Effect of Platelet-Rich Plasma on Peri-Implant Bone Repair: A Histologic Study in Dogs

During the last decades emphasis was given to the study of growth factors1–6 and platelet-rich plasma (PRP) associated with bone grafts aiming to obtain better bone quality7–15 and clinically favor bone healing and chronology.

Platelets elicit the initial cellular responses from neutrophils, macrophages, fibroblasts, and endothelial cells. Carlson16 points out the importance of the platelet-derived growth factor (PDGF) in tissue healing and as a growth factor reserve. The PDGF presents several positive effects in tissue healing, stimulating angiogenesis, and helping to initiate the actions of other growth factors and cells.8 

Clinical studies described enhanced bone healing, both in quantity and quality of newly formed bone, with the use of PRP.2,8,9,17–19 Marx7,20 suggests that PRP does not present osteoinductive capacity as do the bone morphogenetic proteins, but it accelerates the chronology of bone healing. Furthermore, PRP should be prepared at the moment of use because 10 minutes after the gel is activated 70% of the growth factors will have been released, and the amount grows to 100% after 1 hour. Action of the growth factors at the surgical site could extend up to 10 days.

Bone undergoes continuous remodeling, which allows it to adapt to functional demands. Several endogenous substances take part in that process, such as parathormone, vitamin A, and calcitonin. The hormone from the parathyroid glands acts on the metabolism of calcium and phosphate and helps regulate growth factors that are active on osteoblasts and osteoclasts.21 

Recent studies began to emphasize the effects of humoral factors that can influence tissue healing, instead of being concerned only with the morphologic aspects of that process. Those studies point out the effects of platelet-derived factors. Platelets or thrombocytes are discoid anuclear cytoplasmic fragments of megakaryocytes, measuring 2 to 5 µm in diameter with a complex cytoplasm, an irregular surface, and a life span from 7 to 10 days.22 Platelets play a direct role in hemostasis and participate in the inflammatory process and tissue healing.

The study by Gray and Elves23 brought about advancements in the application of PDGFs by showing their effects on the angiogenesis necessary for healing of bone grafts. Nowadays it has been shown that growth factors play a major role in cellular proliferation and differentiation, angiogenesis, extracellular matrix production, and chemotaxis.24 Research on growth factors3,25 and PRP brought new evidence about tissue growth, bone maturation and chronology of bone healing.8,26,27 

After the initial euphoria about the use of PRP to enhance bone formation, whether associated with bone grafts or not, subsequent controversial results28–35 suggest the need for more experimental evidence on the matter. Therefore, this study evaluates the use of PRP in osseointegrated implant sites.

The experimental protocol was approved by the local ethical committee (No. 13/2003). Nine mongrel dogs (2 to 4 years old) with a mean weight of 22 kg were used in the present study. The animals had been previously evaluated by a veterinarian and had received vaccination; vermifugal treatment; and laboratory testing, including routine hematology, creatinine, and alanine aminotransferase at the School of Veterinary Medicine, Jaboticabal–São Paulo State University, Brazil. All were kept in individual cages and fed with proper commercial ration and water ad libitum during the whole experiment except for 12 hours before surgery.

The day before the surgery all animals received oral spiramycin (75 000 IU/kg) and metronidazole (12.5 mg/kg), which were continued for 5 days after surgery. Diet and water intake were suspended for 12 hours before the anesthetic procedure. The animals were then weighed and submitted to general anesthesia for tooth extractions. For that task each animal received intravenous levomepromazine chlorhydrate (1 mg/kg) and a subcutaneous injection of kaprofen (1 mg/kg). Next, intravenous propofol (5.0 to 8.0 mg/kg) was used to induce anesthesia and allow orotracheal intubation. Halothane diluted in an oxygen flux of 30 mL/kg/min was then used to maintain inhalation anesthesia in a partially closed system. Oxygen saturation, carbon dioxide, electrocardiogram, respiratory frequency, noninvasive blood pressure, and temperature were monitored.

Under general anesthesia, supragingival prophylaxis was accomplished for all teeth with an ultrasonic set. Using 0.12% chlorhexidine gluconate followed by saline irrigation for antisepsis, the second and third premolars of each animal were removed bilaterally.

After 60 days under general anesthesia, 2 threaded titanium dental implants (sandblasted acid-etched surface 7.0 mm × 3.75 mm, Titanium Fix, A S Technology, São José dos Campos, Brazil) were inserted in both sides of the mandible. The implantation followed the protocol described by Branemark et al.36 

On the left side PRP was introduced in the surgically prepared implant sites with a Luer syringe in an approximate volume of 1.5 mL. The threaded portion of each implant was immersed into the PRP before implantation. Those 18 implants constituted the experimental group. On the right mandible sites implantation was done without the use of PRP, and these 18 implants served as the control group.

Intravenous blood was collected from each animal before the implantation procedures to obtain an automated platelet count. A minimum platelet count of 1 000 000/µL was required. After general anesthesia, 5.0 mL of intravenous blood were collected in a disposable sterile syringe. The blood was placed in vacuum glass tubes containing 0.5 mL of 10% trisodium citrate solution. The protocol described by Anitua9 was followed, and thus 1 cycle of 1200 rpm centrifugation was performed for 10 minutes at 22°C for platelet separation.

A triphasic solution was obtained in which the superior portion contains few platelets, red cells, and plasma; the intermediate layer contains the PRP; and the lower portion is poor in red cells and growth factors. Most of the red cells lie in the bottom of the tube. The inferior portion of the intermediate layer, 1 to 2 mm above the red portion, where the largest number of platelets are, was transferred with a graduates pipette to an Eppendorf tube to which 50 µL of 10% calcium chloride were added. After 15 to 20 minutes the gel was obtained. To obtain the PRP, centrifugation and surgical procedure were initiated at the same time, so that PRP was inserted into the surgical sockets as soon as it was activated, followed by implant insertion.

Under general anesthesia biopsies were taken after 15, 30, and 55 days, corresponding to 3 dogs and 6 implants for each period for each group for histologic analysis. Implants and adjacent bone were removed en bloc respecting 2.0 mm from the implant.

The specimens were washed and decalcified for 16 weeks in Morse solution. After this the implants were removed from the decalcified blocks. Specimens were then washed for 24 hours in running water and were placed in 20% sodium sulphate for 1 week. They were again washed for 24 hours before automated processing for 48 hours as follows: baths in 70%, 90%, and absolute alcohol and in alcohol and xylol (1∶1), xylol (2 times), and paraffin. Serial 5-µm thick sections were obtained with a microprocessor cut machine (Leica Instruments, Nussloch, Germany) and stained with hematoxylin and eosin. An optical microscope and camera (Olympus BX 51 and Olympus Camedia C 5060 5.1 megapixel; Camedia Master 4.1 version, Tokyo, Japan) were used to acquire images as follows: panoramic (×40), threads (×100), and single thread (×200). The slides were coded so that the examiner who performed the histometric analysis was blinded to treatment group. The histologic and histometric analyses of the groups were done blindly by one pathologist. The central section in each specimen was selected for quantitative assessment of different tissue components: vital bone and marrow. These measurements were expressed as a percentage of the total surface area of the bone core section. The bone to implant contact percentage (BIC%) was obtained according to a mask of the shape of the implant (Figure 1).

A Mann-Whitney U test was used to evaluate differences between groups and a Wilcoxon paired test was used to evaluate the bone to implant contact among the experimental periods. The significance test was 2-tailed and conducted at a 5% level of significance.

Most sections from the control group showed areas of the peri-implant space occupied by dense connective tissue and other areas occupied by delicate bone trabeculae formed by immature bone with presence of numerous osteoblasts (Figure 2). The treated group showed similar peri-implant characteristics (Figure 3).

Some specimens from the control group presented varying histologic characteristics in different areas. Other specimens showed delicate united bone trabeculae in greater amounts than in the previous period. The trabeculae presented reversal lines and evident lamellar disposition with flattened osteocytes (Figure 4). It was possible to observe thread spaces filled sometimes by bone and sometimes by dense connective tissue. Newly formed bone arose from the implant site walls rather than from the region adjacent to the implants. The sections from the treated group showed similar characteristics compared with the control group (Figure 5).

In the control group, bone healing was not altered. Trabeculae were immature and presented numerous rounded and flattened osteoblasts. The bone trabeculae determined areas of medullary tissue (Figure 6). Dense connective tissue was present in proximity to the implant threads. Histologic aspects were variable for the group treated with PRP. The areas adjacent to the implant threads presented a dense connective tissue interface. Incipient mineralization centers were seen in proximity with the dense connective capsule (Figure 7). In some areas thin bone trabeculae were found alternating with areas of bone resorption suggesting enhanced bone metabolism.

The histometric evaluation is shown in the Table. The BIC% increased significantly at 55 days for both groups compared with the BIC% at 15 days (P < .05); however, there was no difference between the groups over the experimental periods (P > .05). The BIC% values for the control group ranged between 33.56 and 67.8.17% while the means values for the PRP group ranged between 33.45% and 68.56% over all periods.

This study failed to show an increase in bone to implant contact around implants placed in PRP in canine mandibles compared with the control group. The data also do not show any morphologic differences between the experimental and control groups. The amount of bone formed at different periods may be related to the maturation period of bone tissue.

In the present study, after 15 days histologic aspects were similar for control and PRP-treated groups. Both showed osteoblastic activity in the peri-implantar spaces that was characterized by bone formation alternating with areas occupied by dense connective tissue in the same interface. Contact ossification at the implant−bone interface was not seen in any of the specimens. Bone formation started in proximity with the bone wall and not from areas adjacent to the implants. This is in accordance with the fact that PRP lacks osteoinductive capacity.7 There was not early acceleration of the healing process as described by others.3 

After 30 days, both groups presented similar histologic responses alternating areas of bone formation with dense connective capsule within the spaces between the implant threads. In the longest observation period of 55 days, dense connective capsule in the treated group was denser and thicker than observed in the control specimens, although a significant difference between the evaluated groups was not found (P > .05). Dense connective tissue proliferation seemed to have been stimulated by the PRP. Immature trabeculae and bone resorption were seen adjacent to the dense connective capsule.

The speeding up of the healing process described previously8,9 was not found for any of the observation periods. In fact, several authors dispute the beneficial effects of PRP.37–40 It is important to point out that the concentration necessary to bring about those effects may be species specific with different ideal platelet counts and resulting concentration of biological factors. This is possibly an important variable in obtaining different results, as is the method of obtaining the PRP. According to Weibrich et al,41 both hypo- and hyperconcentration of platelets worsen bone-healing results.

Plachokova et al42 presented a systematic review of the effects of PRP on bone regeneration. The authors showed that PRP has some beneficial effects on periodontal regeneration; however, the use of PRP on maxillary sinus augmentation obtained weak results. These results could be attributed to different protocols to produce the PRP.

The platelet concentration obtained from different PRP protocols could also explain the different results observed in the aforementioned studies.41,42 Platelet concentration lower than 1 000 000/µL produced worse results43 than higher proportions of platelets (>1 000 000/µL).

It is possible that PRP was partially removed from some areas during implant insertion, while increasing the pressure on the bone walls in others, promoting resorption in the latter case. The net effect can justify the greater dense connective tissue observed in the peri-implant spaces for the treated group compared with the control specimens. Only 1 dog had an increased inflammatory response in the treated and control sites, but there was no implant mobility when the biopsies were taken. This was considered to be an individual response to surgery or postoperative local trauma due to discomfort.

The PRP did not promote better quality or quantity of newly formed bone as found in previous studies.28–35 In fact, the treatment resulted in results similar to those in controls, that is, when prepared implant sites were not treated with PRP before implant insertion. It is important to point out that the periods evaluated in our study (15, 30, and 55 days) are enough to evaluate the effect of growth factors released during 10 days in the surgical site.8 In addition, our results were obtained from decalcified sections, where the implants were removed, showing no percentage of bone to implant contact. This information could have limited our results.

Within the limits of the present study:

1. Treatment with platelet-rich plasma did not favor bone formation.

2. Results did not suggest benefits of treating the prepared implant site or implant surface with PRP before implant insertion.

BIC%

bone to implant contact percentage

HE

hematoxylin and eosin

OM

original magnification

PDGF

platelet-derived growth factor

PRP

platelet-rich plasma