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Blood clot stabilization after different mechanical and chemical root treatments: a morphological evaluation using scanning electron microscopy

Purpose This in vitro study was conducted to evaluate the effects of different debridement techniques and conditioning procedures on root surface morphology and blood clot stabilization. Methods Two debridement techniques (curette [CU] vs. high-speed ultrasound [US]) and 2 conditioning procedures (ethylenediaminetetraacetic acid [EDTA] and phosphoric acid [PA]) were used for the study. Seven experimental groups were tested on root surfaces: 1) no treatment (C); 2) CU; 3) US; 4) CU+EDTA; 5) US+EDTA; 6) CU+PA; and 7) US+PA. Three specimens per group were observed under scanning electron microscopy (SEM) for surface characterization. Additional root slices received a blood drop, and clot formation was graded according to the blood element adhesion index by a single operator. Data were statistically analyzed, using a threshold of P<0.05 for statistical significance. Results The C group displayed the most irregular surface among the tested groups with the complete absence of blood traces. The highest frequency of blood component adhesion was shown in the CU+EDTA group (P<0.05), while no differences were detected between the CU, US+EDTA, and CU+PA groups (P<0.05), which performed better than the US and US+PA groups (P<0.05). Conclusions In this SEM analysis, EDTA and conventional manual scaling were the most efficient procedures for enhancing smear layer removal, collagen fiber exposure, and clot stabilization on the root surface. This technique is imperative in periodontal healing and regenerative procedures.


INTRODUCTION
Obtaining clean, smooth, and decontaminated root surfaces during periodontal surgery is imperative for the organization of new connective tissue re-attachment and enhancement of the tissue healing process [1]. Even though ultrasonic or manual scaling and root planing are the most widely used techniques in clinical settings for the mechanical cleaning of the root surface during periodontal therapy [2,3], they cannot fully decontaminate the dental hard tissue [4]. Moreover, due to mechanical debridement, a compact smear layer is formed in intimate contact with the treated surface, constituted by a mixture of organic and inorganic materials, microbial toxins, and debris [4,5]. From a histological point of view, the presence of a smear layer interposed between the root surface and the adjacent connective tissue could limit the connective tissue re-attachment process [1,4,5].
Chemical conditioning was, therefore, introduced to support mechanical treatment and detoxify the root surface while dissolving the smear layer [1,6]. The exposure of root surface collagen fibrils using demineralizing solutions activates the physiological cascade of processes involved in periodontal healing, restores the biocompatibility of the root [7], and favors clot stabilization in the earlier stages of periodontal regeneration [8]. The exposure of the dentin matrix allows the interaction between the collagen fibrils of the root surface and the fibrin clot that comes from the periodontal wound, resulting in better regenerative outcomes [4,8].
Several chemical conditioning agents have been proposed to improve biological responses during surgical periodontal therapy. Among these solutions, ethylenediaminetetraacetic acid (EDTA), citric acid, phosphoric acid (PA), and tetracycline hydrochloride are the most widely used demineralizing solutions [9]. However, the lack of standardization of the protocols for the use of these chemical agents in the literature precludes the possibility of precisely indicating the best treatment to promote the healing process in periodontal tissues after surgery.
In particular, EDTA has been commonly used in periodontal practice in different concentrations (12% to 24%) thanks to its neutral pH (up to 7) and its chelating properties towards the mineral structure of the tooth, without a risk of damaging the dentin matrix [9,10]. PA, in contrast, has been extensively used in adhesive dentistry and much less in periodontics, even though it is capable of effectively removing the smear layer and opening up the dentinal tubules, possibly creating a suitable substrate for fibrin clot linkage [11,12].
Thus, the present study aimed to evaluate in vitro different debridement techniques (ultrasonic device and manual scaling) and chemical conditioning procedures (EDTA and PA) on root surface morphology and blood stabilization through scanning electron microscopy (SEM) evaluation. The null hypotheses tested were that the type of debridement technique and the conditioning solution would not influence root surface morphology and the adhesion of blood elements to the root surface.

Specimens' collection
Patients with chronic periodontitis and no history of systemic complications who visited the Department of Periodontology of the University of Bologna (Bologna, Italy) were recruited for this study, in full accordance with ethical principles, including the Declaration of Helsinki. The selected patients were carefully informed about the scope of the study, after which they signed a written informed consent form.
The eligible teeth were required to fulfill the inclusion criteria, such as no history of scaling and root planing in the previous 6 months, an absence of caries or restorations in the proximity of the cementoenamel junction (CEJ), proximal attachment loss of 6 mm or more with bleeding on gentle probing, and grade III mobility that predicted an unfavorable prognosis of the tooth, necessitating extraction therapy.
https://doi.org/10.5051/jpis.2102800140 Blood clot stabilization after different root treatments https://jpis.org Three upper premolars were considered suitable for the study, and, after extraction, they were rinsed with saline solution to remove blood, while calculus and other visible debris were hand-removed with curettes (CU). Teeth were then stored in distilled water at 4°C to avoid dehydration until their use, for no longer than 7 days. The protocol was approved by the Ethical Committee of the University of Bologna (Italy; protocol N°: 71/2019/OSS/AUSLBO).

Specimen preparation
Two parallel grooves were made on each tooth using a cylindrical bur mounted on a highspeed handpiece under water irrigation: the first one at the CEJ level and the second one up to 6.5 mm from the first mark, in the apical direction.
The root blocks were longitudinally divided into 2 halves through the root canal, and the pulp tissue was removed with a tweezer, taking care not to touch the walls of the pulp chamber. Each half was randomly assigned to 1 of the following surface mechanical treatments in order to eliminate the contaminated cementum and create a smooth and hard surface: #5-6 Gracey curettes (CU; Hu-Friedy, Chicago, IL, USA) for 1 minute (about 50 apico-cervical traction movements performed) or a high-power ultrasonic device (US; Castellini, Imola, Italy). A single operator performed the debridement procedures. Then, each half specimen was cut lengthwise into several slices (n=6 per half portion, a total of 18 per debridement procedure) (Figure 1). Additionally, 2 slices per tooth (n=6) were obtained from the mesiodistal sides of each root (without mechanical treatment) and used as a control group (Figure 1b).
Each radicular slice, from either the US or CU group, was randomly allocated into 1 of the following surface conditioning procedure groups: 1) irrigation with saline solution (control group); 2) application of 24% EDTA gel (PrefGel, Straumann, Basilea, Switzerland) for 2 https://doi.org/10.5051/jpis.2102800140 The solutions were brushed onto the entire surface of the radicular slices with a clean cotton pellet and the procedures were repeated thrice. After surface conditioning procedures, the specimens were abundantly rinsed with saline solution (0.9% NaCl) 3 times for 5 minutes in a multi-well dish, with a gentle swirling motion using a rotating tabletop shaker at low speed.
One slice per group was set aside for morphological evaluation. Further, to evaluate the influence of surface conditioning on blood adhesion, 1 drop of fresh human blood taken from a healthy volunteer donor was placed on the top of the remaining pre-treated slices. The blood was allowed to clot over the specimens for 20 minutes in a humidified chamber at 37°C. The specimens were finally rinsed in phosphate-buffered saline (3 times×5 minutes each) in multiwell dishes with gentle rotating movements [4]. After rinsing, the specimens were fixed with 2.5% glutaraldehyde in 0.15 M cacodylate buffer solution (pH 7.2) for 4 hours, rinsed in 0.15 M cacodylate buffer, and dehydrated in ascending ethanol series solutions (50%, 70%, 90%, 95%, 100%; 5 minutes each concentration) [13]. The dehydration process was concluded in a carbon dioxide (CO 2 ) critical point drier (CPD 030, BalTec, Pfäffikon, Switzerland).

SEM evaluation
Specimens were mounted on aluminum stubs with colloidal graphite, gold-sputtered, and examined using SEM (Nova Nanosem, 450, FEI Europe, Eindhoven, Netherlands) at a voltage of 15 kV. Images of each sample were obtained at ×1,000, ×4,000, and ×10,000 magnification from the most representative areas. Micrographs of the samples that received blood drops were scored using the blood elements adhesion index (BEAI) score [14] as follows: • Score 0: absence of fibrin network and blood cells; • Score 1: scarcely distributed fibrin network and/or blood cells; • Score 2: moderate number of blood cells and thin fibrin network with poor interlacing; • Score 3: dense fibrin network with rich interlacing and the presence of blood cells.
A single, independent investigator carried out the imaging and scoring of the SEM microphotographs. Data were collected and statistically analyzed. Since data were not normally distributed (

RESULTS
The morphology of the root dentin surfaces after the conditioning treatments, observed on SEM, seemed to be relatively consistent and was characterized by smear layer-covered surfaces with no underlying tubule exposure (Figure 2). Specifically, the surface textures of the C group had a rough appearance, with sparsely distributed traces of plaque and microorganisms (Figure 2A). Similar morphological structures were found in the CU, US, CU+EDTA, and US+EDTA groups, with the presence of sparsely distributed honeycomb filamentous formations (Figure 2B-E). The specimens decontaminated with PA presented a foamy surface (Figure 2F and G). https://doi.org/10.5051/jpis.2102800140 Blood clot stabilization after different root treatments https://jpis.org Representative SEM images used for BEAI grading and the score distribution among the tested groups are presented in Figures 3 and 4, respectively. SEM images of representative radicular slices that received blood application were evaluated by the BEAI and are shown in Figure 5.
Differences in blood cell attachment were observed among the tested groups on SEM microphotographs. The C group showed a surface completely devoid of fibrin network linkage and blood cell adhesion ( Figure 5A). These differences translated to BEAI scores as follows. Among the experimental groups, the CU+EDTA group revealed the highest distribution of a fibrin network, with a statistically significant difference from other groups (100% of score 3, P<0.05). According to pairwise comparisons, the following results were obtained: CU+EDTA (P<0.05) > CU = US+EDTA = CU+PA (P<0.05) > US = US+PA (P<0.05) > C (P<0.05). The C group showed a total absence of blood elements on the root surface and it was the only one to present a score of 0 (32%) among the tested groups (Figure 4).

DISCUSSION
The results of the present study demonstrated that different debridement techniques and conditioning of the root surface influenced its morphology and improved the adhesion of blood elements to the root surface, especially conventional manual scaling combined with EDTA. Hence, the null hypothesis could be rejected.
Root preparation during periodontal surgical therapy aims to decontaminate the infected root surface to make it smoother, harder, more biocompatible, and suitable for the healing process [1,2]. Several studies have shown that the smear layer formed after root surface debridement may prevent the migration and the attachment of the blood elements from the periodontal wound [8,14]. The adhesion and stability of the blood clot between the gingival flap and the root surface are crucial for the healing process, especially in regenerative and mucogingival procedures for the formation of new connective tissue attachment. Thus, chemical demineralizing agents have been introduced to remove the smear layer and promote the exposure of dentin collagen fibrils, which represents the substrate for the linkage with the fibrin clot [10,15]. However, despite the promising results of several in vitro studies, other histological and clinical studies have reported inconsistent results regarding those agents' positive or negative effects [15][16][17][18]. The lack of evidence about real clinical effectiveness also stems from differences in the techniques and experimental procedures [15].
To the best of our knowledge, this is the first study to evaluate the effects of the combination of 2 different techniques of scaling and root planing and 2 different modalities of chemical conditioning. Our findings show that contaminated root surfaces do not represent a proper substrate for the healing process since they are covered by residual tissue, plaque, and calculus that do not promote the adhesion of the blood elements, corroborating previous results [4,[19][20][21]. The samples from the control group showed the worst results according to the BEAI (Figure 4), with only a few blood cell traces without any organization, mixed with fibrous structures and debris (Figures 2A and 5A). Therefore, root debridement is strongly recommended as a crucial procedure for proper periodontal healing.
Root surface instrumentation for 1 minute has shown promising results even without conditioning, although, in accordance with previous studies, scaling and root planing could not fully decontaminate the root surface, further producing a variable amount of debris and smear layer [2,4,8,15,21]. The 2 tested scaling and root planing techniques (manual vs. US) did not lead to relevant morphological differences of the treated surfaces [22,23]. However, in the present study, it was noteworthy that CU attained statistically significant higher BEAI index than US only. Nevertheless, it should be pointed out that the results of 1-minute-long instrumentation of the root can be strongly affected by the operator's manual skill, especially for CU use [3].
Furthermore, in the present study, chemical conditioning increased the biocompatibility of the treated surfaces, leading to better adhesion and organization of the blood clot. The blood clot could adhere to the root surface even without any chemical treatment, but in the samples treated with EDTA or PA, greater interaction between the collagen structure of the dentin and the fibrin clot weave was recorded. The reason could be related to the etching procedures that effectively removed the smear layer and exposed the underlying dentin substrate, promoting links between the fibrils and the fibrin. Thus, we can speculate that the healing process may be more predictable when the mechanical debridement is associated with etching of the root surface, which seems to promote better adhesion of blood cells.
EDTA is one of the most frequently used solutions for this purpose in periodontics at different concentrations and with various application times. It has been previously reported to remove the smear layer while preserving the dentin collagen matrix and avoiding excessive acidification of the surrounding tissues [4,8,9,19,21,22,24]. Blomlöf et al. [25] showed that a 24% EDTA solution was significantly more effective for the removal of the smear layer than lower concentrations. Moreover, gel formulations, such as the one used in the present study, allow better control of etching properties [26]. Nevertheless, the real efficacy https://doi.org/10.5051/jpis.2102800140 Blood clot stabilization after different root treatments https://jpis.org of EDTA in terms of promoting blood cells' adhesion to the root remains controversial [4].
If not properly removed from the root surface, EDTA could have a negative effect on blood clot adhesion. Indeed, EDTA is a calcium chelator, and its residues could slow or inhibit coagulation processes [19,26]. However, the results of the present study demonstrated that these potential drawbacks can be prevented if EDTA is thoroughly rinsed after application. Moreover, the most effective application time remains unclear. Although Gamal et al. reported a positive correlation between the application of EDTA for 4 minutes and the adhesion of the periodontal ligament cells to the root [16], it is reasonable to prefer an application time that is clinically effective, but simultaneously compatible with the surgical timing. The 2-minute application time used in the present study seemed to effectively remove the smear layer and promote blood clot adhesion, while remaining within a clinically acceptable time frame.
PA is widely used in adhesive dentistry to remove the smear layer and demineralize peritubular and intertubular dentin during etch-and-rinse or selective enamel etching bonding procedures [11,12]. Some investigators have proposed also using PA in the periodontal setting, thanks to its ability to remove the radicular smear layer and expose the dentinal tubules, allowing interactions between the collagen fibrils and the fibrin clot in the periodontal wound [11,24]. Both EDTA and PA led to good blood clot stability. Nevertheless, some samples etched with PA showed a greater exposure of dentin collagen fibrils, probably due to its lower pH and, as a consequence, greater etching properties. Our choice to use it for 15 seconds, a common application time in adhesive dentistry, comes from evidence that prolonged contact with the root surface can lead to dentin matrix degradation, as reported after the application of acid substances on the dentin substrate [7,15]. The high etching potential of PA could have interesting clinical implications since it seems to guarantee the exposure of the dentin collagen components in only 15 seconds, thereby saving time during the surgical procedure. However, the effect on the surrounding periodontal tissues remains unclear, and if the gingival flap does not completely cover some areas at the end of surgery, this could lead to dentinal hypersensitivity [24].
In conclusion, the results of this preliminary study confirm the crucial role of scaling and root planing in creating reliable adhesion of blood cells to the root as the first step of the healing process. The additional use of chemical decontaminants, such as EDTA or PA , enhances the interaction between dentinal collagen fibrils and the fibrin clot, suggesting a potential clinical benefit in periodontal reconstructive surgical procedures. Additional in vitro investigations with an increased number of specimens, as well as clinical studies, are needed to validate the procedure further.