4. Materials and Methods
4.1. Type of study
Prospective observational cohort study approved by the Andalusian Biomedical Research Ethics Coordinating Committee (Code US-DTL-2022.1) that complies with all the guidelines of the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects [
42].
This is an observational study whose invasive procedure was the collection of saliva samples from the peri-implant tissues of implants rehabilitated with different diameters of healing and prosthetic abutments.
All patients signed an informed consent form based on this study and understood and accepted the type of treatment carried out on them.
4.2. Patient selection
A series of patients meeting the following inclusion and exclusion criteria were selected:
Inclusion criteria:
Adult patients over 40 years of age
Edentulous patients requiring single crowns
Single edentulous spaces 6 to 8mm in height
Exclusion criteria:
Patients with uncontrolled chronic diseases
Patients with immune diseases
Patients with smoking habits, alcoholism or narcotic drug use
Patients medicated with steroids or bisphosphonates
Patients with active periodontal disease
Patients with poor hygiene habits
4.3. Surgical procedure
Patients were surgically treated with implants (Astra tech EV, Dentsply Sirona S.A., Barcelona, Spain) of 4.2mm or 4.8mm diameter in the upper and lower jaw.
The implants received healing abutments and were subsequently connected with customised abutments (Atlantis, Dentsply Sirona S.A., Barcelona, Spain), which were digitally designed with the software (Atlantis WebOrder, Dentsply Sirona S.A., Barcelona, Spain), (Atlantis Design, (VAD) version 4.6.5, which provided the necessary precision, the combination of biological, anatomical and engineering parameters providing the beneficial conditions for the soft tissue healing and adaptation of the final restoration.
All prosthetic abutments were individualised with a larger diameter than the healing abutment in order to evaluate the pain and inflammatory response of patients after definitive rehabilitation, which was quantified by the presence of biomarkers present in the crevicular fluid such as interleukin 1 beta (IL-1β) proteins in an ELISA test.
4.4. Prosthetic procedure
The material of choice for the preparation of customised abutments was Zirconium Oxide (InCoris ZI meso, Dentsply Sirona, DeguDent GmbH Rodenbacher Chaussee Hanau-Wolfgang, Germany) for CAD/CAM production, the same abutment design was used for all patients, the only variant was the measurement of the available soft tissues and according to that the surface to make the contact with the critical and subcritical profile, what we now know as B,C,E zone [
43].
For the prosthetic crown, zirconium oxide discs were chosen (Cercon XT ML, Dentsply Sirona, DeguDent GmbH Rodenbacher Chaussee Hanau-Wolfgang, Germany) which has 750 MPa of resistance to bending over its entire length, taking the precaution of maintaining a minimum thickness of the restoration of 1.5mm as a margin of error and the advantage of having a 49% translucency in degradation throughout the restoration, obtaining a more aesthetic result.
The cases were sent to the dental laboratory, which continued with the milling process of the single-unit restorations on implants.
The cases were planned to be of the indirect cement-screwing type. Dual-curing self-adhesive resin cement (Relyx unicem 2, 3M ESPE AG, Dental Products Seefeld, Germany) was used because of its dimensional stability and high resistance to microfiltration due to long-term dissolution of the material.
Finally, the single crowns were installed in each of the patients and the pain scale was performed immediately.
4.5. Pain and inflammation scales
These were manually annotated on paper with a template for each patient, which consisted of variables from 0 to 5, for greater patient comprehension, with 0 being no pain and 5 being maximum pain. Specifically, value 0 was assigned to those patients who had no pain at any time during the placement or afterwards, value 1 was assigned to those patients whose pain did not last more than 10 minutes, value 2 to those patients whose pain disappeared before 24 hours, value 3 to those whose pain disappeared between 24 and 48 hours after placement, value 4 to those whose pain disappeared after 72 hours, and value 5 to those whose pain lasted for more than 72 hours.
The inflammation scale was also evaluated, also consisting of variables from 0 to 5, and scored manually on paper with a template for each patient, being 0 no inflammation and 5 maximum inflammation. Specifically, value 0 was assigned to those patients who had no inflammation at any time during or after placement, value 1 was assigned to those patients whose inflammation did not last more than 10 minutes, value 2 to those patients whose inflammation disappeared within 24 hours, value 3 to those whose inflammation disappeared between 24 and 48 hours after placement, value 4 to those whose inflammation disappeared within 72 hours, and value 5 to those whose inflammation lasted for more than 72 hours.
4.6. Sampling
Sample collection was carried out 4 hours after delivery of the rehabilitation to each patient.
The collection of inflammation biomarker samples was taken at 4 sites (mesiobuccal, distobuccal, mesiolingual and distolingual) of each implant, avoiding contaminating the sample with saliva, a sterile paper collection strip (PerioPaper strips, Oralflow, Smithtown, NY) being inserted into the peri-implant sulcus for 30 seconds according to the manufacturer's instructions.
The four strips from each implant were pooled in Eppendorf centrifuge tubes and subsequently stored at -80°C until further processing.
4.7. Biomarker analysis using ELISA technique for IL-1β
The kit used for the ELISA technique was the KIT Quantikine® HS ELISA Human IL-1β/IL-1F2 Immunoassay (Ref. HSLB00D). It is a sandwich-type enzyme immunoassay technique in which the plate is treated with a monoclonal antibody specific for human IL-1β, so that in the presence of IL-1β in the sample, the antibody fixed on it will bind. the plate. On the day of processing, they were thawed and 400μl of Calibrator Diluent RD5T were added, the tubes were homogenized by vortexing so that they came into contact with the solution and centrifuged 3 times at 15,600G for 5 minutes at 4ºC. This elution was divided into 4 aliquots of 100μl, only one being used for the test.
Before ELISA, the samples are diluted by mixing 10μl of sample in 390μl of RD5T buffer (0.025) as indicated in the protocol, thus obtaining a 40x dilution. This step was repeated twice and left at 80x.
In total, 40 samples (in duplicate) and 8 standards (in duplicate) were used, making a total of 96 wells.
The procedure begins by adding 50μl of the RD1-63 test diluent to each well. After this, 100ul of standard or sample are added per well and covered with an opaque adhesive sticker for incubation for 2 hours at room temperature in a plate shaker at 500rpm. Afterwards, it is turned upside down and dried face down on paper. The process is repeated 3 times for 4 washes. Each wash is carried out with 400μl of Wash Buffer (reference 895003 of the kit itself, Ref. HSLB00D). After the last wash, all of the Wash Buffer is removed by inverting the plate and drying on clean paper.
Once this is done, 200μl of Human IL-1β HS conjugate is added to each well and covered again with a new adhesive sticker for incubation for 1 hour at room temperature while shaking. Subsequently, a new wash is carried out. After that, 200μl of Streptavidin Polymer-HRP (1X) is added to each well and it is covered again with a new adhesive sticker for incubation for 30 minutes at room temperature on the plate shaker at 500rpm. The washing is repeated and 200μl of Substrate Solution is added (100μl of color A + 100μl of color B) and incubated for 30 minutes at room temperature on the bench, well protected from light.
Finally, 50μl of Stop Solution is added to each well and resuspended. The colour of the wells then changes from blue to yellow.
The results are read before 30min with a λ= 450 nm. To correct the absorbance, another reading of λ= 570 nm is taken, due to possible imperfections in the plate. The reading was carried out on the Thermofisher MultiScan Go spectrophotometer.
4.8. Interpretation of samples
The protocol that was followed was to take the average of the two readings of each sample or standard. To obtain more precise results, the average was subtracted from the absorbance that gave zero.
A corresponding working standard curve was created according to the absorbances obtained in the standards, representing the absorbances on the Y axis and the known concentration of the standards on the X axis. The X was removed from the equation of the straight line and the calculation was carried out for each absorbance of the samples obtained.
If the samples had been diluted, the measured concentrations were multiplied by the dilution factor.
The minimum detectable dose was 0.033pg/ml.
4.9. Statistical Analysis
The Kolmogorov-Smirnov test has been applied to determine the normality of the numerical variables, concluding that, for the variables under analysis, in no case is the distribution normal.
To cross-check qualitative variables, the Chi2 test was carried out. To determine the groups that make the difference, Haberman's corrected standardized residuals have been used, which has made it possible to obtain the significance of the cells independently. This significance implies that the percentage of the cell is different, statistically, from that corresponding to the total. of the sample.
For the processing of categorical and numerical variables, the Mann-Whitney U has been applied since the variables under analysis do not have a normal distribution.
Given that the target variables follow a non-normal distribution, Sperman's correlation has generally been applied.
Statistical significance has been indicated with the usual format (p<0.05; p<0.01; p<0.001, p<0.0001 and p<0.00001), the lower the figure the greater the significance.