Efficacy of a root conditioning agent on fibrin network formation in periodontal regeneration: A SEM evaluation.

BACKGROUND: Even though numerous biomaterials have been devised and employed for periodontal regeneration, it should be well understood that the root surface receptiveness to clot formation and maintenance during initial periodontal wound healing, decides the nature of the connective tissue attachment. So this study was carried out with the prime objective of assessing the initial wound healing events occurring in vivo after the application of citric acid on to the root surfaces during periodontal regenerative therapy.
MATERIALS AND METHODS: Thirty-two human teeth were used for this in vitro study. Two dentin blocks each measuring 4 × 2 × 1 mm were made from each tooth. These dentin blocks were planed and treated differently with Phosphate Buffered Saline (PBS), citric acid, PBS and fresh human blood, citric acid and fresh human blood and were segregated into eight groups. Finally all the dentin blocks were processed and subjected to a scanning electron microscope study.
RESULTS: In PBS-treated samples, the dentin surface was irregular corresponding to smear layer and the dentinal tubule openings were obscured. Whereas, in those treated with citric acid revealed a smooth dentin surface devoid of smear layer and the dentinal tubular openings were clear. Further samples that were treated with PBS plus blood showed little or no fibrin network formation whereas with those citric acid plus blood showed a fine thick fibrin network formation adhered to dentinal surface.
CONCLUSION: The results of this present in vitro study suggests that use of citric acid as a root conditioning agent has a beneficial effect on initial wound healing events, which are critical for periodontal regenerative therapies.

INTRODUCTION

The aim of periodontal therapy is to restore the tooth supporting tissues affected by periodontal disease to their original architectural form and function. Advances in surgical techniques, and the availability of bone grafts, substitutes and biological modifiers has made periodontal regeneration, clinically feasible.

However, following conventional surgical therapy, the epithelium from the wound margin migrates apically along the root surface resulting in the formation of long junctional epithelium. This long junctional epithelium hinders the cells of the connective tissue, from adhering to the root surface. The pioneering experiments conducted by Melcher et al.[1], have suggested that for regeneration to occur, the periodontal ligament cells and not the gingival connective tissue, epithelial or bone cells should repopulate the root surface. Based on these observations, it is clear that the root surface must serve as a suitable site for cell attachment and fiber development during periodontal regeneration.

Under the impression that demeneralized root surfaces held an induction potential for osteogenesis and cementogenesis, Register et al.[2], carried out studies involving citric acid treatment of root surfaces and reported accelerated healing and new cementum formation on the acid treated root surfaces. Enhanced new attachment formation following citric acid demineralization was thought to occur by one more of the following mechanisms, namely, accelerated cementogenesis, removal of smear layer, widening of dentinal tubules, splicing of collagen fibrils and induction of mesenchymal cell differentiation.

However, the critical events in determining connective tissue attachment occur within a short period of time after the initiation of healing. Fibrin linkage to the root surface collagen, via fibronectin has been reported to be an important mechanism that establishes a cascade of events, ultimately resulting in connective tissue attachment. The formation of this pivotal fibrin linkage depends on the thrombogenecity of the root surface which is enhanced upon root conditioning. This in vitro Scanning Electron Microscope study aimed to assess the effects of citric acid application on smear layer removal, exposure of dentinal tubules and formation of a fibrin network on periodontally healthy and diseased root surfaces.

MATERIALS AND METHODS

Thirty-two human teeth were used for this in vitro study. Sixteen teeth extracted for orthodontic purposes were designated as control, and sixteen periodontally diseased teeth were obtained and designated the experimental group. Periodontal disease was confirmed by eliciting bleeding on probing, measurement of proximal attachment loss >5 mm and radiographical demonstration of bone loss >1/3rd of the root surface. All the selected teeth were caries free and were not subjected to scaling and root planing. Following extraction, they were rinsed immediately with phosphate buffered saline (Merck) - ph 7.4 and individually placed in small sterile containers filled with phosphate-buffered saline.

Preparation of dentin blocks

The teeth from both control and test groups were sectioned as given below. The apical third of the root and the crown portion below the cementoenamel junction was cut and discarded. The remaining mid rood portion of each tooth was sectioned longitudinally buccolingually with a diamond disc (Isomet) under copious irrigation with phosphate buffered saline. Later it was cut cross-sectionally so that blocks of rectangular shape were obtained measuring 4 × 2 × 1 mm. Two dentin blocks were made from each tooth. The above procedure did not induce any change in the root surface of the control and test groups. In total, 64 dentin blocks were obtained from all the 32 teeth and were stored in small sterile containers filled with phosphate-buffered saline at 4°C until further use.

Chemical treatment and blood application

The dentinal blocks were segregated into eight treatment groups [Table 1].

Table 1

Table listing the different treatment groups from A-G

Phosphate buffered saline treatment of the blocks was done by immersing the teeth for 5 minutes in phosphate-buffered saline. Citric acid treatment was done using the burnishing technique which involved the application of saturated citric acid solution (35.25 gm of citric acid in 50 ml of distilled water, ph-1, Merck) with the help of cotton pellets to the external surfaces of the dentin blocks. The cotton pellets were changed every 30 sec.

Finally all the blocks were subjected to three 5 minute washes in phosphate-buffered saline. The blood application was carried out on 32 dentin blocks. Fresh human whole peripheral blood obtained from a healthy human donor with informed consent was applied and allowed to clot on the blocks for 20 minutes. Following this the blocks were subjected to three, five minute washes in phosphate buffered saline. Finally all the dentin blocks were processed and subjected to a scanning electron microscope study.

Preparation for scanning electron microscope study

Immediately after final rinsing, the dentin blocks were fixed in 1% formaldehyde phosphate-buffered saline solution (Merck) for 15 minutes. Then all the blocks were rinsed and incubated for 10 minutes in 0.02M glycine (Merck) in phosphate-buffered saline. The samples were post fixed in 2.5% glutaraldehyde (Merck) in phosphate-buffered saline for 30 minutes and dehydrated through graded ethanol series - 25%, 50%, 75%, 95% and finally 3 exchanges of 100% alcohol. The blocks were mounted on stubs and sputter coated with gold and observations were made with a scanning electron microscope (Philips XL30 E scanning electron microscope) at ×800 and ×3200 magnifications.

RESULTS

In total, 64 dentin blocks were analyzed after appropriate treatment by a scanning electron microscope. The samples belonging to group A (Healthy teeth + phosphate-buffered saline treatment) showed an irregular amorphous layer of smear covering the dentinal surface and tubules [Figures 1 and 2]. In contrast the application of citric acid to the control teeth (Group B) rendered a clear surface free of smear layer with patent dentinal tubules, their openings clearly visible. [Figures 3 and 4]. Similar results were obtained for groups E [Figures 5 and 6] and F (E: phosphate-buffered saline-treated experimental teeth, F: citric acid-treated experimental teeth). The citric acid treatment of the diseased root surfaces also helped in removal of the smear layer and exposure of the orifices of dentinal tubules to the same extent as that of control teeth [Figures 7 and 8]. Control teeth that were treated with phosphate buffered saline and subjected to application of blood (Group C) demonstrated a dentinal surface that was covered by clumps of erythrocytes with a few isolated platelets [Figures 9–11]. In contrast control teeth that were treated with citric acid and subjected to application blood (Group D) demonstrated many erythrocytes with fewer clumps, isolated active platelets and a thick fibrin network [Figures 12–14]. Similar results were obtained for group G (experimental teeth-phosphate-buffered saline treated and subjected to blood application) [Figures 15–17] and group H (experimental teeth-citric acid treated and subjected to blood application) with group H demonstrating a thick fibrin network compared to group G which had predominantly erythrocytes with sparse fibrin [Figures 18–20].

Figure 1

Healthy root sample treated with PBS (×800). The picture shows and irregular amorphous surface (smear layer). The dentin surface and the dentinal tubules (←) were obscured by smear layer

Figure 2

Photomicrograph of similar sample as Figure 1 (×3200). Dentin surface was irregular and mostly covered by smear layer. Dentinal tubule (←) openings were not clearly seen

Figure 3

Healthy root sample treated with citric acid (×800). The dentin surface was clear and smooth with no smear layer. The openings of dentinal tubules (←) were clearly visible

Figure 4

Photomicrograph of similar sample as Figure 2 (×3200). Dentin surface appears smooth and was devoid of smear layer. The dentinal tubules (←) openings were clearly seen

Figure 5

Diseased root sample treated with PBS (×800). The surface was irregular and covered with smear layer. The dentinal tubule (←) openings were not clear

Figure 6

Photomicrograph of similar sample as Figure 5 (×3200). Dentin surface is irregular and the openings of dentinal tubules (←) are not clearly visible. Sparsely distributed microbes were seen

Figure 7

Diseased root sample treated with citric acid (×800). The surface shows no smear layer except for some debris. The dentinal tubule (←) openings were clearly visible

Figure 8

Photomicrograph of similar sample as Figure 6 (×3200). Dentinal tubules (←) openings were seen

Figure 9

Healthy root sample treated with PBS plus blood (×800). The dentin surface was covered by clumps of erythrocytes (O). Few isolated platelets () were visible. There was no fibrin network formation

Figure 11

Photomicrograph of similar sample as Figure 9 (×3200). Erythrocytes () were seen covering the surface. Few platelets () were observed. Little amount of fibrin network () seen

Figure 12

Healthy root samples treated with citric acid plus blood (×800). The surface was covered with many erythrocytes (). Activated platelets were seen. There was a clear fibrin network () formation observed at the base

Figure 14

Photomicrograph of similar sample as Figure 10 (×3200). Many erythrocytes () were seen covering the surface. Aggregating activated platelets () were observed. Clear thick fibrin network () seen

Figure 15

Diseased root sample treated with PBS (×800). Erythrocytes () were observed covering the dentin surface. Few platelets () were seen with little fibrin network () formation

Figure 17

Photomicrograph of similar sample as Figure 15 (×3200). Erythrocytes () were seen all over the surface with few platelets (). No fibrin network () was seen

Figure 18

Diseased root sample treated with citric acid plus blood (×800). Thick fibrin network () was seen all over the surface with cells (erythrocytes () entrapped)

Figure 20

Photomicrograph of similar sample as Figure 16 (×3200). A very fine thick network of fibrin () was seen with erythrocytes () entrapped

Photomicrograph of similar sample as Figure 10 (×3200). A thick fine fibrin network was clearly observed. Erythrocytes () were seen entrapped within this fibrin network()

Figure 10

Photomicrograph of similar sample as Figure 9 (×3200). Clear erythrocytes () were seen covering the surface. An isolated, activated platelet () was observed. No fibrin network seen

Photomicrograph of similar sample as Figure 16 (×3200). The surface shows a spread out fibrin network () formation on the dentin surface with few cells () entrapped

Figure 16

Photomicrograph of similar sample as Figure 15 (×3200). Erythrocytes () were seen all over the surface with few platelets (). Little fibrin network () was seen

DISCUSSION

The traditional mode of periodontal surgical therapy was focused on treating the soft wall of the periodontal pocket in order to achieve pocket eradication. Although these procedures may aid in reducing or eliminating the pocket, they do little to regenerate the lost Periodontium. Recent advances in periodontal regeneration, based on treating the diseased root surface viz planing, removal of plaque, calculus and root bound toxins have been devised and employed. But the ability to make the root surface disease free and conductive for the formation of a new connective tissue attachment has always been a challenging task.

In this regard, it is suggested that the initial clot formation and attachment of the fibrin network to the root surface is utmost critical for the final culmination in new attachment. Again, for this pivotal fibrin attachment, conditioning of the root becomes mandatory. In our study, it was observed that there was no difference in clot formation and fibrin network stabilization between the healthy control and diseased experimental teeth. The difference arose only between the citric acid treated group and phosphate-buffered saline treated group. While the citric acid treated teeth exhibited a more prominent fibrin network with activated platelets and sparse erythrocytes, the phosphate-buffered saline treated group showed only sparse fibrin with more erythrocyte clumps. The results demonstrate clearly that citric acid usage favors clot formation and retention on the root surface - an important step in successful regeneration as compared to the untreated root surface.

Our scanning electron microscope investigation was carried out with the prime objective of assessing the fibrin network formation on healthy and diseased roots. The root conditioning protocol used by us was a 5-minute application of saturated aqueous citric acid with a burnishing technique to demineralize the root surface and remove the smear layer. The surface demineralization by citric acid causes the dentinal collagen matrix, exposed to blood to get coated with a variety of plasma proteins.

It is suggested that the properties of the above mentioned plasma protein layer on the root determines the type of cell migration and response (Horbett et al.,[3]. The protein layer formation is followed by the adherence of platelets and deposition of fibrin forming a network into which cells can migrate and get trapped. It has also been found that on a root surface without well exposed collagen fibers, the fibrin network only opposes but cannot get attached (Polson and Proye[4], Hanes, Polson and Ladenheim[5]. On the other hand, a well-demineralized root surface with exposed collagen fibrils is considered highly thrombogenic. To this, the fibrin network appears to dynamically attach and remain (Proye and Polson).[6]

The attachment of fibrin strands to collagen fibrils of the root surface has been shown to be facilitated by the availability of fibronectin and factor XIII a in the wound environment. An oriented fibrin network attached to demineralized dentin may serve to mechanically inhibit the apical migration of epithelium and simultaneously enhance (Sobel and Gallin[7]), providing a structural scaffold (Polson and Proye[8]) resulting in true regeneration.

If formation of the fibrin network is considered so important, then questions may be raised on the anticoagulant effect of root conditioners. In this regard also citric acid scores over other agents. A Transmission Electron Microscope (TEM) study by Wikesjo et al.,[9] has shown that citric acid has no. adverse effects on fibrin polymerization and attachment to the root surface.

As mentioned earlier, our healthy and diseased samples treated with citric acid maintained a thick fibrin network while the phosphate-buffered saline treated samples retained a very thin fibrin layer. The possible reason for the finding could be that repeated rinsing might have dislodged the clot on the phosphate-buffered saline treated root surfaces while the citric acid group would have strongly adsorbed the fibrin mesh work.

The present study findings are also in agreement with the extensive studies done by Polson et al.,[4], Delazari et al.,[10], and Baker et al.,[11], who have shown the efficiency of citric acid conditioning in forming and retaining a stable blood clot.

In vivo studies by Nilveus and Egelberg[12], on the canine furcation model and critical size supra-alveolar periodontal defects have also displayed the efficiency of citric acid conditioning to support new attachment formation.

The observations from the present study emphasize the importance of understanding molecular events that occur during periodontal wound healing. If essential mechanisms such as clot formation and fibrin linkage are well established by successful root conditioning performed by the clinician, predictability and feasibility of periodontal regenerative procedures would definitely improve.