sequential bone healing of immediately loaded mini-implants: … · 2017. 11. 26. · original...

ORIGINAL ARTICLE

Sequential bone healing of immediately loadedmini-implants: histomorphometric andfluorescence analysis

Glaucio Serra,a Liliane S. Morais,a Carlos Nelson Elias,b Marc A. Meyers,c Leonardo Andrade,d Carlos A. Muller,e

and Marcelo Mullere

Rio de Janeiro, Brazil, and San Diego, Calif

Introduction: Mini-implants are often immediately loaded for orthodontic treatment; however, changes in in-terfacial tissues caused by early loading and its effects might compromise the mini-implant’s function. Thepurpose of this study was to compare the healing of interfacial tissues 1, 4, and 12 weeks after the placementof titanium-alloy mini-implants in New Zealand rabbits; some of the implants were loaded immediately andothers were left unloaded. Methods: Eighteen animals were used in the experiment. Each received 4 titaniumgrade 5 mini-implants (2.0 3 6.0 mm), 2 of which were immediately loaded with 1 N of force. Tissue healing wasverified at 1, 4, and 12 weeks after placement. Four different fluorescent molecules were injected into the rab-bits to label calcium deposition. After the rabbits were killed, mineralized bone samples with the mini-implantswere removed, fixed, cut, stained, and observed with bright-field, polarized, and fluorescence microscopy.Results: After 12 weeks of healing, higher bone contact and bone area were observed than after 1 or 4weeks, regardless of loading. Differences between the loaded and unloaded groups were not observed(P \0.05) at 1 and 4 weeks. The bone deposition rate was higher in the loaded group. Conclusions: The1-N immediate force application did not compromise bone formation around mini-implants. (Am J OrthodDentofacial Orthop 2010;137:80-90)

Rigid bone anchorage has been developed sincesuccessful use of conventional dental implantsin orthodontics.1-5 Changes in the design were

proposed to overcome the disadvantages of conven-tional dental implants such as limitations of placementsites, invasive surgical procedures, postoperative pain,and difficulty of attaching elastics or springs.6-10

First, changes were made to the dimensions of theimplant. Length reduction was proposed, and then thesize and design were completely changed, creating

a PhD student, Department of Mechanical and Aerospace Engineering, Univer-

sity of California-San Diego; Phd student, Engineering Military Institute, Rio de

Janeiro, Brazil.b Professor, Military Engineering Institute, Department of Mechanical Engi-

neering and Materials Science, Rio de Janeiro, Brazil.c Professor, Department of Mechanical and Aerospace Engineering, University

of California-San Diego.d Professor, Biomedical Science Institute, University Federal of Rio de Janeiro,

Rio de Janeiro, Brazil.e Researcher, Oswaldo Cruz Institute, Department of Animals Experimentation,

Rio de Janeiro, Brazil.

Supported by CAPES and CNPq, Ministry of Education and Culture, Brazil

(Process numbers 472449/2004-4, 400603/2004-7, and 500126/2003-6).

The authors report no commercial, proprietary, or finacial interest in the prod-

ucts or companies described in this article.

Reprint requests to: Glaucio Serra, Av. Nossa Senhora de Copacabana 1355, Apt

805, Copacabana, Rio de Janeiro. RJ, Brazil; e-mail, [email protected].

Submitted, July 2007; revised and accepted, December 2007.

0889-5406/$36.00

Copyright � 2010 by the American Association of Orthodontists.

doi:10.1016/j.ajodo.2007.12.035

80

specific systems.7,11 Miniplates, mini-implants, and on-plants were the most used rigid orthodontic bone an-chorage systems.6,12,13 Mini-implants became widelyused because they met orthodontic needs: eg, widerange of placement sites, simplified surgical proceduresand lower costs.8,10 However, these design changes re-sulted in higher failure rates than conventional dentalimplants.14-17 In this context, some factors must be con-sidered that might jeopardize the success of the mini-implants. Screw diameter of 1 mm or less, inflammationof peri-implant tissues, thin cortical bone at the place-ment site, micromovement during the first healingphase, and the timing and modulus of loading are possi-ble causes for failure of mini-implants.14,16,18,19

Some researches have analyzed the effects of earlyloading in the healing process, and the protocol formini-implant loading has been discussed. Wehrbeinand Diedrich5 proposed a healing time of 25 weeks be-fore load application. After that healing period, the im-plants were successfully loaded during 26 weeks with2 N of force without problems. Some researchers postu-lated that, when the load is placed prematurely, there isno uniform intimate bone-implant contact because ofthe interplay of fibrous tissue.20,21 On the other hand,a review by Szmukler-Moncler et al22 summarizedthat micromovement is more harmful than the load dur-ing the early healing phase. Saito et al11 thought that

mailto:[email protected]

Fig 1. Experimental implant: cylindrical screw of tita-nium alloy. a, hexagonal head with 3.4 mm height; b, ac-tive area, 6.0 mm length and 2.0 diameter, with 0.51 mmbetween the top of the pitches; c, machined threadedsurface.

American Journal of Orthodontics and Dentofacial Orthopedics Serra et al 81Volume 137, Number 1

18 weeks was long enough for biologic fixation capableof resisting up to a load of 2 N for 24, 28, or 32 weeks.Aldikacti et al23 applied a 2-N load after 6 weeks ofhealing. They used a long loading period of 52 weeks,and the mini-implants maintained stability throughoutthe study. In 2005, some authors proposed an early load-ing protocol for mini-implants after just 1 or 2 weeks ofhealing.10,24 Yao et al24 suggested that 2 weeks shouldbe enough for soft-tissue healing. Kim et al10 loadedthe mini-implants after 1 week and had a 9% failurerate after 10 weeks of continuous loading.

Immediate loading has also been described in clini-cal studies.17,25 Park et al25 applied 2 N of force for 36weeks immediately after placing mini-implants in pos-terior sites. They obtained a success rate of 90%. Mo-toyoshi et al,17 using an immediate load in the sameregion and with the same force, had a success rate of85.5%. The consensus in the early load protocol isthat even experienced clinicians can have failures andthat the removal of the mini-implant after the treatmentis uneventful.6,9,26-28 Limited information is availableabout the effects of immediate loading on the biologicsequences of healing during the early phases of tissueintegration, when the primary mechanical stability ofmini-implants must be substituted for stability obtainedthrough biologic means.

Our aim in this study was, therefore, to evaluate theinterface reactions at early and late stages of osseointe-gration around mini-implants immediately loaded with1 N. We used bright-field, polarized, and fluorescencemicroscopy for this evaluation.

MATERIAL AND METHODS

The protocol for ths animal study was approved bythe standing ethics committee on animal research of Os-waldo Cruz Foundation (Rio de Janeiro, Brazil), and allprocedures were conducted in accordance withCanadian Council of Animal Care guidelines.

Eighteen 6-month-old male New Zealand white rab-bits, weighing 3.0 to 3.5 kg, were used. The surgicalprocedures were common to all animals and consistedof placement of 4 mini-implants in the left tibial meta-physes of each animal. All surgeries were performedunder sterile conditions in a veterinary operating room.

The titanium-aluminum-vanadium alloy mini-im-plants had a cylindrical screw design and a hexagonalhead (6.0 3 Ø 2.0 mm; Conexao Sistemas de Proteses,Sao Paulo, Brazil). They were machined by turning,cleaned, passivated with nitric acid, and sterilized by25 Gy of cobalt radiation (Fig 1).

The rabbits were acclimatized for a month in a vivar-ium before the surgical procedure. Immediately beforethe surgery, the animals were anesthetized with intra-muscular injection of tiletamine (5 mg/kg) and zolaze-pan (5 mg/kg) followed by continuous delivery of 2%halothane and isofluthane throughout the surgery. Thehair on the medial surface of the upper portion of theleft leg was clipped and the skin was cleansed with io-dinate surgical soap. An incision approximately50 mm in length was made parallel to longitudinalaxis of the tibia, in the medial aspect. The periosteumwas elevated by using sterile surgical techniques, andthe bone was denuded. Four implantation holes about5 mm apart were drilled with a 1.6-mm drill under pro-fuse sterile saline-solution irrigation and at low rotaryspeed. The mini-implants were threaded at the first cor-tex of the tibia with their longitudinal axes parallel toeach other and perpendicular to the external corticaltibia. After placement, the 2 central mini-implantswere immediately loaded with reciprocal forces. Anickel-titanium closed-coil spring was attached to themini-implant’s head, providing 1 N of force. Then theperiosteum was closed with resorbable sutures, andthe skin was sutured.

After the surgical procedures, each animal had 4mini-implants, 2 loaded and 2 unloaded, for a total of72 mini-implants. Thirty mini-implants were used inthis study, and the other 42 were analyzed by removal

Table I. Experimental design

New Zealand

rabbits (n)

18

Mini-implants (n) 48 2.0 3 6.0 mm

Load Immediate 1 N

Time of healing 1, 4, and 12 weeks

Placement torque 18 samples

Histologic analysis 30 samples

Groups (n 5 5) 1wUn, 1wLo, 4wUn,

4wLo, 12wUn, 12wLo

1wUn, 1 week unloaded; 1wLo, 1 week loaded; 4wUn, 4 weeks un-

loaded; 4wLo, 4 weeks loaded; 12wUn,12 weeks unloaded;

12wLo,12 weeks loaded.

82 Serra et al American Journal of Orthodontics and Dentofacial Orthopedics

January 2010

torque test and scanning electron microscope analysis inthe first part of this study (Table I).

The experimental design was delineated to analyze3 periods of healing: 1, 4, and 12 weeks. In each assess-ment period, there was 1 group loaded and another un-loaded in a total of 6 groups (6 samples per group). Inthis manner, after each proposed time, 6 rabbits werekilled by exsanguination.

Within 30 minutes after the rabbit’s killing, theplacement surgical procedure described above was per-formed on each animal’s right leg; a new mini-implantper rabbit was placed for a total of 18 mini-implants.The maximum placement torque was measured witha manual torque meter (Table I). Thus, 48 mini-implantswere used, from which 30 had the sequential interfacereactions analyzed and 18 had the maximum torqueinsertion measured.

Quadruple polychromatic fluorescence labeling wasperformed after the second postoperative day (Fig 2).The sequential administration of fluorescent dyes variedbetween the groups and followed the topographic local-ization and the rate of new bone formation. The animalsreceived intramuscular injections of oxytetracycline(15 mg/kg of body weight), calcein blue (30 mg/kg ofbody weight), alizarin-complexone (30 mg/kg of bodyweight), and xylenol orange (90 mg/kg of body weight)diluted in 2% sodium carbonate solution.

After sample preparation, the slides were analyzedby using a fluorescence microscope (BX 51WI, Olym-pus, Tokyo, Japan) with filters with wavelengths of450 to 490 nm (blue filter) and 340 to 380 nm (violet fil-ter). The distance between the label marks was mea-sured, and the mineral appositional rate (MAR) wascalculated in micrometers per day.27 The MAR wasmeasured in both sides of the unloaded samples and inthe tension and compression sides of the loaded samples(2 measurements in each area).

After the healing period of 1, 4, or 12 weeks, the an-imals were killed, and the left leg of each rabbit was care-fully sectioned and removed. The closed-coil springs

were cut, and the tibia block specimens containing themini-implants and at least 2 mm of surrounding tissuewere dissected. The specimens were fixed (4% parafor-moldehyde solution in 0.1 mol/L sodium phosphatebuffer, pH 7.2), rinsed in the same buffer, and dehydratedin a graded ethanol series until absolute. Thereafter, theblocks were embedded in methylmethacrylate (Techno-vit 7100, Heraeus Kulzer, Dormagen, Germany) andsectioned in the longitudinal plane with a microtome(Exakt Medical Instruments, Oklahoma City, Okla).The thick slides were ground and polished to about 50mm for fluorescent microscopic examination. Subse-quently, the slides were stained with 2% toluidine bluefor the bright-field microscopic examination and the his-tomorphometric measurements. Each tibia block samplewas prepared to result in 1 central slide.

Histologic analysis was carried out by bright-fieldpolarized light transmission microscopy. The micropho-tographic images were acquired by using 10, 20, and 40objectives with a digital camera (EOS D-30, Canon, To-kyo, Japan) interfaced with a computerized system andthe microscope. Before sample observation, a standardslide consisting of a linear gridline in micrometers wasphotographed for scaling the images. After obtainingthe images, the measurements were made on each sideof the bone within 1 mm2 of the mini-implant surface,which corresponded to the bone interface at the first 2screw threads. The Image J Launcher software (Java ver-sion 1.1.4. for Windows, Microsoft, Redmond, Wash)was used to measure and convert pixels to micrometers.

Two static variables were measured. The fractionalbone-to-implant contact (% BIC) consisting of the lin-ear bone-to-implant contact (mm) of the total of themini-implant linear surface (mm) in the analyzed areaand the fractional bone area (% BA) consisting of thetotal bone area (mm2) of the total area analyzed (mm).

Statistical analysis

Statistical analyses for reporting means and stan-dard deviations of data from MAR, % BIC, and % BAwere performed for all groups. To quantify the signifi-cance of the differences, the data were evaluated by2-way analysis of variance (ANOVA) followed by thepost-hoc Tukey test and the t test (P \0.05).

RESULTS

The 48 mini-implants were placed without macro-deformation or fracture. The rabbits had no complica-tions such as infection or leg fracture during the healingprocess.

All mini-implants monocortically placed into therabbits’ tibia bones were clinically immobile after the

Fig 2. Polychromatic fluorescence labeling: O, oxytetracycline; C, calcein blue; X, xylenol orange; A,alizarin-complexone. †Day of killing.

Fig 3. Bone deformation (BD) and microfractures (MF)present mainly in 1-week groups. Original magnification,10 times.


surgical procedure. At sample preparation, 3 mini-im-plants exhibited excessive clinical mobility and wereconsidered lost samples, yielding an overall successrate of 90%. These 3 failed mini-implants were in the1-week loaded, 4-week unloaded, and 12-weekunloaded groups.

The maximum placement torque was between108.02 and 84.40 N mm (mean, 98.33 N per millimeter;SD, 6 9.52 N mm).

By polarized light, no micrographic signs of chronicinflammation at the interface at any healing intervalwere observed. The analysis showed monocortical fixa-tion for all mini-implants. Bone deformations and mi-crofractures were observed primarily in the 1-weekgroups; even so, the native bone around the mini-im-plants was preserved throughout the experiment (Fig 3).

Corticalization was observed in some samples in the4-week loaded group. The increase of cortical thicknesswas found only in the endosteal bone region, indicatingprimary bone formation in this area. Corticalization wasalso observed in the 12-week groups with periosteal andendosteal bone formation (Fig 4).

After 1 week of healing, the mini-implants were em-bedded into the bone with the top and the peripheral por-tions of the pitches of the screw in close contact with thenative bone. The portions between the pitches werefilled by wound tissue, and no histologic differencewas found between the loaded and unloaded samples(Fig 5, A and B). Thus, the native lamellar bone was pre-dominant, and it was not jeopardized by the immediateloading or the placement technique in this initial healingphase, providing mechanical stability for the mini-im-plants during the first healing period. In the 4-weekgroups, the relationship between the native bone, the

mini-implant, and the interfacial tissue was maintained.The wound-tissue aspect was distinct between theloaded and the unloaded samples. The latter seemedto be less dense than the former, indicating a modifiedhealing process (Fig 5, C and D). After 12 weeks ofhealing, there was significant bone formation betweenthe threads of the mini-implant in both the loaded andunloaded samples. Then most of the region initiallyfilled by wound tissue was replaced by newly formedbone. There was no difference in the histologic findingsbetween the compression and the tension sides of theloaded mini-implants; however, the tendency of differ-ences between the loaded and unloaded samples wasmaintained. The loaded samples exhibited moreorganized tissues (Fig 5, E and F).

Fig 4. Sequential corticalization: A, absence of bone deposition in endosteal or periosteal sides at 1week; B, first sign in the endosteal region in the 4-week groups (white arrows); C, at 12 weeks, theincrease of bone thickness was in both the endosteal and periosteal sides.

Fig 5. Histologic results. A and B, Interfacial tissue (IT) and preserved native bone (NB) in close con-tact with the mini-implant (M-i) surface; original magnification, 20 times. C and D, Slight difference inthe interfacial tissue of the loaded and unloaded sample; original magnification, 20 times. E and F,Bone penetrating in the internal pitch area. Newly formed bone is more organized with lamellar aspectin the loaded group; original magnification, 40 times.


January 2010

The amount of osseointegration quantified by directbone-to-implant contact (% BIC) and the area of boneobserved between the threads of the screw (% BA) arelisted in Tables II and III. The % BIC results ranged

from 37.03% to 39.73% in the 1-week and 4-weekgroups, increasing to 66.01% and 70.96% after 12weeks of healing (Table II). Similarly, the % BA resultsindicated significant increases in the 12-week groups,

Table II. Histomorphometric values: % BIC rate

Histomorphometric values:% BIC rate (Mean 6 SD)

Healing time Unloaded Loaded

1 week 39.73 6 5.7a 37.68 6 6.1a

4 weeks 37.03 6 5.0a 38.76 6 3.4a

12 weeks 66.01 6 10.9b 70.96 6 7.1b

Term Two-way ANOVA interaction: 1 3 2

Load Loaded 3 unloaded / P 5 0.495

Time 1w 3 4w /P 5 0.927

1w 3 12w /P 5 0 .0001

4w 3 12w /P 5 0 .0001

Mean values with the same superscript letters are not significantly dif-

ferent (P .0.05). Mean values with different superscript letters indi-

cate statistically significant differences (P \0.05).

1w, 1-week groups; 4w, 4-week groups; 12w,12-week groups.

Table III. Histomorphometric values: % BA rate

Histomorphometric values: % BA rate (Mean 6 SD)

Healing time Unloaded Loaded

1 week 66.40 6 3.3a 69.57 6 2.6a

4 weeks 64.45 6 7.3a 70.35 6 3.7a

12 weeks 86.13 6 5.5b 87.14 6 4.2b

Term Two-way ANOVA interaction: 1 3 2

Load Loaded 3 unloaded / P 5 0.128

Time 1w 3 4w /P 5 0.998

1w 3 12w /P 5 0 .0001

4w 3 12w /P 5 0 .0001

Mean values with the same superscript letters are not significantly dif-

ferent (P .0.05). Mean values with different superscript letters indi-

cate statistically significant differences (P \0.05).

1w,1-week groups; 4w, 4-week groups; 12w,12-week groups.

Table IV. Comparisons of means of the tension and compression sides

t test for independent samples

Time Area % BIC mean 6 SD % BA mean 6 SD % BIC % BA

1 week Compression 37.10 6 6.78 70.32 6 3.25 P 5 0.8105 P 5 0.4576

Tension 38.27 6 5.11 68.89 6 2.29

4 weeks Compression 39.16 6 4.85 72.56 6 3.21 P 5 0.7330 P 5 0.0577

Tension 38.36 6 1.43 68.14 6 3.10

12 weeks Compression 70.52 6 6.78 87.18 6 2.23 P 5 0.8578 P 5 0.9780

Tension 71.40 6 8.18 87.10 6 5.98


ranging from 64.45% to 70.35% after 1 and 4 weeks ofhealing, reaching 86.13% and 87.14% in the 12-weekgroups (Table III). The statistical analysis confirmedthat, although there was no significant bone growth inthe 1-week and 4-week groups, significant formationof new bone was indicated by both % BIC and % BA af-ter 12 weeks, regardless of loading (Tables II and III).The compression and tension areas did not show signif-icant differences at any healing time (Table IV).

A striking result came from the 2-way ANOVAanalysis, which proved that the load did not enhanceor impair the healing process in terms of the histomor-phometric parameters (% BIC, P 5 0.495; % BA,P 5 0.128). Also, the healing time had a significant in-fluence, in which the 12-week groups showed a relevantincrease in bone around the mini-implants (% BIC,P 5 0.0001; % BA, P 5 0.0001) (Tables III and IV).

Fluorescence microscopy images showed a gradualincrease of deposition of the dyes. In the 1-week groups,no specific marks could be seen in the bone around themini-implants (Fig 6). Bone deformation and micro-fractures were clearly identified in the slides of this pe-riod. Aweakly stained bone was observed after 4 weeks;however, some loaded samples had an unspecific aliza-rin-complexone dye, which was the last fluorescence la-

bel administered. This label was restricted to theendosteal region, indicating the beginning of the min-eral deposition just in that region of the loaded sample.The 12-week groups showed well-defined labels in allperi–mini-implant bone, in both the endosteal and peri-osteal regions, allowing for rate measurements (Fig 6).

The regions labeled by fluorochromes were easilyidentified in all 12-week specimens (Fig 7); however,the calcein blue dye was not visible when the blue filterwas used. It was visible with the violet filter. Neverthe-less, because of the need to have all labels in the sameview to proceed with the MAR calculations, thesedata were discarded. The statistical analyses showedsignificant differences between the unloaded and loadedgroups (P 5 0.024 and P 5 0.008, respectively),whereas the tension and compression sides of the loadedgroup had no significant difference (P 5 0.934)(Table V). This indicates accelerated healing in theloaded sample without a difference between the areasof tension and compression.

DISCUSSION

These findings indicate that immediate loading didnot compromise the healing process in the tissues

Fig 6. Fluorescence microscopy. A and B, Longitudinal section of mini-implant and surroundingbone after 1 week of healing without dye deposits. Bone deformation (BD) and microfractures (MF)are shown in both the load and unloaded groups. C and D, the 4-week groups. Unspecific aliza-rin-complexone dye marked in the endosteal region of the loaded sample (A). E and F, the12-week groups. Great level of fluorochrome labeling shown in all surrounding bone, regardlessthe loading. CCS, Closed-coil spring; M-i, mini-implant. Original magnification, 4 times.


January 2010

around the mini-implants, although the quality and therate of bone formation had been altered. The analysisof the undecalcified sections provided an overview ofthe sequential healing evolution, since the primary me-chanical stability of the mini-implants obtained by thefriction and close contact with native bone until thelate biological attachment provided by the new boneformation at the bone–mini-implant interface. The fluo-rescence analysis allowed for comparison of the effectsof the immediate load application to the bone formationrate as well as to the topographic localization of theearly healing events.

The first prerequisite for the success of implants isminimal damage to the host tissues during the surgicalprocedure.1,2,5 Copious irrigation to prevent superheat-ing during drilling, preservation of the periosteal tissue,and maintenance of cleanliness are well-described pre-cautions. Furthermore, Motoyoshi et al17 described therecommended implant placement torque in the range

of 50 to 100 N mm. They concluded that low valuesare insufficient for establishing mechanical stability,and high values could generate excessively high stresslevels, resulting in degeneration of the interfacialbone. In our study, the mean placement torque was98.3 N mm, indicating a good relationship between sta-bility and interfacial stress. Additionally, all sampleswere clinically stable after placement, and the healingprocess resulted in osseointegration of most samples af-ter the maximum experimental interval. Thus, we as-sumed that the surgical technique and the postsurgicalconditions were suitable for the immediate loading se-quential analysis.

Within the framework of the time of loading, theproposed healing interval before orthodontic force ap-plication has been gradually decreased since the firstprotocol described.10,11,23,24 Now some experienced re-search groups have described a success rate of about90% with an immediate orthodontic loading

Fig 7. Well-defined fluorochrome labels used in theMAR calculations: A, alizarin-complexone; X, xylenol or-ange; O, oxytetracycline. Original magnification, 40times.

Table V. MAR rate values compared with 1-wayANOVA and post-hoc Tukey test

GroupMineral appositional

rate (mean 1 SD)

12wUn (n 5 16) 2.96 1 0.8 mm/day

12wLo compression

(n 5 10)

3.68 1 0.5 mm/day

12wLo tension (n 5 10) 3.78 1 0.5 mm/day

ANOVA P 5 0.00035

Post-hoc Tukey test 12wUn vs 12wLo compression: P 5 0.024

12wUn vs 12wLo tension: P 5 0.008

12wLo compression vs 12wLo tension:

P 5 0.934

12wUn,12 weeks unloaded; 12wLo,12 weeks loaded.


protocol.10,16,17,25 Even so, they suggested that immedi-ate loading could be used with no compromise in thestability of the mini-implants. In agreement, we founda 10% fail rate, and the lost samples were not relatedto immediate loading. On the contrary, Costa et al20

and Melsen and Costa21 postulated that prematureloading resulted in fibrous tissues in the interface.Szmukler-Moncler et al22 and Cope29 addressed thesecontroversial conclusions by suggesting that micromo-tion is more detrimental than premature loading, and,when these forces are acting simultaneously, the resultscould be misinterpretated. Orthodontic loading could beapplied up to threshold, above of which it would dam-age to the osseointegration process.16,22,29,30 The over-loading limit depends on the quality and quantity ofnative and newly formed bone and on the implant de-sign. Isidor,30 using finite-element analysis, concludedthat loads resulting in interfacial strains higher than6700 m are harmful to the healing process. The transferof stress at the bone–mini-implant interface after loadapplication is influenced by implant design and surfacegeometry.31 Load concentration tends to occur at thefirst threads of the screw-design implants after lateralforce application, possibly resulting in bone resorptionaround these areas.19,32 Buchter et al16 demonstratedthat immediate tip forces higher than 9 N resulted in cer-vical resorption around the mini-implant or failure ofthe mini-implant. Oyonarte et al31,33 compared thebone response in the proximity of machined-threadedimplants and porous-surface implants after orthodonticloading. They found significant cervical bone resorptionaround the machined-threaded implants and concludedthat implant geometry resulted in high stress concentra-tion and, consequently, bone loss. In our study, bone re-sorption around the first threads of the mini-implant

after 1, 4, or 12 weeks of loading was not found. Be-cause we did not apply a finite-element model to com-plement our in-vivo study, we could not correlate theloading areas with the histologic response, but it couldbe suggested that the 1 N force associated with themini-implant design used in this study did not reachthe overloading limit.

Hoshaw et al34 stated that the placement process ofscrew-shaped implants causes microdamage in the adja-cent bone, and the damage concentration decreases after4 weeks of healing. Despite the different dimensionsand loading protocol, in our study, many microfracturesand bone deformation in the 1-week groups were alsofound. After 4 weeks of healing, some were still present,but they were not found in the 12-week groups. Thebone modeling and remodeling processes not only filledthe interface with newly formed bone but also regener-ated bone in the regions of damage.

Regarding the histomorphometric findings, no sig-nificant statistical difference was found between the1-week and 4-week groups in terms of % BIC and %BA values, regardless of the immediate loading. Similarto our previous study, in which the removal torquevalues increased in the 12-week groups, the % BICand % BA means increased significantly after 12 weeksof healing; however, no statistical difference was foundbetween the loaded and unloaded groups.35 Moreover,the areas under tension and compression also had no sta-tistical difference, signifying that the immediate loaddid not influence the amount of osseointegration.

The increase of the static histomorphometric values(% BIC and % BA) after several weeks of healing is welldescribed, not only for the delayed loading protocol butalso for the immediate loading protocol.3,33,36,37 Never-theless, bone formation at the areas of tension and com-pression remains controversial. Consistent with ourfindings, Wehrbein and Diedrich5 observed no micro-morphologic differences between the compression and


January 2010

tension side of the implants. The implants were loadedafter a healing period of 25 weeks. In addition, Melsenand Lang38 stated that the quantity of osseointegrationwas not influenced by the applied load (1-3 N) after12 weeks of healing. On the contrary, Buchter et al,37

in an immediate loading study, analyzed bone responsearound 2 geometrically different mini-implants loadedwith tip forces varying between 1 and 9 N. They con-cluded that forces of 5 or 6 N resulted in increasedbone–mini-implant contact. The force variation de-pended on the geometry of the mini-implant. Wehrbeinet al7 suggested that bone generation activity close toimplants could be influenced by the magnitude of forceand the quality of the bone. They concluded that the 1-Nforce did not cause an increase in bone deposition in al-veolar bone, but 2-N forces resulted in moderate boneapposition in the midsagittal palatal bone, especiallyin the areas under compression. Such bone formationdepends on several clinical factors including the magni-tude of the force, the quality of the supported bone, andthe geometry of the implant. The different experimentalprotocols used in these studies could explain the variedfindings.

The most interesting finding of our study was relatedto the bone deposition rate. The rate was significantlyhigher in the loaded implants than in the unloaded im-plants after 12 weeks (P 5 0.024 and P 5 0.008, respec-tively). The areas under compressive and tensile stresseswere not statistically different (P 5 0.934). The bone re-generation process could be accelerated by early biome-chanical stimulation. This was suggested by Sarmientoet al,39 who provided experimental radiographic, histo-logic, and mechanical evidence during the fracture heal-ing process. In the implant field, Rubin et al40 tested theearly biomechanical stimulation in porous titanium-al-loy implants and found that exposure to low-amplitudemechanical strain during the healing period enhancedprecocious biologic fixation. In addition, increasedbone deposition has been found in the bone aroundthe loaded implants when compared with bone sur-rounding unloaded implants.5,7,8,27,33 In this study, thebone deposition rate was higher in the loaded groups.Hence, it can be suggested that healing was acceleratedby early loading. Supporting this suggestion, the wovenbone produced along the endosteal surface is the pri-mary response to the cortical healing defects, and, inour study, some endosteal corticalization was seen inthe loaded samples after 4 weeks.2,41,42 Moreover, fluo-rescence alizarin-complexone dye was observed in theendosteal areas of these samples.

However, the accelerated healing process did not re-sult in greater osseointegration (% BIC or % BA) in theloaded group after 12 weeks. Huja and Roberts27 de-

scribed the continuous and accelerated remodeling pro-cess within 1 mm of the loaded implant surface asa possible mechanism whereby the loaded implant main-tains the integration with less mineralized bone. A highrate of bone remodeling produces incompletely mineral-ized lamellar tissue in contact with the implant surface.This mechanism has been suggested to be important toprevent microdamage and crack accumulation at the in-terfacial bone.22,27,33,43,44 Thus, it could be suggestedthat immediate loading influenced the quality of thenewly formed bone, quickly producing less mineralizedbone. This could be correlated with the findings of thefirst part of this study, in which a lower removal torquevalue was found in the 12-week loaded group comparedwith the unloaded 12-week group.35 The well-describedeasy removal of mini-implants in clinical studies couldbe also related to this suggestion.9,25,45 Quantificationof the bone mineral content in bone-tissue growth underimmediate loading conditions is necessary to confirmthis hypothesis.

Self-drilling mini-implants have recently been de-scribed.10 Self-tapping mini-implants were used in thisstudy. The choice between these 2 mini-implant typesshould be made carefully. The main differences betweenthem are the immediate postsurgical conditions: the self-drilling mini-implant has more bone–mini-implant con-tact and better primary stability. On the other hand, theimplant placement torque could be higher dependingon the characteristics of the host bone. It could generatea high stress level, resulting in local ischemia and necro-sis of the bone at the interface.17 Thus, the results of thisimmediate-loading study should not be extrapolated toself-drilling mini-implants.

Some important features—eg, soft-tissue inflamma-tion around the mini-implants, exposure to the oral en-vironment, and variations in cortical thickness—caninfluence the healing process but were not consideredin this animal study. So, the extrapolation of these re-sults to clinical applications should be done with cau-tion. Our data showed that osseointegration wasreached regardless of immediate loading. It can be sug-gested that the immediate loading protocol resulted inaccelerated healing and modified new bone formationwithout jeopardizing the stability of the mini-implantsduring the experimental period.

CONCLUSIONS

1. The titanium-aluminum-vanadium mini-implantswere appropriate as anchorage for 1 N of immediateloading.

2. The immediate 1-N load caused no significantchanges in the amount of osseointegration


(histomorphometric parameters) after 1, 4, or 12weeks of healing (P \0.05).

3. The bone deposition rate was higher in the loadedgroups than in the unloaded groups, indicating ac-celerated healing.

We thank Lars Bjursten for helping with the manu-script and Jared Braden Goor for helping with theoptical microscope analysis (Department of Bioengi-neering, University of California-San Diego); theNational Science Foundation Ceramics Program(Linnette Madsen, Director) for travel and laboratorysupplies; and Conexao Sistemas de Proteses, Sao Paulo,Brazil, for supplying the mini-implants used in thisstudy.

REFERENCES

1. Turley PK, Kean C, Schur J, Stefanac J, Gray J, Hennes J, et al.

Orthodontic force application to titanium endosseous implants.

Angle Orthod 1988;2:151-62.

2. Roberts EW, Smith RK, Zilberman Y, Mozsary PG, Smith RS.

Osseous adaptation to continuous loading of rigid endosseous

implants. Am J Orthod 1984;86:95-111.

3. Roberts EW, Helm RF, Marshall JK, Gongloff RK. Rigid endo-

sseous implants for orthodontic and orthopedic anchorage. Angle

Orthod 1989;59:247-56.

4. Roberts EW, Marshall KJ, Mozsary PG. Rigid endosseous implant

utilized as anchorage to protract molars and close an atrophic

extraction site. Angle Orthod 1990;60:135-52.

5. Wehrbein H, Diedrich P. Endosseous titanium implants during and

after orthodontic load—an experimental study in dog. Clin Oral

Implants Res 1993;4:76-82.

6. Kanomi R. Mini-implant for orthodontic anchorage. J Clin Orthod

1997;36:763-7.

7. Wehrbein H, Glatzmaier J, Yildirim M. Orthodontic anchorage

capacity of short titanium screw implants in the maxilla. An

experimental study in dog. Clin Oral Implants Res 1997;8:

131-41.

8. Ohmae M, Saito S, Morohashi T, Seki K, Qu H, Kanomi R, et al. A

clinical and histological evaluation of titanium mini-implants as

anchors for orthodontic intrusion in the beagle dog. Am J Orthod

Dentofacial Orthop 2001;119:489-97.

9. Paik CH, Woo YJ, Kim J, Park JU. Use of miniscrews for inter-

maxillary fixation of lingual-orthodontic surgical patients. J

Clin Orthod 2002;36:132-6.

10. Kim JW, Ahn SJ, Chang YI. Histomorphometric and mechanical

analyses of the drill-free screw as orthodontic anchorage. Am J

Orthod Dentofacial Orthop 2005;128:190-4.

11. Saito S, Sugimoto N, Morohashi T, Ozeki M, Kurabayashi H,

Shimizo H, et al. Endosseous titanium implants as anchors for me-

siodistal tooth movement in the beagle dog. Am J Orthod Dento-

facial Orthop 2000;118:601-7.

12. Chung KR, Kim YS, Linton JL, Lee YJ. The miniplate with tube

for skeletal anchorage. J Clin Orthod 2002;36:407-12.

13. Janssens F, Swennen G, Dujardin T, Glineur R, Malevez C. Use of

an onplant as orthodontic anchorage. Am J Orthod Dentofacial

Orthop 2002;122:566-70.

14. Miyawaki S, Koyama I, Inoue M, Mashima K, Sugahara T, Ta-

kano-Yamamoto T. Factors associated with the stability of tita-

nium screws placed in the posterior region for orthodontic

anchorage. Am J Orthod Dentofacial Orthop 2003;124:373-8.

15. Choi BH, Zhu SJ, Kim YH. A clinical evaluation of titanium mini-

plates as anchors for orthodontic treatment. Am J Orthod Dento-

facial Orthop 2005;128:382-4.

16. Buchter A, Wiechmann D, Koerdt S, Wiesmann HP, Piffko J,

Meyer U. Load-related implant reaction of mini-implants used

for orthodontic anchorage. Clin Oral Implants Res 2005;16:

473-9.

17. Motoyoshi M, Hirabayashi M, Shimizu N. Recommended place-

ment torque when tightening an orthodontic mini-implant. Clin

Oral Implants Res 2006;17:119-4.

18. Favero L, Brollo P, Bressan E. Orthodontic anchorage with spe-

cific fixtures: related study analysis. Am J Orthod Dentofacial

Orthop 2002;122:84-94.

19. Chen F, Terada K, Hanada K, Saito I. Anchorage effects of palatal

osseointegrated implant with different fixation: a finite element

study. Angle Orthod 2005;75:593-601.

20. Costa A, Raffaini M, Melsen B. Miniscrews as orthodontic an-

chorage: a preliminary report. Int J Adult Orthod Orthognath

Surg 1998;13:201-9.

21. Melsen B, Costa A. Immediate loading of implants used for ortho-

dontic anchorage. Clin Orthod Res 2000;3:23-8.

22. Szmukler-Moncler S, Salama H, Reingewirtz Y, Dubruille JH.

Timing of loading and effect of micromotion on bone-implant in-

terface: review of experimental literature. J Biomed Mater Res

1998;43:193-203.

23. Aldikacti M, Acikgoz G, Turk T, Trisi P. Long-term evaluation of

sandblasted and acid-etched implants used as orthodontic anchors

in dogs. Am J Orthod Dentofacial Orthop 2004;125:139-47.

24. Yao CC, Lee JJ, Chen HY, Chang ZC, Chang HF, Chen IJ. Max-

illary molar intrusion with fixed appliances and mini-implant an-

chorage in three dimensions. Angle Orthod 2005;75:754-60.

25. Park HS, Lee SK, Knon OH. Group distal movement using im-

plant anchorage. Angle Orthod 2005;75:602-9.

26. Park YC, Lee SY, Kim DH, Jee SH. Intrusion of posterior teeth

using mini-screw implants. Am J Orthod Dentofacial Orthop

2003;123:690-4.

27. Huja SS, Roberts E. Mechanism of osseointegration: characteriza-

tion of supporting bone with indentation testing and backscattered

imaging. Semin Orthod 2004;10:162-73.

28. Eriksen FE, Axelrold WD, Melsen F. Bone histomorphometry. 1st

ed. New York: Raven Press; 1994.

29. Cope JB. Temporary anchorage devices in orthodontics: a para-

digm shift. Semin Orthod 2005;11:3-9.

30. Isidor F. Histological evaluation of peri-implant bone at implants

subjected to occlusal overload or plaque accumulation. Clin Oral

Implants Res 1997;8:1-9.

31. Oyonarte R, Pilliar R, Deporter D, Woodside DG. Peri-implant

bone response to orthodontic loading: part 1. A histomorphomet-

ric study of the effects of implant surface design. Am J Orthod


32. Vasquez M, Calao E, Becerra F, Ossa J, Enr�ıquez C, Fresneda E.

Initial stress differences between sliding and sectional mechanics

with an endosseous implant as anchorage: a 3-dimensional finite

element analysis. Angle Orthod 2001;71:247-56.

33. Oyonarte R, Pilliar R, Deporter D, Woodside DG. Peri-implant

bone response to orthodontic loading: part 2. Implant surface ge-

ometry and its effect on regional bone remodeling. Am J Orthod


34. Hoshaw SJ, Brunski JB, Cochran GVB. Mechanical loading of

Branemark implants affect interfacial bone modeling and remod-

eling. Int J Oral Maxillofac Implants 1994;9:345-60.


January 2010

35. Serra GG, Morais LS, Elias CN, Meyers MA, Andrade L,

Muller C, et al. Sequential bone healing of immediate loaded

mini-implants. Am J Orthod Dentofacial Orthop 2009 (in press).

36. Deguchi T, Takano-Yamamoto T, Kanomi R, Hartsfield JK,

Roberts WE, Garetto LP. The use of small titanium screws for or-

thodontic anchorage. J Dent Res 2003;82:377-81.

37. Buchter A, Wiechmann D, Gaertner C, Hendrik M, Vogeler M,

Wiesmann HP, et al. Load-related bone modeling at the interface

of orthodontic micro-implants. Clin Oral Implants Res 2006;17:

714-22.

38. Melsen B, Lang NP. Biological reactions of alveolar bone to or-

thodontic loading of oral implants. Clin Oral Implants Res

2001;12:144-52.

39. Sarmiento A, Schaeffer JK, Beckerman L, Latta L, Enis JE. Frac-

ture healing in rat femora as affected by functional weight-bear-

ing. J Bone Joint Surg Am 1977;59:269-75.

40. Rubin CT, McLeod KJ. Promotion of bone ingrowth by fre-

quency-specific, low-amplitude mechanical strain. Clin Orthop

Relat Res 1994;298:165-74.

41. Lopes CC, Junior BK. Histological findings of bone remodeling

around smooth dental titanium implants inserted in rabbit’s tibia.

Ann Anat 2002;184:359-62.

42. Lange GD, Putter CD. Structure of the bone interface to dental im-

plants in vivo. J Oral Implantology 1993;19:123-35.

43. Huja SS, Katona TR, Burr DB, Garetto LP, Roberts WE. Micro-

damage adjacent to endosseous implants. Bone 1999;25:217-22.

44. Klokkevold PR, Johnson P, Dadgostari S, Caputo A, Davies JE,

Nishimura RD. Early endosseous integration enhanced by dual

acid etching of titanium: a torque removal study in the rabbit.

Clin Oral Implants Res 2001;12:350-7.

45. Melsen B, Petersen JK, Costa A. Zigoma ligatures: an alternative

form of maxillary anchorage. J Clin Orthod 1998;32:154-8.

sequential bone healing of immediately loaded mini-implants: … · 2017. 11. 26. · original...

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