2.3.2. Histological Analysis: Qualitative and Quantitative

The histological sections were qualitatively and quantitatively investigated in a light microscope (L.M., Eclipse Nikon ME600, Nikon, Tokyo, Japan) by two of the authors: C.J. and D.R. Measurements were performed with the NIS-Elements D 64-bit software (version 3.2, Nikon Metrology, SARL, Lisses, France) using a 10× objective. Light microscopic (LM) images of the histological features were obtained with a Nikon DS-Ri1 camera (Nikon Instruments Inc. Meville, NY, USA). The first implant-bone contact was ascertained with a 40× objective.

The distance from the implant top to first bone contact was measured on both sides of each implant (anterior and posterior) sides. The difference between the distances for control versus test (silk ligatures) samples in the proximal tibia and for the cotton versus silk samples in the femur, were analyzed using non-parametric Wilcoxon Signed Rank with the pair considered as the control and test samples from the same rabbit. Statistical significance was set for *p* < 0.05. A level of 0.05 was selected for the α-error and the statistical significance was set for *p* < 0.025 after Bonferroni's adjustment for multiple calculations. A qualitative investigation of the marginal hard and soft tissues was also performed.

#### *2.4. Gene Expression Analysis—qPCR*

The samples for the gene expression analysis originated from the soft tissues that were in direct contact with the implant heads in the distal tibias and bone tissue that was adherent to the implants in distal tibia and constituted four study groups (soft tissues specimens from implants with and without ligature and bone specimens from implants with and without ligatures). A total of 32 samples (20 soft tissues and 12 bone tissue) were analyzed. The soft tissues were cut with sterile, disposable tissue punches of 6 mm diameter (1 punch per specimen) and dissected from the implant surface with a curette (Miltex, Inc. York, PA, USA).

The specimens were immediately placed in separate sterile plastic tubes containing RNA*later* solution (Ambion, Inc., Austin, TX, USA), for fixation. The samples were then stored at 4 ◦C overnight and further stored at −20 ◦C until processing.

#### 2.4.1. mRNA Isolation

(This step is performed to purify the mRNA from the samples.)

mRNA isolation and qPCR amplification were performed at TATAA Biocenter, Gothenburg, Sweden.

Before mRNA isolation and extraction, the samples were thawed on ice. The samples were extracted using Qiazol (Cat.No 79306) and the RNeasy mini kit (Cat.No 74104) (Qiagen GmbH, Venlo, Netherlands) according to manufacturer's instructions.

Sample concentrations where determined using spectrophotometry (Dropsense, Trinean, Pleasanton, CA, USA) and RNA integrity was analyzed using capillary electrophoresis (Fragment Analyzer, Thermo Fisher Scientific, Waltham, MA, USA).

After extraction, the RNA was cleaned of inhibitory factors using the RNeasy MinElute Clean up kit (Qiagen GmbH, Venlo, Netherlands, Cat no. 74204).

#### 2.4.2. Reverse Transcription (RT)

(This step produces complementary DNA (cDNA) to the mRNA isolated from the respective samples.)

RNA was reverse transcribed in single 20 μL reactions on all 32 samples.

Samples where first normalized to 33.33 ng/μL to reach a quantity of 500 ng RNA per tube that was loaded into the reaction. The RT was performed according to manufacturer's instructions with TATAA Grandscript cDNA synthesis kit Cat.No A103 (TATAA Biocenter AB, Gothenburg, Sweden), with the following concentration: 1 μL RT enzyme, 4 μL reaction mix, 15 μL normalized sample RNA.
