2.4.3. Assays Design and Validation

(This step is performed to create short DNA fragments that are later used to determine each specific region of DNA to be copied, in this case the regions that encode the proteins of interest for the study.)

Eleven assays where designed and validated by TATAA (according to SOP 009 ver 1.2 SOP 001 ver1.4) (Validation plan qPCR assay validation). A list of all assays can be found in Table 1. For validation, a control genomic DNA (gDNA) from male rabbit (Zyagen) and a pool of all the cDNA samples was used. Seven-point standard curves in 10-fold dilutions were run for all the assays, in quadruplicates. The annealing temperature was set to 60 ◦C for all assays. PCR products; gDNA, cDNA, selected standard point and NTC were then run on Fragment Analyzer using the DNF-910-33-DNA 35-1500bp kit (Thermo Fisher Scientific, Waltham, MA, USA) to check the product lengths and primer specificity. The qPCR data for the assay validation was analysed with GenEx software (version 6, MultiD Analyses AB, Göteborg, Sweden) to calculate the efficiencies and performance of the assays with a confidence interval of 95%.



ACTβ, β-actin (reference gene); ALPL, alkaline phosphatase; ARG1, arginase 1; C5aR1, complement C5a Receptor 1; CD11β, macrophage marker CD11β; CD19, B-lymphocyte surface protein CD19; CD4, T-cell surface glycoprotein CD4; CD8, T-cell transmembrane glycoprotein CD8; GAPDH, glyceraldehyde 3-phosphate dehydrogenase (reference gene); IL8, interleukin 8 receptor alpha; LDHA, lactate dehydrogenase A (reference gene); MCP1, monocyte chemotactic and activating factor; NCF1, neutrophil cytosolic factor; OC, osteocalcin; CTSK, Cathepsin K; FGF2, fibroblast growth factor 2; IL1β, interleukin 1 beta; IL6, interleukin 6; VEGFα, vascular endothelial growth factor alpha; TNFα, tumor necrosis factor alpha; TRAP, Triiodothyronine receptor auxiliary protein.

#### 2.4.4. Real-Time Quantitative Polymerise Chain Reaction (RT-qPCR)

(This final step monitors the amplification of the selected DNA segments in real time and enables the researcher to quantify the amount of each DNA segment at the start of the reaction.)

The cDNA samples were diluted 10× to have enough volume and were then analyzed using TATAA SYBR GrandMaster® Mix Cat. No. TA01-625 (TATAA Biocenter AB, Gothenburg, Sweden). Five μl of TATAA SYBR Green Master Mix, 0.4 μL of Primer (forward & reverse), 2.6 μL of nuclease-free water and 2 μL of cDNA templates were used for each reaction mix. All pipetting was performed by a pipetting robot (EpMotion 5070, Eppendorf, Germany). Duplicate NTCs were included for all the assays and cDNA samples were run in duplicate reactions. Universal ValidPrime assay, Cat No. A107P (TATAA Biocenter AB, Göteborg, Sweden) was used to compensate for possible gDNA contamination.

The quantification was performed using the LightCycler 480 (Roche, Basel, Switzerland) and detection was performed in the SYBR channel. Cq values were based on the second derivative maximum threshold method. Inter-plate calibrator, IPC Cat. No. IPC250 (TATAA Biocenter AB, Göteborg, Sweden) was run on each plate to be able to correct for inter-run differences. qPCR raw data were pre-processed and analyses with GenEx software (version 6, MultiD Analyses AB, Gothenburg, Sweden) and were thereafter imported in Qbase+ software (version 3.1, Biogazelle, Zwijnaarde, Belgium) for calibrated normalized relative quantification of the gene expression. Three assays were used as reference genes (ACTB, GAPDH and LDHA) and their quality as reference genes was assessed with the GeNorm algorithm.

#### 2.4.5. Statistical Analysis

The gene expression results were reported as calibrated normalized relative quantities (CNRQ). Mean and 95% confidence intervals were reported for each assay for each group.

The difference in mean between the test (silk ligated implants) and the control (pristine Ti-implant) groups in the soft tissue samples and between the test and control groups in the bone samples were analyzed using non-parametric Wilcoxon Signed Rank. The test and control samples from the same rabbit were considered as paired. The level of α-error was set to 0.05 and statistical significance was adjusted according to Bonferroni´s method for multiple testing; therefore, it was set to *p* < 0.0027.

#### **3. Results**

#### *3.1. Clinical Appearance at Sacrifice*

The tissue harvesting procedure is shown in Figure 3. Uneventful healing and primary wound closure were achieved in all cases. Dissection of the tissues revealed no pus or other signs of infection around the implants. Exposure of the bone adjacent to the implants revealed small saucerization-like defects around most ligated implants and sometimes callus formation lateral to the ligatures. Some control implants presented with callus formation lateral to the implants, but no saucerization-like bone defects were noted.

**Figure 3.** (**a**) The soft tissue covering a tibial control implant (superior) and test implant (inferior) after removal of skin and subcutaneous tissues. (**b**) A small saucerization-like defect (green arrow) visible after removal of the silk ligature around the inferior implant and callus formation (black arrow) around the superior control implant.

#### *3.2. Histological Results*

#### 3.2.1. Histomorphometrical Results

One implant with a cotton ligature was excluded from the histomorphometrical analysis due to a superior displacement of the ligature and unfavorable implant location that engaged the dorsal femoral bone plate and showed non-union of the implant on that side. The distance from the implant top to first bone contact was significantly longer for test implants ligated with silk compared to controls (*p* = 0.007) (Figure 4). This difference was due to the combined effect of bone resorption inferior to the ligatures in the test implants and also bone gain due to callus formation adjacent to protrusive implant tops in some control implants. The difference between implants ligated with silk or cotton was not statistically significant (*p* = 0.37).

**Figure 4.** Box-plot showing the distance from implant top to first bone-to-implant contact for implants with silk ligatures (Silk) and pristine implants (Control).
