WITHDRAWN: Pilot Testing and Histomorphometric Evaluation of a Novel Mini Implant System(n)

doi:10.21203/rs.3.rs-4878672/v1

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WITHDRAWN: Pilot Testing and Histomorphometric Evaluation of a Novel Mini Implant System(n)

https://doi.org/10.21203/rs.3.rs-4878672/v1

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The design of a mini-implant is a key element affecting the implant's initial stability, ability to sustain load and achieve successful osseointegration. This study aims to present the findings of pilot testing of a novel mini-implant technology (‘n’).

The novel mini-implant (‘n’) is made of titanium grade V, having a trapezoidal thread pattern and a transverse hole of 0.8 mm diameter. This novel mini-implant (‘n’) was inserted in a test group of 3 male New Zealand white rabbits. A control group of 3 male New Zealand white rabbits was inserted with a similar number of control mini-implants. One animal in the test group additionally received a bone-inducing graft (DFDBA GRAFT) in the transverse means. The animals were sacrificed after 42 days and bone tissues were fixed. The histomorphometric findings were evaluated using BIC, BAFO, and cortical bone thickness.

There was no mortality among the studied animals. Mean BIC% and BAFO (%) were 7.88% (95% CI: 5.10% - 10.66%) and 10.33% (95% CI: 5.86%-14.81%) higher among test animals. The woven bone formation was found to be higher among the test group as compared to the control group at 1mm, 1 – 2mm, and 3 – 4mm distance from the implant site. Higher BIC and BAFO were observed in the animals that received the bone-inducing graft.

This study proposes a novel design of mini-implant (‘n’) which is found to be safe in this pre-clinical trial and has the potential to achieve higher primary and secondary stability.

Health sciences/Health care

Health sciences/Medical research

Physical sciences/Engineering

Physical sciences/Materials science

Mini-implants

Orthodontics

Pilot study

Osseointegration

BIC

BAFO

Mini implants as anchoring devices are a standard procedure in orthodontics.
The implant design is a key element affecting implant’s initial stability, its ability to sustain load and achieve successful osseointegration
This article introduces a novel mini implant design that showed higher BIC and BAFO
The novel mini implant system has potential to achieve higher primary and secondary stability in pre-clinical trial

Anchorage,defined as the nature and degree of resistance to displacement offered by an anatomic unit, is a crucial consideration for a successful orthodontic treatment.¹The use of mini-implants for anchorage reinforcement has increased dramatically over the past decade. In general, mini-implants are simple to place in a short time, have low cost, and can be loaded immediately.^2,3Retention of mini-implants depends on multiple factors like implant dimensions, implant surface characteristics, insertion angle, insertion torque, site of insertion, soft tissue characteristics, bone quality, risk of inflammation, and root proximity.^4–6 For any successful implantation, whether dental implants or orthodontic mini-implants, stability is necessary. Primary stability is defined as implant stability immediately after insertion, whereas secondary stability is due to ongoing bone remodelling as a result of osseointegration around the mini-implant.⁷By definition, osseointegration is direct bone apposition on the implant surface.⁸ Osseointegration depends on the surgical technique, geometrical design of the implant, material biocompatibility, and loading conditions.^9,10

A systematic review by Reynders et al has reported success rates of mini-implants ranging from 0% to 100%, with most studies reporting success rates greater than 80% if mobile and displaced implants are considered successful.¹¹Another meta-analysis reported a 13.5 percent (95% CI 11.5–15.9) overall failure rate of mini-screw.¹²The mini-implants are known to displace during the orthodontic loading and fail to remain completely stationary in position when subjected for several months to load.^13,14 This suggests that, although currently available mini-implants have good primary stability, there is a need to enhance their secondary stability to ensure long-term success. The key element affecting the implant’s initial stability and its ability to sustain load and achieve successful osseointegration is implant design.¹⁵ This article introduces a novel mini-implant technology (termed as ‘n’) that has the potential to enhance secondary as well as primary stability. The present study shares the histomorphometric findings of pilot testing of novel mini-implant technology (‘n’) in New Zealand white rabbits and compares it with the currently available mini-implant system.

DESIGN OF NOVEL MINI-IMPLANT (’n’)

We developed a new mini-implant system (‘n’) made of the titanium grade V which is 11.5 mm long and 2.2 mm in diameter for the first segment and 1.9 mm for the second segment which gradually decreased towards the apex (Figures 1 and 2). The mini-implant system contains a transverse hole of 0.8 mm diameter to allow regeneration tissues to pass through.

The new mini-implant system(n) was compared with a commercial mini-implant which is cylindrical and measuring 2 mm in diameter and 10 mm in length but no transverse hole.

A total of six New Zealand white rabbits were divided into two groups of three animals:A control group for commercialmini-implants and A test group for novelmini-implants(‘n’).All the animals were of male sex. The experimentation on animals was carried out conforming to the guidelines set forth by regulatory bodies.^16,17

Intramuscular injections ofketamine (40 mg/kg) and xylazine (10 mg/kg) were used to anesthetize the animals. The tibia bone was selected as the site for implantation. The surgical site was swabbed with betadine solution and a small incision was made in the skin and muscle at certain locations to expose the bone. The cortex of the tibia was exposed and holes with proper diameter (for test and control mini-implants) were drilled using low drilling speed. A totalof ten control group mini-implants were implantedin three animals (Animal no. 1, 2, and 3) whereas, fifteen test group mini-implants(n) were implanted inanother three animals (Animal no. 4, 5, and 6).(Fig. 3a, b, c, d). One of the three animalsin the test group (Animal no. 6) was additionally implanted with five bone graft samples (DFDBA BONE GRAFT FROM TATA MEMORIAL HOSPITAL TISSUE BANK, MUMBAI) to observe the effect of bone graft on bone-to-implant contact (BIC), bone area fraction occupancy (BAFO) and cortical bone thickness.

After implantation, the muscles of all six animals were sutured with absorbable sutures, and the skin with non-absorbable sutures. The sutured areas of individual animals were treated with betadine and antifungal powder. Individual animals were treated with analgesics and antibiotics for 7 days after treatment. All the animals were fedad libitum identical standard diet. Throughout the experimentation, every animal implanted with the test or control mini-implant was inspected periodically for their health by a veterinary doctor.

After 42 days, all the animals were sacrificed using an overdose of intravenous thiopentone sodium. The implant locationswere inspected macroscopically for tissue responses such as haemorrhage, oedema, and encapsulation.

The bone blocks were fixed overnight in 10% neutral buffer formalin in toto. After 24 hours, control and test mini-implants(‘n’) were carefully removed without damaging the surrounding bone tissue. Bony tissues were subjected to decalcification by concentrated nitric acid followed by grossing. The tissue specimens were then kept in automated tissue specimens (graded acetone, xylene, and paraffin wax). Tissue blocks were prepared by embedding them in paraffin wax and thin sections of 3–5 µm were made by rotary microtome. Slides were then stained and examined under a microscope in the scanner and 10x views to evaluate histomorphometry in different locations viz, upper one-third, middle one-third, and lower one-third regions of the mini screw.

The histomorphometric changes were recorded by capturing longitudinal sectionsof each implantwitha Nikon Eclipse E200 microscope and the images were analysed by ImageJ software.^18–20The histomorphometricfindings were determined using cortical bone thickness, bone-to-implant contact (BIC), and bone area fraction occupancy (BAFO) in the test(‘n’) and control mini-implant system. BIC was evaluated based on how much of the implant surface is touching bone on a microscopic level and graded as a percentage. BAFO was calculated as the percentage of area within the implant threads occupied by visibly distinguishable bone.^10,15

The study protocol was reviewed and approved by the Institutional Ethics Committee of the Research Centre through IAEC protocol no. “RRC/IAEC/07/2022/035”. This research centre is approved for experiments on laboratory animals by “The National Committee for The Purpose of Control and Supervision of Experiments on Animals” (CPCSEA, New Delhi) and the Food and Drug Administration of the State Government.

This study is reported in accordance with ARRIVE guidelines.

STATISTICAL ANALYSIS

The data was described using mean and standard deviation (SD) for quantitative variables. Mean differences and 95% confidence interval (CI) of these differences were calculated using IBM SPSS version 25.

All the animals showed normal wound healing and there was no loss of implants in any of the test or control animals. There was no animal mortality throughout the study orany evidence of haemorrhage, oedema, or encapsulation at the implant site.

Microscopic changes observed in animals are shown by histopathological images in Fig. 3. Histological examination of Animal 1 showedresidual material surrounding nascent bone growth with limited infiltration in the metaphysis. Animal 2 showedfocal congestion in the bone. Animal 3 exhibited osseous degeneration in the bone. Animal 4 showed Minimal infiltration at the distal metaphysis of bone, denoted by an arrow. An increased lacuna was seen in the bone of Animal 5. Woven bone developments werereported in the bones of Animal 6.

It was observed that BIC% in control animals ranged from 43.45–49.29% in three regions with an average BIC% of 46.19% (SD ± 2.32%). BIC% in test animals ranged from 49.32–59.24% with an average BIC% of 54.07% (SD ± 3.16%). Thus, the mean BIC% among test animals was found to be 7.88% (95% CI of difference 5.10%- 10.66%)higher compared to control animals (Table 1). Among test animals, animal no. 6 received an additional bone graft. BIC% in this animal was 57.76%, 55.23%, and 54.25%in the upper region, middle region, and lower region respectively, which was higher than the other two test animals in the upper and middle region but not in the lower region.

Table 1

BIC % in Test and Control Animals
Animal no.	Group	BIC %			Mean BIC % (SD)
Animal no.	Group	Upper Region	Middle Region	Lower Region	Mean BIC % (SD)
1	Control	45.12%	43.45%	47.28%	46.19% (± 2.32%)
2	Control	48.47%	49.29%	45.37%
3	Control	46.31%	48.07%	42.40%
4	Test	54.62%	51.37%	59.24%	54.07% (± 3.16)
5	Test	51.24%	49.32%	53.68%
6	Test (with Bone Graft)	57.76%	55.23%	54.25%

Mean BAFO in control animals was 49.43% whereas mean BAFO in test animals was 59.76%. There was a 10.33% higher BAFO (%)(95% CI of difference 5.86% − 14.81%) among test animals compared to control animals (Table 2).

Table 2

BAFO (%) in Test and Control Animals
Animal no.	Group	BAFO %	Mean %
1	Control	48.60%	49.43%
2	Control	50.17%
3	Control	49.52%
4	Test	59.60%	59.76%
5	Test	57.17%
6	Test	62.52%

Bone formation distant to the mini-implant was measuredby cortical bone thickness at 1mm, 1–2mm, and 3–4mm distances. Mean cortical bone thickness in the test group was 888.13 µm at 1 mm, 759.03 µmat 1–2 mm distance, and 749.81 µm at 3–4 mm distance. Among control animals, mean cortical bone thickness was 750.53 µmat a 1 mm distance, 709.11µm at a 1–2mm distance, and 732.81 µm at a 3–4mm distance. Cortical bone thickness was comparatively higher at 1 mm distance in both test animals and control animals compared to 1–2mm and 3–4 mm distance. The woven bone formation was higher among the test group as compared to the control group at all three distances of measurements (Table 3).

Table 3

Cortical Bone Thickness and Its Means at 1 mm, 1–2 mm and 3–4 mm
Animal No.	Group	1 mm	Mean	1–2 mm	Mean	3–4 mm	Mean
1	Control	762.61	750.53	729.22	709.11	724.49	732.81
2	Control	741.63		694.32		739.18
3	Control	747.37		703.81		734.77
4	Test	873.20	888.13	693.40	759.03	706.23	749.81
5	Test	886.34		764.53		749.67
6	Test	904.87		819.17		793.55

This study evaluated a new design(‘n’) of mini-implantto understand its safety and histomorphometric outcomes like bone-implant contact, bone area fraction occupancy, and cortical bone thickness in comparison to conventional mini-implantsamong New Zealand white rabbits. There was no mortality among test animals who received new mini-implant nor there wasoedema, necrosis, or inflammatory reaction at the site of implantation. The novel mini-implant ‘n’ was found to be safe in this pre-clinical pilot testing.

Owing to its intraosseous conical shape and novel (trapezoidal thread pattern) thread pattern, this design is proposed to achieve greater primary stability.²¹

Following the insertion of an implant or TAD, relatively immature new bone grows quickly at the mini-implant interface, eventually replacing a complex but stronger bone along the interface.²²Histopathological analyses of the current study suggested that after 6 weeks of implantation, BIC% and BAFO were higher among test animals compared to control animals which suggests better production of new woven bone in test animals than control animals. Within the 1 mm of the mini-implant in test animals, the amount of cortical bone thickness is also greater(Table 3). The novel design is provided with a traversing means (TM) of 0.8 mm (Fig. 1). We hypothesized that the woven bone would additionally travel through the 0.8 mm TM, giving enhanced secondary stability to the mini-implant which will improve resistance to loading. Further, we observed that BIC and BAFO were higher in animal 6 which were layered with bone-inducing means (DFDBA GRAFT).

The proposed novel design of mini-implant (‘n’) is safe and haspromising potential to achieve higher primary and secondary stability in this pre-clinical trial. The test mini-implant’s unique design makes it possible to put in additional bone grafts. However, further studies are needed to precisely estimate insertion torque and confirm the primary and secondary stability of the new implant technology.

DATA AVAIBILITY

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

DECLARATION OF CONFLICT INTERESTS

There are no conflicts of interest concerning research, authorship, and publication of articles.

FUNDING

The author received no financial support for the research.

APPLIED PATENT NUM

INDIAN PATENT APPLIED: Ref. 202221053745

PCT INTERNATIONAL APPLICATION: PCT/IN2023/050393

CDSCO REGISTRATION RECEIVED: License Number: MFG/MD/2018/000079

ACKNOWLEDGMENT

I acknowledge the support provided by Ribosome Research Centre, Surat for conducting animal study and Nebula Surgical, Rajkot for providing the novel implants. I also acknowledge Dr. Nilesh Fichadiya, Assistant Professor in Community Medicine, P. D. U. Medical College, Rajkot for his support in data analysis and critical appraisal of manuscript.

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No competing interests reported.

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WITHDRAWN: Pilot Testing and Histomorphometric Evaluation of a Novel Mini Implant System(n)

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