically significant difference in the intra-group analysis(P > 0.05). (E) Periostin: *statistically significant difference between the groups, P < 0.05; no statistically significant difference in the intra-groupanalysis (P > 0.05). (F) PLGF: lowercase letters indicate a statistically significant difference among the periods of evaluation within each group,P < 0.05; no statistically significant difference between the groups (P > 0.05).

osteoblastogenesis-related markers at thefinal time point evaluated (120 days). Theanalysis showed that implants insertedwith conventional torque promoted aug-mented release of BMP-9 at day 120 whencompared to implants inserted with lowtorque (P < 0.05; Fig. 4D). BMPs aremembers of the transforming growth fac-tor beta family and play important roles in

the processes of bone formation and stemcell differentiation30. BMP-9 is one of themost highly osteogenic BMPs, and alsopromotes the differentiation of mesenchy-mal stem cells into osteoblasts31,32. Thepresent study results showed that thegreater torque promoted BMP-9 release,indicating osteoblastic activity during thelate phase of osseointegration.

In agreement with these BMP-9 results,higher periostin levels were also observedat 120 days for the implants inserted withconventional torque when compared tothose inserted with lower torque(P < 0.05; Fig. 4E). Periostin is a mole-cule expressed by osteocytes in bone thatis capable of modulating Wnt-beta-cateninsignalling. The levels of periostin mRNA

656 Verrastro Neto et al.

and protein have been shown to be up-regulated during fracture healing, particu-larly in proliferating osteoblast cells33. Itis speculated that periostin plays a role inthe recruitment of cells of the osteoblasticlineage to the site of repair34.Importantly, periostin shows higher ex-

pression under mechanical stress and ten-sion35,36. In a study by Bonnet et al.,periostin mRNA and protein were foundto be overexpressed in the mouse tibiaunder axial compression37. Thus, it issuggested that up-regulation of this osteo-blastic factor in implants inserted withhigher torque could be related to the aug-mented loading at the bone–implant inter-face.Another relevant finding in this study is

related to the increased levels of PLGF, animportant angiogenic-related factor in-volved in bone formation and repair pro-cesses38,39. For implants inserted withconventional torque, PLGF was higherin the late stage of repair (120 days) whencompared to the earlier stages (7 and 30days) (P < 0.05; Fig. 4F). Interestingly, anin vitro study by McCoy et al. demonstrat-ed that PLGF is a mechano-regulatedmarker, the expression of which is propor-tional to both the magnitude and durationof the mechanical stimulus applied40.Thus, the elevated peri-implant levels ofPLGF in the implants inserted with con-ventional torque at the final time point(120 days) may also be in response tothe augmented load at the bone–implantinterface, in agreement with the up-regu-lation of periostin in this experimentalgroup at the same time point.Some studies have tried to establish the

ideal insertion torque to improve bonehealing during immediate-loading rehabil-itation. An experimental study in dogs byRea et al. showed that a higher mineral-ized bone-to-implant contact was ob-served when a low torque(approximately 30 N cm) was used com-pared with a high insertion torque(>70 N cm) in immediately loadedimplants41. In line with this, Testoriet al. stated that a minimum insertiontorque of 30 N cm is required for success-ful immediate-loading protocols from aclinical point of view9. Nevertheless, froma molecular point of view, the biochemicaldata from the peri-implant fluid analysedin the present study demonstrated that areduced torque approach (average inser-tion of 19.18 N cm) provided additionalbenefits in terms of the release of bone andvascular mediators when compared to theconventional torque approach (averageinsertion of 46.04 N cm). When com-bined, the findings of this study suggest

that osteoblastic activity may predomi-nantly be observed following insertionwith low-level torque, especially duringthe first days of bone repair after implan-tation, considering the up-regulation ofVEGF and OPG in the peri-implant fluidseen during this period. On the other hand,data from the analyses demonstrated thatconventional-level torque was related toup-regulated BMP-9 and periostin at thefinal evaluation time point, suggesting anosteoblastic influence only during late pe-ri-implant repair, whereas osteoclasticevents were predominant when the higherinsertion force was used during surgicalimplant placement. While these relevantmolecular findings may highlight themechanisms involved in peri-implantbone repair in the earlier stage of osseoin-tegration after the implantation of imme-diate-loading implants, further studies arenecessary to determine precise clinicalguidelines.The preliminary outcomes from this

investigation suggest some biologicalmechanisms that could explain the posi-tive effects of lower insertion torque dur-ing initial peri-implant healing. Theresults of this study provide a basis forfuture studies on the possible relevance ofthe surgical technique for implant place-ment with low or high insertion torque,and to better understand the bone andvascular changes around the implants.This information could be used to deter-mine the best treatment strategy and meth-od of osteotomy preparation to benefitimplant loading and lead to quicker pros-thesis rehabilitation.Considering the points discussed, the

results of this study confirm the suggestedhypothesis that different levels of torquefor the insertion of dental implants maymodulate the release of angiogenesis- andbone-related markers.

Funding

Nothing to declare.

Competing interests

Nothing to declare.

Ethical approval

This study was approved by the universityethics committee(44161215.3.0000.5512).

Patient consent

Not required.

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Address:Fabiano Ribeiro CiranoDepartamento de OdontologiaUniversidade Paulista (UNIP)Av. Dr. Bacelar,1212Vila ClementinoSao Paulo 04026-002BrazilTel.: +55 11 5586 4000Fax: +55 11 5586 4000E-mail: fabiano.cirano@docente.unip.br