Material and methods

This study was approved by the Institutional Animal Care and Use Committee (protocol approval number 03.10.2013-89) of the University of Marmara, Istanbul, Turkey. A total of 81 young adult Sprague-Dawley rats (348 ± 29 g, 9-10 weeks old) were obtained and acclimatized in the University Laboratory Animal Science Centre. The animals were divided into the following groups: donor group (killed for the preparation of PRP; n = 5), high-platelet concentration injection group (hPRP, n = 38), and moderate platelet concentration injection group (mPRP, n = 38). In the latter 2 groups, 5 time points were studied on days 3, 7, 14, 21, and 60. Both the hPRP and mPRP injection groups consisted of 8 animals per time point, except for the day-60 groups, which had 6 animals each. The animals were fed a soft rat diet to prevent any damage to the intraoral appliance and water ad libitum. To limit the influence of interanimal variation in response to the PRP injection, a split-mouth design was used, and the right side of each animal’s maxilla served as the experimental side (hPRP-E and mPRP-E), whereas the left side served as the control side (hPRP-C and mPRP-C) without receiving any PRP injection. All procedures were completed under general anesthesia with intraperitoneal administration of a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg).

Orthodontic tooth movements were achieved with superelastic closed-coil springs (9 mm; G&H Wire Company, Franklin, Ind) placed between the maxillary molars and incisors as previously described. The superelasticity of the coil springs ensured a constant force of 40 cN, and no reactivation was performed during the experimental period. The closed-coil springs were attached to the maxillary first molars and incisors with a stainless steel ligature wire (0.008 in; G&H Wire Company). To improve the fixation of the appliance, a 0.5-mm hole was made by using a metal bur with a slow-speed hand piece. The hole was drilled from the distal surfaces of the incisors, through the teeth, perpendicular to the longitudinal axis of the incisor at the gingival level. A steel wire was inserted through the hole and then tied. The free ends of the ligatures on both molars and incisors were covered with glass ionomer cement (Multi-Cure Glass Ionomer Orthodontic Band Cement; 3M Unitek, Monrovia, Calif) to prevent slipping and gingival irritation. The mandibular incisors were trimmed weekly with a coarse diamond bur to ensure that they were out of occlusion and not at risk of breaking the appliance.

The production of PRP began with a 10-mL homologous blood sample that was withdrawn from the donor animal via cardiac puncture. One milliliter of the blood sample was set apart to determine the concentration of platelets and leukocytes in whole blood. The remaining 9 mL was mixed with an anticoagulant (3.8%, 1-mL sodium citrate) to prevent clotting. The blood sample was centrifuged at 113 g for 5 minutes to separate the plasma containing the platelets from the red cells. The plasma was drawn off the top and centrifuged for an additional 2 minutes at 3772 g to separate the platelets. Then, suitable platelet concentrations were achieved by the resuspension of PRP with plasma (high platelet concentration, 5 times the concentration in whole blood; moderate concentration, 2 times the concentration in whole blood).

The concentrations of platelets and leucocytes in whole blood and the PRP were analyzed automatically (Genius KT Veterinary, Shenzhen Genius Electronics, Guangdong, China) using modified software for animal probes. Triple measurements were performed, and the mean value was recorded as the final measurement.

After each animal’s appliance was placed in a certain subgroup (example: 3-day hPRP-E, n = 8; 3-day mPRP-E, n = 8), a donor rat was killed for the preparation of fresh PRP. The concentrations were measured, and 10 μL of PRP was injected by means of a microsyringe (Hamilton Robotics, Bonaduz, Switzerland) into the buccal vestibular mucosa next to the mesial root of the right first molar of each rat while it was under general anesthesia. All injections were volumetrically equivalent, and the determined volume limit of 10 μL was the limit that the tissue could accept without repulsion. Injections were done only once at day 0 and not repeated again. The remaining PRP samples from both the hPRP-E and mPRP-E groups were stored at −4°C for future biochemical analysis to determine the level of platelet-derived growth factor- _ββ (PDGF _ββ ) at both high and moderate PRP concentrations.

Tooth movement distances were measured by palatal superimposition of 3-dimensional (3D) digital models as previously described. Silicone (Express XT Quick; 3M ESPE, Seefeld, Germany) impressions of the rats’ maxillary dentitions at baseline and at every observational time point before they were killed were directly scanned with a 3D scanner (3 Shape R 700; CadBlu Dental, Chicago, Ill). The validity and precision of the scanner was 0.05 mm. Reconstructions of 3D models were performed by 3 Shape Scanniorthodontics 2008 (CadBlu Dental), and the pretreatment and postmesialization models were superimposed on the area of the palatal rugae. After superimposition, the amount of tooth movement was calculated by using Mimics software (version 12.11; Materialise, Leuven, Belgium). The midpoints of the mesial marginal ridge of the maxillary right and left first molars were marked on the pretreatment and posttreatment models. The distances between these points were measured. All measurements were repeated 3 times by the same investigator (A.G.) blinded to the treatment groups.

PRP samples stored at −4°C were brought to room temperature, and to ascertain the concentration of PDGF _ββ , the hPRP and mPRP samples were assayed in a rat PDGF _ββ immunoassay (Cusabio Biotech, Wuhan, China) as described by the manufacturer. The minimum detectable dose was 31.25 pg per milliliter. Measurements for each sample were made in duplicate. In brief, the microplate provided in the kit was precoated with an antibody specific to PDGF _ββ . Standards and samples were added to each well for 2 hours of incubation at 37°C. A biotin-conjugated antibody specific for PDGF _ββ added to the well and incubated again at 37°C for 1 hour. After they were washed 3 times, the wells received avidin conjugated to horseradish peroxidase solution and incubated for 1 hour at 37°C. After they were washed 5 times with the wash solution, the TMB (3, 3′, 5, 5′-Tetramethylbenzidine) substrate solution was added to each well, and the wells were incubated for 15 to 30 minutes at 37°C in the dark. The reaction was stopped by adding stop solution, and the intensity of the color was measured spectrophotometrically at 450 nm using a microplate reader (RT-2100C; Rayto Life and Analytical Sciences, Shenzhen, China). The data were analyzed via 4-parameter logistic curve fitting using CurveExpert Basic (version 1.40; CurveExpert and GraphExpert Software, Madison, Ala.).

Eight animals from each group were killed on days 3, 7, 14, and 21, and 6 animals from each group were killed on day 60. After that, the maxillae were removed, stripped of soft tissues, and prepared for histology. Tissue samples were fixed in 10% neutral formaldehyde in 0.1 mol of phosphate buffered saline solution (pH = 7.4). Samples were decalcified with Morse solution (10% sodium citrate and 22.5 % formic acid) for 4 to 5 weeks, dehydrated in an alcohol series, divided in 2 hemimaxillar halves, and embedded in paraffin. Paraffin blocks were sectioned longitudinally (10 μm thick) parallel to the long axis of each tooth, from mesial to distal aspects. As a second step, every tenth tissue sample section was selected through a set of consecutive sections. Choosing the first section was done in a systematic random manner. Sampled sections were stained with Masson’s trichrome techniques.

A semiautomated stereology workstation for image and stereologic analyses composed of a CCD digital camera (Microfire 1600 × 1200P; Optronics, Goleta, Calif), image capture card (ATI FireGL; Advanced Micro Devices, Camberley, United Kingdom), personal computer, and computer-controlled motorized specimen stage (BioPrecision Group, Howthorne, New York), a microcator (Heidenhain, Traunreut, Germany), and a light microscope (DM 4000B; Leica, Wetzlar, Germany) were used. As software, Stereo Investigator (version 7.0; MicroBrightField, Williston, Vt) was used. The measurements were made using a C Plan X10 objective (Leica) (NA = 0.22).

The Cavalieri principle was used to estimate the volume of the alveolar bone using the previously described stereologic workstation. A point counting grid (d = 100 μm) was applied for volume estimation to obtain maximum efficiency. The representative areas per point (a/p) were 1000 μm ² for each point. After applying the point counting grid to the sampled sections in a systematic random fashion, the numbers of points hitting alveolar bone in the sample were counted. Results were used for the estimation of alveolar bone volumes using the following formula:

where, t is the mean section thickness; a/p represents the area of each point on the point counting grid; and Σ p is the total number of points hitting the sectioned area. Then, the total volume of sampled sections from each rat hemimaxilla was multiplied by the section sampling fraction to estimate alveolar bone volume.

The efficiency of sampling and convenient point density for volume estimation were checked by estimation of coefficient of error and coefficient of variation as previously described ( Tables I and II ).

Table II

Intraradicular alveolar bone coefficients point numbers for volumetric measures in the mPRP group

mPRP	Day 3 mPRP-E/mPRP-C		Day 7 mPRP-E/mPRP-C		Day 14 mPRP-E/mPRP-C		Day 21 mPRP-E/mPRP-C		Day 60 mPRP-E/mPRP-C
	n = 6		n = 6		n = 6		n = 6		n = 6
Section thickness (μm)	10		10		10		10		10
Number of sampled sections	13	14	15	15	14	13	13	13	14	13
Mean intraradicular alveolar bone point number	2047	2117	1932	2004	1491	1668	1192	1169	2034	2061
CE intraradicular alveolar bone	0.007	0.005	0.005	0.007	0.008	0.007	0.005	0.008	0.008	0.006
Mean intraradicular total point number	4376	4603	4621	4713	5179	4374	4491	4343	4868	4744
CE intraradicular total region	0.008	0.005	0.007	0.008	0.007	0.005	0.005	0.008	0.007	0.006

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