Treatment of endodontically induced periapical lesions using hydroxyapatite, platelet-rich plasma, and a combination of both: An in vivo study.

AIM AND
OBJECTIVES: To evaluate bone regeneration in endodontically induced periapical lesions using Hydroxyapatite, Platelet-Rich Plasma (PRP), and a combination of Hydroxyapatite and Platelet-Rich Plasma for a period of one year.
MATERIALS AND METHODS: Twenty systemically healthy patients of both genders between the ages 20 and 40 years were included. To qualify, the patient had to have a tooth where non-surgical root canal therapy had failed, periapical radiolucency was present, and periapical root end surgery was required. The bony defect had to be confined to the apical area, with the bone covering the entire root surface coronally, with an intact lingual cortical plate. Patients were randomly divided into four groups, with five patients each, as follows: Group I - Replacement with Hydroxyapatite, Group II - Replacement with PRP, Group III - Replacement with PRP and Hydroxyapatite, and Group IV - Control group with no substitutes. The patients were evaluated both clinically and radiographically.
RESULTS: The radiographic evaluation revealed that Group I patients showed complete bone regeneration with evidence of a trabecular pattern, at the end of one year, Group II patients showed complete bone regeneration at the end of nine months, Group III patients showed complete bone regeneration at the end of six months, and Group IV patients showed bone regeneration, which was not satisfactory even after one year.
CONCLUSIONS: The PRP and Hydroxyapatite combination facilitated better and faster bone regeneration when compared to PRP alone.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

The success of endodontic therapy depends on complete periapical repair and regeneration. Most of the time teeth with periapical lesions heal satisfactorily after non-surgical endodontic intervention.[1] However, we do come across cases with persisting symptoms and infection that require periradicular surgery in order to remove the pathological tissues and to eliminate the source of irritation, and promote healing.[2] Abramovitz et al., have discussed the case selection for apical surgery and non-surgical re-treatment. They have reported that treatment of 24.5% of the cases was impossible without surgical therapy.[3]

Bone grafts and bone regeneration materials are being used with varying degrees of success. All these approaches are known as regenerative therapies.[45] The key to tissue regeneration is to simulate a cascade of healing events that, if coordinated, can result in the completion of integrated tissue formation. This is possible only with the use of growth factors, extracellular matrix, and the use of bone morphogenetic proteins instead of routinely used synthetic bone grafts,[6] because synthetic bone grafts induce regeneration by osteoconduction, whereas, these biological modulators induce regeneration by osteoinduction.

The need for these biological modulators resulted in the development of Platelet-Rich Plasma [PRP] by Whitmen et al., in 1997.[7]

Platelet-Rich Plasma is the component of blood with concentrated platelets in a limited volume of plasma.[8] This autologous plasma is a rich source of growth factors and its application is thought to be an effective way of inducing tissue repair and regeneration.[910] Marx et al., in 1998, reported the first clinical dental results with PRP and they suggested that adding PRP to bone grafts accelerated the rate and degree of bone formation. Platelet membranes had been shown to stimulate the mitogenic activity of human trabecular bone cells.[11] The proliferation rate of human osteoblast-like cells was increased, up to a certain level, by adding thrombocytes.[12]

Recent studies have indicated the stimulator influence of PRP in collagen biosynthesis and proliferation of osteoblastic cells.[1314] These in vitro results have supported the current assumption that the clinical use of PRP might enhance bone regeneration.[9-14] The aim of this in vivo study was to evaluate the bone regeneration in endodontically induced periapical lesions using Hydroxyapatite, Platelet-rich plasma, and a combination of both, for a period of one year.

MATERIALS AND METHODS

Clearance from ethical committee was obtained for performing this in vivo study. Twenty systemically healthy patients of both genders, between the ages 20 and 40 years, were included. The procedure was explained to the patients in their own language and informed consent was obtained from them. To qualify, the patient had to have a tooth where non-surgical root canal therapy had failed, periapical radiolucency was present, and periapical root-end surgery was required. The bony defect had to be confined to the apical area with the bone covering the entire root surface coronally and had an intact lingual cortical plate. The patients were randomly divided into four groups of five patients each: Group I — Replacement with Hydroxyapatite (Periobone G, Top notch health care product, India. Batch No: PB01040006), Group II — Replacement with PRP, Group III — Replacement with PRP and Hydroxyapatite, and Group IV — Control group with no substitutes.

Procedure for procuring PRP by manual isolation technique[15]

Ten milliliters of venous blood was drawn from the patient and transferred into two test tubes containing Ethylene diamine tetra acetic acid (EDTA) as an anticoagulant. The blood was centrifuged at 5000 rpm for 15 minutes. The blood and plasma formed two layers; the supernatent being plasma. The plasma was then aspirated with a micropipette and transferred to a sterile test tube without an anticoagulant. The samples of plasma were subjected to a second centrifugation at 2000 rpm for 10 minutes, which allowed the precipitation of the platelets (0.8 to 1.2 ml) to fall onto the bottom, while the surface Platelet Poor Plasma was discarded using a micropipette. The PRP was transferred to a dappen dish and stored at room temperature until further use. Just before placing the PRP into the bony cavity, a small amount of bovine thrombin (DADE BEHRING Test Thrombin Reagent, Germany) and a few drops of 10% Calcium Chloride [obtained by mixing 1 g of calcium chloride in 10 ml of distilled water] were added to the PRP to form a gel in a few seconds [Figure 1]. This preparation of the PRP gel was conducted by another operator during the surgical procedure.

Figure 1

Platelet-Rich plasma gel

The first step of the treatment plan was to complete the root canal therapy in cases where dry canals existed and in others obturation was done on the day of surgery. The surgical protocol included a routine medical history followed by blood investigations. The surgical procedure included reflection of a full thickness mucoperiosteal flap by sulcular incision and two relieving vertical incisions. Debridement of tissues at the defect site was followed by irrigation with sterile saline solution. Root planing was performed on the root surface under the apical portion of the root. No root resection was done. For Group I patients, the Hydroxyapatite crystals were mixed with the patients’ own blood and thoroughly compacted into the bony defect until it reached the level of the periphery of the bone margins. Care was taken not to spill the graft. For Group II patients, the PRP gel was carefully placed into the cavity till the entire cavity was filled [Figure 2]. For Group III patients, the bottom of the defect was filled with PRP. Then Hydroxyapatite crystals were mixed with the remaining PRP and were packed into the defect as a second layer. For Group IV patients, the defect was not replaced with any substitutes.

Figure 2

Bony cavity filled with Platelet-Rich plasma gel

Wound closure was obtained with a 3-0 black silk suture. Analgesics (Combiflam [Aristo] {ibuprofen 400 mg + paracetamol 325 mg} twice a day), Antibiotics (Amoxycillin 500 mg [Alembic pharma] twice a day) and 0.2% Chlorhexidine mouthwash (hexidine, ICPA Health products), was prescribed for five days post surgically. The sutures were removed after seven days. The patients were reviewed at regular intervals of one week, and one, three, six, nine, and twelve months. During the review, radiographs were taken using the paralleling cone technique (RWT Rontgenfilmhalter mit Langtubus, KKD, D-7090 Ellwangen). These follow-up visits included routine intraoral examinations and professional plaque control. All these cases were evaluated clinically and radiographically. Clinically, the patients were assessed for edema, postoperative pain, signs of infection, untoward reaction, and wound dehiscence.

RESULTS

Clinically, all cases showed moderate edematous swelling 24 hours after the surgery, which gradually subsided over a period of five days. The patients did not complain of any unusual or severe pain. There were no signs of infection, untoward reaction, wound dehiscence or extrusion of material in any of the patients.

Radiographically, Group I patients showed complete bone regeneration, with evidence of a trabecular pattern at the end of one year, Group II patients showed complete bone regeneration at the end of nine months, Group III patients showed complete bone regeneration at the end of six months and Group IV patients showed bone regeneration, which was not satisfactory even after one year [Figures 3–10].

Figure 3

Group I — Follow-up radiographs, hydroxyapatite

Figure 10

Group IV – Follow-up radiographs

Group I — Follow-up radiographs

Group II — Follow-up radiographs, Platelet-Rich Plasma

Group II — Follow-up radiographs

Group III — Follow-up radiographs, combination of Platelet-Rich Plasma & HA

Group III — Follow-up radiographs

Group IV — Follow-up radiographs, control

DISCUSSION

Today, while non-surgical endodontics is considered as the primary mode of treatment, the decision to intervene surgically is open to deliberations. The success of an endodontic procedure depends upon complete healing of the lost periapical tissues. Most periradicular lesions heal uneventfully after conventional endodontic treatment. However, some may require surgical intervention to remove the pathological tissue and simultaneously eliminate the source of irritation that could not be removed by the orthograde root canal treatment.[16] Bone regeneration after surgical intervention takes place in a very slow manner. Hence, to enhance these processes a number of bone substitutes are being tried out. The objective of using a bone graft is to achieve successful and complete healing of the bone. Bone grafting is the most common form of regeneration therapy. A variety of materials are available for bone regeneration, which are highly osteoconductive or osteoinductive like, freeze dried bone graft, bioactive glass, emdogain, PTR polymer, MTA, tricalcium phosphate, and octacalcium phosphate .[81718]

Blood and blood products have been used for over a century for various purposes, including the use of serum and individual components. Since the early twentieth century, various functions of platelets have been known, such as, hemostasis and platelet-plug formation. Research regarding the active use of growth factors present within the platelets started in the middle of twentieth century. These studies began to explore the fact that platelets could be used to modulate regeneration, and to repair and heal the tissues.[710]

In 1997, Whitman et al., introduced PRP.[7] In 1998, Marx et al., showed PRP as an autologous source of Platelet Derived Growth Factor (PDGF) and transforming growth factor-b (TGF) that was obtained by sequestering and concentrating platelets by gradient density centrifugation. This method showed a 338% increase in platelet concentration. PRP increased the density of bone when placed along with the bone grafts.[19] Platelet gel was prepared by blood collection preoperatively, and was prepared by another operator during surgery.

Platelet rich plasma contain various growth factors namely PDGF, transforming growth factor β, insulin-like growth factor, and epidermal growth factor. These factors play a prime role in bone regeneration[2021]

The various advantages of PRP are:

Improved support of tissue healing

More rapid mineralization of collagen in bone repair

Earlier stability of grafts because of the sticky consistency of fibrin component

Cytokines and growth factors brought to the site

Formation of a firm nonfriable clot

Clevatage of fibrinopeptides A and B from a molecule caused by thrombin, resulting in the formation of fibrin monomer,

Thrombin activated factor XIII that in turn allowed for a linkage in the presence of ionized calcium

No potential for disease transmission

Less thrombogenic than untreated graft material

Minimal adhesion formation resolving in four to six months

Enhanced osteoconduction: Osteoblasts were normally non-motile cells, in that, they would not move across a distance greater than 0.4 mm (400 microns), PRP enhanced the fibrin network, and the movement of osteoblasts along the fibrin network was enhanced resulting in a woven bone formation, much earlier than normal. The initial layer was a very thin (1 – 2 microns) plate. This phase would last for three months

Cellular activity: Bone morphogenetic Protein (BMP) acted as a ‘switch’ through which the reception as well as bone formation occurred and acted as a modulator

Stem cells in the graft started the cascade of osteogenesis[10]

Hydroxyapatite is used, as it helps in osteoconduction. Osteoconduction is a process wherein the bone is formed by the in-growth of capillaries and osteoprogenitor cells from the recipient bed into, around, and through a graft. These Hydroxyapatite crystals when used act as a scaffold upon which new bone is deposited, which is then followed by a slow resorption of the graft. PRP is used, as it helps in better healing and faster regeneration, because of the biological modulators and also helps in osteoinduction. PRP acts as an osteoinductive material because osteoinduction is a process whereby new bone is produced in an area where there was no bone earlier, where one tissue or its derivative causes another undifferentiated tissue to differentiate into a new bone. Calcium Chloride is added to nullify the effect of the anticoagulant EDTA, and Bovine thrombin is added to gellate the plasma. As only small grains of thrombin are added, it does not produce any antigenic response, as proved by Neil Winter Bottom et al., in the year 2002. A number of methods have been used for the procurement of PRP, such as:

Electromedic 500 gradient density cell separator (Medtronics) by Marx et al., in 1998

Smart PreP by Bhanot and Alex, in 2002

ELMD 500 transfusion system by Whitman and Green, in 2002.

Automated systems like PLACON 7 and Hemonetics MCS

The method used in this study to procure PRP and PRP Gel was based on the test-tube or Manual isolation techniques (vacutainer system) by Sonnenleit and Sullivan et al.[15]

Evaluations of all these cases were carried out both clinically and radiographically. Radiographic evaluation in Group I (Periobone G) showed increased radio-opacity immediately, due to the compaction of the bone graft into the defect. In the first month there was reduction in volume of the material due to initiation of resorption of the bone graft. In the third month there was diffuse radio-opacity due to increased calcification of the graft. In the sixth and ninth months, bone regeneration was seen, but not complete, due to partial resorption of the graft. In the first year there was complete bone regeneration due to osteoconduction as explained earlier. In Group II (PRP) the immediate, first week and first month postoperative radiographs showed a radiolucency similar to that of the preoperative radiograph, as PRP was a radiolucent material. In the third and sixth months bone regeneration was evident, but not complete, due to the presence of biological modulators as discussed earlier. In the ninth month bone regeneration was complete. In Group III (PRP and Periobone G) there was an increase in radio-opacity immediately, due to the compaction of the bone graft into the defect. In the first month there was reduction in volume of the material due to initiation of resorption of the bone graft. In the third month there was evidence of bone regeneration, but not complete, due to partial resorption of the graft. In the sixth month bone regeneration was complete because of the combined effect of Periobone G and PRP.[22] In Group IV (Control) there was incomplete bone regeneration, even at the one-year follow-up, due to nonavailability of bone substitutes to induce osteoconduction.

In this study the grafts did not produce any adverse reactions. Wound healing and primary closure of the surgical site were good. Radiographic evaluation revealed favorable results.

Today's understanding of the bone science recognizes the pivotal role of the growth factor in the clinical bone grafting success. This study elucidates the mechanism of action and the points of influence that the fundamental growth factors of PDGF and TGF-β exert on bone regeneration. The amplification of PDGF and TGF-β through the technique of platelet sequestration and concentration into PRP is seen as an available and practical tool for enhancing the rate of bone formation and the final quality of the bone formed.

CONCLUSIONS

Under the conditions of this in vivo study, it can be concluded that:

The use of bone substitutes helps in rapid and qualitative bone regeneration when compared to natural healing

Hydroxyapatite crystals facilitated bone regeneration, but not to the extent of the other two tested groups (PRP and Combination)

Plasma-Rich Platelets with biological modulators showed better results, but a combination of PRP and Hydroxyapatite showed the best results

In a clinical situation the use of bone substitutes will definitely enhance periapical regeneration