1. What data in particular will be shared?
   Response - All of the individual participant data collected during the trial, after de-identification.
   


2. What additional supporting information will be shared?
   Response -  Study Protocol
   Response -  Statistical Analysis Plan
   Response - Informed Consent Form
   Response - Clinical Study Report
   
   
3. Who will be able to view these files?
   Response - Researchers whose proposed use of the data has been approved by an independent review committee identified for this purpose.
   


4. For what types of analyses will this data be available?
   Response - To achieve aims in the approved proposal.
   


5. By what mechanism will data be made available?
   Response - Proposals should be directed to [shivank0810@gmail.com].
   


6. For how long will this data be available start date provided 22-06-2024 and end date provided 22-06-2027?
   Response - Beginning 9 months and ending 36 months following article publication.
   


7. Any URL or additional information regarding plan/policy for sharing IPD? 
   Additional Information - NIL

1.      INTRODUCTION

1. Origin of proposal:

One of the most effective methods for managing edentulous areas is to use dental implants for both cosmetic and functional repair. Implants undergo osseointegration when they are inserted into the bone. The longevity of these implants is proportional to the degree of osseointegration. Advances in implant design, restorative techniques, and patient’s desire for shorter treatment time prompted the concept of the immediate loading protocol [1, 2], even though the original surgical protocol suggested submerging an implant post- placement and maintaining a non-loaded environment for 3-6 months [3].

Conventional or delayed loading refers to implant loading that occurs in more than two months after placement; early loading refers to loading that occurs between one week and two months after placement; and immediate loading refers to loading that occurs within the first week after placement. These definitions were established in 2008 at the ITI Consensus Conference [4].

2. Novelty:

In immediate non-functional loading, the placement of prosthesis is done within 72 hours of implant placement. The prosthesis also does not make contact with the opposing arch. Starting prosthetic treatment right after implant placement significantly cuts down the overall treatment duration. Thus, the modified restoration would still be involved in the masticatory process, but the mechanical loading stress is reduced [5]. Consequently, the current investigation opted for immediate non-functional loading.

The two main types of dental implants, depending on their design, are Tissue level Implants & Bone level implants. The benefits of atraumatic recovery under submerged conditions are enjoyed by bone-level implants, which are often implanted in a two-stage surgery. Whereas, tissue-level implants are usually placed in a single surgical procedure, preserving the smooth neck area above the crest for soft tissue attachment. There is no microgap at the level of the crestal bone in tissue-level implants, in contrast to two-piece implants [6].

There is, however, only a limited level of evidence on bone remodeling around different types of implants when comparing bone-level to tissue-level implants.

CBCT scans can objectively evaluate bone quality before surgery, it will provide predictable information regarding the primary stability of implants and enable an appropriate treatment plan to be made in advance [7, 8, 9, 10].

While the concept of immediate loading in implant dentistry is not a recent development, there is a notable lack of studies comparing biomarker levels between immediately loaded tissue & bone level implants over various time frames to assess the progression of bone healing. In the present study we will quantify and assess the level of osteocalcin as a potential biomarker for bone formation as Osteocalcin is essential for controlling metabolism, promoting bone mineralization, and keeping the calcium ions equilibrium in the body.

3. Applicability:

Treatment decisions concerning the timing of loading and its impact on bone health can be informed by the study’s comparison of non-functional immediate loading in tissue-level and bone-level implants, which offers insights into the healing process.

Clinicians will be able to use the results of studies comparing bone-level and tissue-level implant designs to help them choose the best option for their patients, taking into account factors such crestal bone resorption and remodelling.

Current procedures support the use of CBCT scans to evaluate bone quality anticipating implant stability and design treatment options, which will lead to better results with less risk.

Osteocalcin is a biomarker in GCF surrounding implants. This biomarker is helpful in assessing the bone growth and bone healing in various implant types.

4. Outcome:

The study will shed light on the topic by comparing Non-Functional Immediate Loading in Tissue Level and Bone level implants, as well as by measuring bone density and bone loss in both types of implants, and by determining the presence of Bone Biomarker (Osteocalcin) derived from Gingival Crevicular Fluid.

2. AIMS AND OBJECTIVE

i. Aim: The aim of this study is to assess and evaluate the presence of Bone Biomarker (Osteocalcin) derived from Gingival Crevicular Fluid, during Non-Functional Immediate Loading in Tissue Level and Bone level Implants.

ii. Objectives:

● Primary objective: To examine and quantify the presence of Bone Biomarker (Osteocalcin) derived from Gingival Crevicular Fluid around Tissue Level and Bone Level Implants, comparing outcomes of Non- Functional immediate loading protocol.

● Secondary objective: To evaluate the bone density and bone loss surrounding Tissue Level and Bone Level Implants subjected to Non- Functional immediate loading.

MATERIAL AND METHOD:

i. Study design:

● In- vitro/In-vivo/Ex-vivo/Survey - In vivo

● Type of Study – Clinical Research

In this present split mouth study we will include 30 patients having bilateral edentulous area in maxillary premolar region. Detailed information about the study’s objectives, procedures, potential benefits, and associated risks will be provided to each participant.

General information, such as name, age, and gender, will be recorded for each participant. A random assignment of implant sites will be performed using lottery method, resulting in two groups

Group I encompasses sites in patient receiving tissue level implant with Non-Functional immediate loading and subsequent PISF (Peri-implant Sulcular Fluid) collection at 2 week & 3 months.

Group II will include sites in patient receiving bone level dental implant with Non-Functional immediate loading and subsequent PISF (Peri-implant Sulcular Fluid) collection at 2 week & 3 months.

All participants will undergo thorough clinical and radiographic evaluations, which includes orthopantomogram (OPG) and cone-beam computed tomography (CBCT) prior to which informed consent will be obtained.

ii. Sample selection:

● Inclusion criteria -

a) Age exceeding 21 years and below 60 years

b) Overall good health;

c) Absence of systemic diseases that could impact bone metabolism and wound healing;

d) No regular intake of medications for a minimum of 3 months preceding the treatment;

e) Patient’s willingness and ability to adhere fully to the study protocol; and

f) The provision of written informed consent.

g) ISQ value greater than 60

● Exclusion criteria –

a) History of head or neck radiation therapy;

b) Immune-compromised status, including HIV infection or chemotherapy within the past 5 years;

c) Pregnancy or lactation patients during any part of the study;

d) Poor oral hygiene motivation,

e) Psychological or psychiatric issues

f) Patients having intake of Calcium and Vitamin D supplements

● Discontinuation criteria:

a) Patient is unwilling to participate

b) Patient did not turn up on follow up appointment

iii. Sampling:

Sample size/ number of participants: 30 in each group (Total – 60)

● At site - Sardar Patel Post Graduate Institute of Dental & Medical Sciences ( 30 samples in each group )

● In India – Lucknow, Uttar Pradesh

● Globally – N/A

❖ Control Group – N/A

❖ Study Group -

● Group I – Will comprise of sites in 30 patients who will undergo Tissue level implant placement which would be immediately non-functionally loaded and subsequent collection of PISF at 2 weeks & 3 months will be carried out.

● Group II - Will comprise of sites in 30 patients who will undergo a Bone level implant placement which would be immediately non-functionally loaded and subsequent collection of PISF at 2 weeks & 3 months will be carried out.

Justification for the sample size chosen:

In a previous study by Vianna et al. [17], it was reported difference in bone loss pre-implant and 24 months post-operatively was 0.75±1.12 mm in tissue level group and 0.70±0.72 mm in bone level group. In the present study we also targeted a similar outcome. The sample size was calculated from study by Charan and Biswas. [18]

Where:

2 n ≥ 2*SD* (𝑍𝛽+𝑍𝛼/2)

𝑑2

SD: Pooled Standard deviation from previous study= 1.12+0.72= 1.84 Zβ= 0.84

Zα/2= 1.96

d= Mean from previous study= 1.45  

Putting in the formulae

n ≥ 2*1.84*(0.84+1.96)2/(1.45)2

≥ 25.24

~25 in each group.

Assuming a data loss of 10% and rounding off to nearest multiple of 2, the proposed minimum sample size is 28 patients in each treatment group; however, for purpose of statistical comparison we will include 30 patients in each group. Total – 60.

iv. Methodology:

In this split mouth study we will include 30 patients having bilateral edentulous area in maxillary premolar region which will be divided into 2 groups. The patients will be selected by random allocation.

● Surgical phase:

The standardised aseptic surgical process rules will be followed for the insertion of the dental implants. The site will be anesthetized with 2% lignocaine and 1:80,000 adrenaline before the insertion. The procedure involves releasing incision that will be used to raise a full-thickness flap exposing the crest and vestibular limit of the bone.

While the osteotomy is being prepared, a large quantity of cold saline solution will be continuously irrigated. As per the instructions provided by the manufacturer, Group I Tissue Level implants will be placed up to the screw level, with a slim 2.2 mm neck sticking out from the bone crest level whereas Group II Bone Level implants which will be placed directly to the bone crest level, Every precaution will be made to ensure that the periodontal tissue of neighbouring teeth is not compromised. Suturing of the flap after implant placement will be done without tension.

For 10 days, participants will be instructed to rinse their mouths with a 0.1% solution of Octenidine twice daily. They will also be given oral antibiotics (500 mg of amoxicillin, three times daily) and analgesics (600 mg of ibuprofen) for five days [16].

Immediate non-functional loading protocol will be applied to both implants within 48 hours. Clinical evaluation of each implant’s stability measurement, as defined by the Implant Stability Quotient (ISQ), will follow implant surgery. At following appointments, we will use Resonance Frequency Analysis (RFA) equipment (Osstell; Integration Diagnostics AB, Sweden) and temporarily remove the prosthetic abutment and superstructure in order to carry out this examination. A respectable ISQ value of 60 or higher will be accepted.

● Fabrication of Prosthesis:

In order to facilitate immediate nonfunctional loading of implants, a single step double mix impression technique will be utilized after connecting the implant to the open tray transfer coping.

Open tray coping will be attached to the implant analogue and cast will be fabricated with G-mark and Die Stone.

On the articulated casts; Temporary titanium abutment will be placed & screw retained temporary crowns will be fabricated from Bis-Acrylic Composite material. The temporary restoration will be designed with the occlusal surface in mind so that it does not come into contact with the opposing dentition in either the static or functional stages. Verifying the emergence profile and proximal connections is the next step before screwing in the temporary crowns with a 15-Ncm torque and sealing the access hole with composite.

● PISF sampling and transportation:

From all the implant sites Peri-implant Sulcular fluid (PISF) will be carefully collected at 2 weeks and 3 months post-surgery. Cotton rolls will be utilized to isolate the implant sites in order to guarantee accuracy in the sample collecting process. The PICF will be collected using gingival fluid collection strips. The strips will be carefully placed into the sulcus to a depth of 1 mm and will be held there for 30 seconds. We shall discard strips that come into contact with blood contamination. The next step is to use tweezers to carefully remove the strips, and then the strips will be placed in Eppendorf tubes containing 100 μL of phosphate buffer saline with protease inhibitor. All the sample containing tubes will be transported in sealed, leak proof puncture resistant bags on dry ice at -80º C in insulated containers. These tubes will be used for further processing until then the samples will be kept at a temperature of -80º C. Also, a calibrated electronic instrument will be used to quantify the amount of peri-implant sulcular fluid (PISF) absorbed on each strip.

● Quantification of osteocalcin:

Thawing and storage at 4°C will be done on the stored samples before analysis. Next, the samples will be vortexed at 3500 RPM for one minute, and then centrifuged at 12,000 RCF for two minutes. Total protein concentrations will be measured using the collected supernatant. Following the instructions provided by the manufacturer, the samples will be analyzed using enzyme- linked immunosorbent assay (ELISA) kits that are commercially available. Quantitative assessment of OCN protein in both immediately non- functionally loaded implants will be performed using an ELISA.

● Marginal bone loss and Bone density:

All patients will undergo pre-operative CBCT for radiographic evaluation. Subsequent CBCT will also be done at 2 weeks and 3 months post-operatively for determination of bone density and bone loss.

In vivo imaging software will be used for analysis of CBCT scans and bone density will be measured in HU (Hounsfield unit) value. The site of interest will be chosen using the spatial coordination tool (x, y). The x-coordinate, which varies horizontally, will be positioned tangentially, while the y-coordinate, which varies vertically, will be kept constant.

Peri-implant bone level (PBL) will be found by calculating the linear distance between two points: the implant platform’s most coronal point and the point where the bone meets the implant. A two-point assessment will be conducted on the implant’s mesial and distal sides to determine the vertical distance between the platform’s most coronal point and the point of maximum bone-to-implant contact.

T0 (at primary immediate implant loading) and T1 (post final implant loading).