DOI: 10.31038/JDMR.2020334

Abstract

The purpose of this study was to evaluate the local biological effects and bone regeneration efficacy of a polyphenols-enriched ceramic bone filler. To this end, a test article (NBR_Purple) a biphasic phosphate ceramic plus polyphenols from grape pomace and the same material without polypohenols (NBR_White), were implanted in the medial condyle of the femur bone of rabbits for 56 days. A control article of clinical use (Ostim ®), was implanted as the first control condition. There was a second control by performing the same defect at the same location but without any implanted material (void condition). Histological examination at the end of the test period shows statistically significant improvement of bone regeneration by the polyphenol-enriched material over the same material without polyphenols, supporting literature data on the involvement of polyphenol molecules in bone regeneration pathways.

Keywords

Bone filler, Polyphenols, In vivo test, Osteointegration, Histology

Introduction

Replacement of bone loss or reconstruction of bone defect is still a clinical challenge. Different type of bone substitutes are used in clinical practice for treating bone defect caused by trauma, osteoporosis or dental pathology such as periodontitis [1,2]. The mode of action of the biomaterials available on the market mainly relies on mechanical support and is thus limited to providing a functional scaffold for cell adhesion. Synthetic bone fillers, mostly based on calcium phosphate materials, have been widely used due to their good reproducibility, biocompatibility, non-immunogenicity, and also because they offer the opportunity of advanced material engineering [3,4]. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are widely used in bone tissue engineering as bone filler particles [5,6].

Polyphenols are chemical compounds synthetized by plants, widely diffused in the vegetable kingdom. They exert a defense action. Grape is among fruits which contain high quantity of polyphenols, in particular red grape [7,8]. The involvement of different classes of polyphenols in molecular pathways that can involve the regeneration of bone tissue is widely studied [9-13]. A role for polyphenols in the local control of inflammatory diseases leading to bone loss, such as periodontitis or osteoporosis, has been discussed in the literature [14-23]. Coupling of polyphenols to ceramic bone fillers could spawn a new generation of materials for bone regeneration, endowed with mechanical support and biological stimulation properties. In this work we describe the in vivo outcomes of a calcium phosphate bone filler containing polyphenols from red grape pomace, implanted for 56 days in the medial condyle of the femur bone of rabbits, showing that the presence of polyphenols enhances bone regeneration properties of the biomaterial.

Materials

All chemicals were analytical-reagent grade and were purchased from Sigma-Aldrich. Red grape pomace was purchased from a local winery producer (Croatina grape from ALEMAT, Penango, AT, Italy). Ultra-pure (MilliQ) water was used for the preparation of aqueous solutions. Commercially available bone filler Ostim ® has been purchased on the market.

NBR_White

HA was used for enhancing the mechanical strength of scaffold, whereas β-TCP for its degradability; they were mixed in a percentage of 50 wt.%, respectively, to reach an optimum compromise between the two properties. The ceramic scaffolds were prepared by mixing HA and β-TCP powders (47 wt.%) with a binding agent (poly (vinyl alcohol), 3 wt.%), and ultrapure water (50 wt.%) to obtain a ceramic slurry. Dolapix CE 64 was added as a dispersing agent (1 wt.% of the solid load). The polyurethane (PU) sponge impregnation method was used to obtain a macroporous ceramic scaffold [24,25]. A commercial PU sponge slab (45 ppi) of 200 x 200 x 10 mm³ was soaked into the ceramic slurry for 90 s, followed by compression along the transverse plane (40 kPa) and left at room temperature for 5 min before repeating the cycle. Impregnation/compression cycles were repeated for 3 times. The ceramic-coated PU sponge was left to dry overnight at 37°C and sintered in a furnace at 1100°C for 12 h in air (heating rate 5°C/min), in order to obtain a porous HA/βTCP slab of 200 x 200 x 10 mm³ (a volumetric retention of 24 % was calculated). The obtained porous slab was grinded by a jaw crusher (BB 50, Retsch) and sieved in order to obtain a 300 μm – 1000 μm porous grained granulate (ceramic granulate, CG). To make the material injectable, the obtained particles were mixed with a 0.5 % v/v solution of Collagen from porcine source in a rate of 90:10 granulate:collagen solution.

Preparation of Polyphenols Rich Pomace Extract

Croatina grape pomace was received in dry form from the producer and stored at -20°C under vacuum until the beginning of the extraction process. In order to make them suitable for the extraction process, grape pomace were first washed with acidified water, dried in a circulating-air oven (37°C ± 5°C) and grinded in a bladed mill (GM 200, Retsch). The milled grape pomace (300 g) were extracted in 2000 mL of 50:50 acetone:water (v/v) by using an automatic extractor (TIMATIC Micro C). The extraction cycle is fully automatic and alternates a dynamic phase, performed at a programmed pressure, and a static phase in which a forced percolation is generated, which, thanks to the programmable recirculation, ensures a continuous flow of solvent to the interior of the plant matrix thus avoiding over-saturation. Next, the extracted solution was concentrated under reduced pressure in a rotavap and maintained in fridge between 2-4°C. Full characterization of the polyphenols enriched extracts is provided in reference [26]. As reported in the just quoted paper, main identified components include gallic acid (9.42 µg/ml), caffeic acid (1.59 µg/ml), cumaric acid (0.21 µg/ml), quercetin (2.06 µg/ml), rutin (0.19 µg/ml), malvidin-3-glucoside (37.2 µg/ml). This deeply purple-stained croatina grape pomace extract shows a total anthocyanin content of 446.7 µg/ml [26].

NBR_Purple

NBR_Purple was produced by soaking the ceramic granulate with the Polyphenols rich pomace extract in a percentage of 40 % and 60 % respectively, followed by a 24 h of evaporation process which allows the ceramic granulate to be enriched with the polyphenol molecules (Pph). Then, the ceramic granulate functionalized with polyphenols were mixed with 0.5 % v/v collagen solution from porcine source, in the ratio 90:10, to make the material injectable.

Study Design

Review and approval by the Ethical Committee of the Universidad Politécnica de Valencia were obtained prior to conduct of the study. For each rabbit, one defect measuring approximately 2 mm x 5 mm deep was created in each medial condyle at a distal position of the femur. A second defect measuring approximately 2 mm x 10 mm deep was created in each medial condyle at a proximal location of the femur (Figure 1). For that reason, each animal had four defect sites, two on each medial condyle. Three different materials and a void were implanted on each animal. The response of NBR_Purple material (test article) was compared to NBR_White (control article), Ostim® and void. Ostim® is a synthetic nanocrystalline HA bone graft, containing about 65% water and 35 % nanostructured HA particles. Evaluations were conducted at 56 days.

Figure 1: Proximal and distal locations of bone defects at the medial condyle of rabbit femur.

This study had two end-goals:

1. Evaluation of local biological effects after implantation based on the requirements of ISO 10993-6:2007 (Annex E) [27].
2. Performance study by the analysis of % area occupied by trabecular bone.

Distribution of materials in distal and proximal sites at the right and left femoral condyle are reported in Table 1.

Table 1: Distribution of materials in distal and proximal sites.

		Right condyle		Left condyle
Animal #	Cage #	Distal	Proximal	Distal	Proximal
481	2	NBR_Purple	NBR_White	Ostim	Void
482	3	Void	NBR_Purple	NBR_White	Ostim
483	4	Ostim	Void	NBR_Purple	NBR_White
484	5	NBR_White	Ostim	Void	NBR_Purple
486	7	Void	NBR_Purple	NBR_White	Ostim
487	8	Ostim	Void	NBR_Purple	NBR_White
488	9	NBR_White	Ostim	Void	NBR_Purple
489	10	NBR_Purple	NBR_White	Ostim	Void

Animal Test System

Eight New Zealand White rabbit were used for the study. Characteristics Of the animals are reported in Table 2. The rabbit is widely used for evaluation articles intended for clinical implantation. The medial condyle of the femur provides a cancellous bone site, which will mimic the bone sites of clinical use. The rabbit is a small common laboratory species with bones of sufficient size to provide meaningful data and it is accepted by ISO 10993-6:2007 as experimental animal [27]. Any response to the implanted article can be graded and compared to that of a control article.

Table 2: Test system characteristics.

Species	Rabbit (Oryctolagus cuniculus)
Breed	New Zealand White
Source	ISOQUIMEN
Sex	Female
Body Weight Range	3.6 kg to 4.0 kg at implantation
Age	Approximately 20 weeks at implantation
Other conditions	Nulliparous, skeletally mature
Acclimatation period	2 weeks at IBV animal facilities
Number of animals	Eight
Identification method	Ear tag and cage card

The rabbits were weighed. Pre-anaesthetic and anaesthetic procedures are described in Table 3. The analgesic protocol is described in Table 4. Each rabbit received an intramuscular injection of the antibiotic enrofloxacin at 10 mg/kg. After the anaesthetic had taken effect, rabbits were clipped free of fur over the lateral and medial aspects of the rear legs from the wing of the illium to the tarsus. The surgical sites were painted with povidone iodine and draped.

Table 3: Pre-anaesthetic and anaesthetic procedures.

Phase	Product	Via	Dosis [mg/kg]	Concentration [mg/mL]
Pre-anaesthesia	Xylazine Clorhydrate	IM	3.12	20
Pre-anaesthesia	Ketamine Clorhydrate	IM	17.5	100
Anaesthesia-Induction	Propofol	IV	3	1%
Anaesthesia-maintenance	Propofol	IV	21 mg/kg/h	1%

Table 4: Analgesic procedure.

Phase	Product	Via	Dosis [mg/kg]	Concentration [mg/mL]	Frequency
Pre-surgical	Butorphanol tartrate	IM	0.4	10	Once
Post-surgical immediate	Fluxin meqlumine	SC	1	50	Once
Post-surgical immediate	Butorphanol tartrate	IM	0.4	10	Once
Post-surgical deferred	Butorphanol tartrate	IM	0.4	10	3 days after surgery

Implantation Procedure

The surgical site was draped, using sterile technique, the medial aspect of the distal epiphysis of the femur over the medial condyle was exposed through a routine surgical approach. Following exposure of the bone, an initial first pilot hole was created, using a drill with an approximate 1 mm bit, centred between the distal border of the epiphysis and the insertion of the medial head of gastrocnemial muscle. Using a power drill with an approximate 2 mm drill bit, the hole was enlarged to approximately 2 mm in diameter. This distal defect had an approximate depth of 5 mm, which it was controlled with a depth gauge placed in the drill. The material was implanted following the distribution shown in Table 1. A second defect (proximal site) was created in a similar way approximately 2-3 mm away from the first implantation site. In this proximal site, the implanted material follows the distribution shown in Table 1. The proximal defect had an approximate depth of 10 mm. The articles were placed in the bone defect to fill the void and remain flush with the cortical surface. The fascia and subcuticular layer were closed with 4-0 absorbable suture and the skin was closed with an intradermal suture technique. The day of implantation was designated as Day 0. A similar procedure was followed to create the other two bone defects on the contralateral medial condyle. The materials were implanted following the distribution shown in Table 1. Two materials on each medial condyle and four materials per animal were implanted.

Each rabbit was moved to a recovery area and was monitored for recovery from anaesthetic. Once sternal recumbency was achieved, each rabbit was returned to its cage. Analgesic protocol is shown in Table 4.

Rabbit were observed daily for general health, especially at the incision site. Body weights were recorded for all animals prior to implantation, and weekly thereafter and prior to termination.

Terminal Procedures

At 56 days after implantation, the eight rabbits were euthanized. Rabbits were weighed and each rabbit was euthanized with an intravenous injection of sodium pentobarbital-based euthanasia solution. The bone implant sites and adjacent muscle tissue were examined macroscopically and the observations were recorded. Any adverse observations at the implant sites were described. Each femur was dissected free and removed. Femurs were cut as appropriate to allow the fixative (ethanol 70%) to penetrate the bone tissue for proper fixation.

Histological Procedures

Histological procedures based on undecalcified bone samples embedded in PMMA to be cut with rotary microtome are published elsewhere [28]. The defect sites with implants in place were removed by making transverse cuts through the bone proximal and distal to each implant site, taking care not to disturb the sites themselves. Each bone section was labelled to indicate its original location. From 4 to 7 slides for each block was prepared as a transverse section of the bone through the diameter of the defect and stained with toluidine blue at pH 3.5. The identity (animal number, left/right) of each bone section was maintained during processing. Each slide shows a transverse section of the medial condyle with both implantation sites (Figure 1).

The slides related to NBR_Purple and NBR_White were provided to a pathologist for the evaluation of local biological effects after implantation following the criteria described in Annex E of ISO 10993-6:2007 [27]. Six fields (400x magnification) were analysed for each of the slides, totalizing between 24 and 42 fields for the defects analysed of each independent specimen. The histological evaluation was done following standardized procedures of our laboratory. The quantification of the biological effects has been done following the instructions of the ISO 10993-6:2007 standard. In this study 16 different specimens corresponding to an epiphyseal section from both the left and right extremities of rabbit femur have been included. The specimens were toluidine blue‐stained.

Evaluation of Local Biological Effects

Microscopic analysis revealed two areas of interest in each of the specimens included one closer to the medullary canal (proximal region) and the other one localized further from that (distal region). In the bone defects, the testing biomaterials have been implanted. In some cases the defects were left empty to serve as control. The cavities have a spheroid shape and the diameter ranges from 1.10 to 4.28 mm. In the surgical entry, a thickening of fibrotic dense connective tissue could be observed, which in some cases presented biomaterial fragments, probably due to the surgical procedure. In these cases connective organized structures associated with these fragments could be observed. This connective tissue is characterized by fibroblasts and macrophages accumulation as well as by an increased vascularization.

NBR_Purple Material

NBR_Purple material was implanted in 8 specimens, 4 of them corresponding to right and 4 to left condyles. In 4 specimens the implant area is located in the proximal region, while in the other 4 in the distal region. Between 7 and 4 slides of each specimen were included in this study.

NBR_White Material

This biomaterial was implanted in 8 specimens, 4 corresponding to right and 4 to left condyles. In 4 specimens the biomaterial was implanted in the proximal region and in the other 4 in the distal region. 4‐6 slides for each of the specimens were included in this study. Quantitative analysis was performed at 400 x magnification in 6 representative fields of each of the slides included in the study.

Performance Study

Digital pictures were obtained with a video camera (Sony, Exwave HAD) attached to a macroscope (Wild, M420) with a resolution of 97.5 pixel/µm at 7.8 x magnification. The squared region of interest was defined as centred at the middle point of the implant and with a 4-mm side, which it is twice the size of the bone defect diameter. Image processing techniques based on mathematical morphology was implemented in programming macros to specifically threshold the trabecular bone to calculate % area occupied by this tissue [29,30] (ImageJ, 1.49d, Rasband, W.S., U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014). Five slides of every condyles (left and right) including all materials (NBR_Purple, NBR_White, Ostim, void) were studied.

Statistical Analysis

Statistical analysis of the % area occupied by trabecular bone was conducted. To perform means comparison the method employed is one-way analysis of means (not assuming equal variances) and the pairwise comparisons between materials using t tests with pooled standard deviations (R, version 2.15.1, The R Foundation for Statistical Computing). Statistical analysis of bone regeneration within the defect is presented as box plot. The box represent the 25 and 75 percentiles and the horizontal line in the box represents median value. Lines outside the box represent minimum and maximum values [31]. Average value is represented as a blue point.

Results

Macroscopic Observation

All animals gained weight over the course of the study. Eschar and skin lesions associated with the incision lines were observed for most incisions. These skin lesions were considered a common occurrence associated with surgery at this site in rabbits and did not represent any adverse reaction to the tested biomaterials (Table 5). In the weeks following the surgery, incision sites in all animals were perfectly closed and furs cover the waxed area. Only animal 481 on day 1 showed an opened end that needed a second suture following the standard suture procedure. In this case, skin wound evolution was also good. An antiseptic was applied to the sites to prevent any possible infection and all sites healed without further complications.

Table 5: Macroscopic observation.

Animal number	Left leg	Right leg
481	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
482	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
483	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
484	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
486	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
487	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
488	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal
489	Implant site and muscle were macroscopically normal	Implant site and muscle were macroscopically normal

Microscopic Evaluation

Evaluation of Local Effects After Implantation

Osteointegration efficacy was evaluated implanting NBR_Purple material in the medial condyle of the femur bone of rabbits for 56 days. NBR_Purple was compared with a NBR_White bone filler, without polyphenols extract, and a commercial bone filler Ostim ®. At 56 days of implantation, a non-irritant response (index=0.0) was seen with the test (NBR_Purple) as compared to the control bone filler (NBR_White) following the comparison procedure proposed by Annex E, ISO 10993-6.

In Figure 2 are reported the percentage of area occupied by trabecular bone after 56 days of implantation, for NBR_Purple, NBR_White, Void and Ostim material, at distal site. NBR_Purple and Ostim showed the best results (42.9 ± 10.6 and 40.7 ± 5.9, respectively) followed by NBR_White (32.0 ± 9.2) and Void (28.3 ± 8.1). For NBR_Purple, there were significant differences with NBR_White and Void and there were no significant differences between NBR_Purple and Ostim (Tables 6 and 7).

Figure 2: Box and whiskers plot of the four articles analysed at the distal site.

Table 6: Statistics descriptive at distal site.

Distal
Statistic Description
Material	Valid N	Minimum	Maximum	Mean	St. Dev.
NBR_Purple	22	32.11	62.48	42.92	10.65
Ostim	21	32.3	57.22	40.67	5.95
NBR_White	21	21.41	45.91	32.04	9.19
Void	20	14.47	39.51	28.28	8.15

Table 7: Pairwise comparison at distal site using t test with pooled standard deviation.

	NBR_Purple	NBR_White	Ostim
NBR_White	< 0.001	–	–
Ostim	0.39773	0.00187	–
Void	< 0.001	0.16914	< 0.001

Figure 3 shows the percentage of area occupied by trabecular bone at proximal site, Ostim showed the best results (25.7 ± 10.6) followed by NBR_Purple (20.3 ± 10.1) and NBR_White (12.9 ± 2.2). Tables 8 and 9 report the statistical analysis among the bone filler.

Figure 3: Box and whiskers plot of the four articles analysed at the proximal site.

Table 8: Statistics descriptive at proximal site.

Proximal
Statistic Description
Material	Valid N	Minimum	Maximum	Mean	St. Dev.
NBR_Purple	20	3.63	33.13	20.26	10.11
Ostim	21	7.35	40.52	25.73	10.62
NBR_White	20	7.43	17.40	12.92	2.88
Void	21	7.75	15.71	11.96	2.16

Table 9: Pairwise comparison at proximal site using t test with pooled standard deviation.

	NBR_Purple	NBR_White	Ostim
NBR_White	0.00294	–	–
Ostim	0.02309	< 0.001	–
Void	< 0.001	0.68414	< 0.001

In all cases, higher value of area occupied by trabecular bone are observed at distal site by comparison with proximal site. This observation could be due as it is an area with more density of trabecular bone. Besides, proximal zone is very close to the beginning of the medullary canal and the dispersion of the material in this canal could be more frequent than expected.

Figure 4 reported the histological images for the specimens’ number 487I, NBR_Purple bone filler. An area of 3.20 mm of diameter with several new-formed spongy bone tissue could be observed. This tissue contained small and isolates biomaterial deposits. In some locations, fatty tissue and macrophages could be observed. Several bone-forming and bone-resorption cells could be observed (Figure 4A-4C).

Figure 4: Histological section of NBR_Purple implanted in sample 487I.

The same sample, 487I, is represented in Figure 5 for NBR_White. Histological images of this sample presented a 3.40 mm diameter spheroid area with small biomaterial deposit surrounded by fatty tissue, which presented in some locations a moderate inflammatory infiltrate along with venous sinuses.

Figure 5: Histological section of NBR_White implantend in sample 487I.

Discussion

Polyphenols are attracting more and more attention in the field of bone regeneration thanks to their anti-oxidant and anti-inflammatory properties [15,32,33]. In particular, in the field of oral health, clinical evidence has shown that flavanoids have beneficial effect on periodontitis [17-19,34], an oral inflammatory disease of polymicrobial origin that causes the disruption of gingival connective tissue and the alveolar bone supporting the teeth. Commercially available bone filler materials work through a simple scaffolding effect, providing osteoconduction. The new approach involving the biomolecular modification of biomaterials aims at enhancing the host tissue response through biologically active molecules delivered from the device or linked to the device surface [35-38]. In this broad scenario, the use of polyphenols from different source is widely investigated [32,39-45]. Grape pomace is an interesting source of polyphenols, and polyphenols-rich pomace extract are of particular interest due to the heterogeneity of the mixture, which makes these extract extremely interesting, since it contains a large number of polyphenols classes. Potentially beneficial effects of polyphenols have been reported in various study. For example, cell biology and in vivo rodent model studies [34,46,47] have demonstrated that flavanoids regulated the inflammatory response in periodontal components, and preserve effects on periodontal ligaments and alveolar bone tissues [48]. Another class of polyphenols, proanthocyanidins, has been shown both in vitro and in vivo to have a protective effect against oxidative stress and periodontitis [15,49]. The role of polyphenols in molecular signalling mechanisms behind bone anabolism has been recently reviewed [50] and potentials of polyphenols in bone-implant devices have been described [51].

Based on existing literature evidences, in the present work, a polyphenols-rich pomace extract combined to a biphasic phosphate ceramic bone filler was tested in vivo and compared to the same material without polyphenols and a couple of other controls. The study demonstrates in vivo that the presence of polyphenols enhances the osteointegration of the calcium phosphate bone filler. The results show that there is an higher value of the area occupied by trabecular bone after 56 days by the bone filler with polyphenols (NBR_Purple) and the bone filler without polyphenols (NBR_White). These results, support the several studies that have investigated the ability of polyphenols to improve and maintain bone health, showing clear positive effects on bone regeneration.

Conclusion

The study here reported demonstrates that at 56 days after implantation at distal and proximal sites in rabbit femoral condyles, the polyphenols-enriched bone filler NBR_Purple shows higher value of trabecular bone regeneration than those of the same material without polyphenols NBR_White. Assuming that the trabecular bone regeneration in an empty defect (Void condition) could be considered as physiological trabecular bone regeneration after a bone defect creation, NBR_Purple showed a 1.5 times regeneration enhancement at distal site and 2.1 times regeneration enhancement at proximal site. Present in vivo data support the role of polyphenols molecules in the stimulation of bone regeneration mechanisms.

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DOI: 10.31038/JDMR.2020341

Abstract

Stabilisation splints (S.S.) are one of the most commonly used tools in the treatment of temporomandibular disorders (TMD), bruxism or both.

The T Scan III computerised occlusal analysis technique accurately displays the sequence of occlusal contacts in closing and excursion movements and allows for the quantification of occlusion and disclusion times and the distribution of percentages of occlusal forces in the arch.

This study aims to assess the changes that occur in occlusion after the installation of a stabilisation splint.

A sample of 42 patients who attended the UCM Temporomandibular Disorders and Orofacial Pain Specialist Clinic was selected. These were diagnosed with TMD, myofascial pain and or sleep bruxism and treated with stabilisation splints (S.S.).

Records were taken in maximum intercuspation, right and left laterality, and in protrusion before and after installation of the stabilisation splint.

After the installation of the stabilisation splint, there was a decrease in occlusion and disclusion times. This difference was statistically significant in occlusion times at maximum intercuspation and disclusion movements of right and left laterality and protrusion.

As for the percentage of force, the right and left dental arch tend to be balanced after the installation of an S.S. The same is true between the premolar and molar sectors.

In conclusion, stabilisation splints produce significant changes in occlusion when analysing disclusion times in right, left laterality and protrusion movement. Stabilisation splints tend to balance occlusion forces between the premolar and molar sectors and between the two dental arches.

Keywords

T scan III system, Stabilisation splints (SS), Temporomandibular disorder (TMD)/bruxism

Introduction

TMDs are a subgroup of craniofacial pain problems involving the temporomandibular joint (TMJ), masticatory muscles and musculoskeletal structures of the head and neck. Symptoms related to TMDs predominate in young adults and women, with a ratio of females to males of up to 4:1, according to some studies [1].

Bruxism is a common disorder characterized by involuntary squeezing or grinding of the teeth, due to repetitive activity of the chewing muscles. Prevalence ranges from 8 to 31% [2].

Bruxism, as a whole, correlates positively with the presence of TMD pain [3]. Its role, however, in the aetiology of pain in TMDs is relatively unknown. Awake bruxism and sleep bruxism have been observed to have a different association with pain in TMDs [4]. Observational studies by Rossetti 2008 showed that awake bruxism is a greater risk factor for developing myofascial pain in TMDs than sleep bruxism [5] Conversely, Blanco Aguillera et al. and Fernandes et al. found that the pain in TMDs was associated with the sleep bruxism [6].

There are many studies on different aspects of TMDs, bruxism and myofascial pain. Few, however, have objectively evaluated the changes a stabilising splint produces in occlusion to facilitate good orthopaedic stability. In this sense, computerised systems that allow dentists to record these changes dynamically have been available to them for years.

Maness developed the T-Scan system for computer occlusal analysis in 1987, which performs real-time measurements of occlusal force percentages using an intraoral sensor.

Digital occlusal analysis (T-Scan III, Version 8, Tekscan, Inc. S. Boston, MA, USA) provides measurable variations in occlusion time and force percentages, accurately showing the occlusal contact sequence in closing and excursion movements [7]. This T-Scan system detects whether an occlusal force in one tooth, within a set of opposite dental contacts, is greater than, equal to, or less than the occlusal forces that occur in other teeth. It provides, with great precision, the force of occlusal contacts by providing qualitative and quantitative data.

The T-Scan is also a dynamic record, in which mouth closure is recorded in real-time from the initial contact to the maximum intercuspation (MIP) and also the excursion movements initiated from the MIP [7,8].

A complete occlusal analysis with T-Scan can detect abnormalities in the centring and intensity of occlusal forces, showing functional asymmetry that can be related to muscle dysfunction.

Physiological occlusion of maximum orthopaedic stability, which is the desired objective with an occlusal splint, must include several characteristics, such as; 1. There must be simultaneous bilateral contacts during mandibular closure in maximum intercuspation; 2. There must be disclusion of all back teeth during excursion mandibular movements; and 3. There should be no premature contact when the jaw is closed in a stable musculoskeletal position.

Until the development of T-Scan technology, recording of occlusion and disclusion sequence had not been possible, and therefore its times could not be accurately measured. Instead, clinicians had other methods for determining simultaneous contacts, such as observing the distribution and size of joint paper marks along the dental arches with the teeth in contact or listening to the sound of tooth contact and subsequent disclusion. The T-Scan I was the first device capable of measuring occlusal contact time in fractions of 0.01 seconds [9].

This study is designed to answer the following question: To what extent do stabilisation splints optimise occlusal forces and redistribute them in the dentition of patients with any or all of the following conditions; TMD, bruxism, myofascial pain?

Records obtained with the T-Scan III Computer Occlusal Analysis System have been objectively and dynamically analysed and evaluated in patients with TMD or bruxism or both, both in maximum intercuspation, right/left laterality, and protrusive motion, before and after inserting a stabilisation splint.

Material and Method

This study is a cross-sectional analysis of records with and without a splint, in which the medical history and computerised occlusal analysis of an individual patient before and after the installation of a stabilisation splint are evaluated.

A sample of 42 patients who attended our faculty’s Temporomandibular Disorders and Orofacial Pain Specialist Clinic was selected. These were diagnosed with one, various or all of the following; temporomandibular muscle disorders, myofascial pain, sleep bruxism. They, therefore, required treatment with stabilisation splints.

The selection method was by a non-probabilistic sampling of consecutive cases seeking maximum randomness. A previous study was conducted to determine the mean sample size, taking into account epidemiological studies estimating that approximately 5% of the population suffers from TMDs. It was determined that 37.24 records were required to be within acceptable margins of error and confidence level. A 95% confidence level was assumed.

Subjects over the age of 18 of both sexes, suffering from either or; muscular TMD, night bruxism, and ‘grinding’ patients, were included. All were informed about the purpose of the study and signed the informed consent. This study was supported by the Ethical Committee for Clinical Research of the San Carlos Clinical Hospital. ANNEX.

A specific and detailed medical history of TMD was made, following DC/TMD [10] criteria, and a Paesani questionnaire was filled out to detect bruxism [11]. Once the splint had been made and tested, the T-Scan III records were taken, first without the splint and then after placement in the patient’s mouth.

The computerised occlusal analysis technique, T-Scan (T-Scan III for Windows, Tekscan Inc., South Boston, MA), allows the clinician to quantitatively evaluate occlusal contacts and record occlusion during continuous jaw movement [12].

Each patient’s personal data is entered; the graph for each patient’s upper dental arch will thus be customised. The mesiodistal diameter of the central incisive allows us to extrapolate this diameter to all the teeth of the arch. Four records will be taken in maximum intercuspation, right laterality, left laterality and one last recording in protrusion. All these records are taken prior to the installation of the splint and are registered again with the splint installed.

The program processes the data and displays the charts in 3D or 2D (Figure 1).

Figure 1: Below is the “Force vs. Time” graph. This graph illustrates the change in occlusal force percentages over time, both in the middle of the left arch (green line) and in the middle of the right arch (red line). The horizontal axis of the Force vs. Time graph indicates the elapsed time, while the vertical axis indicates the change in the percentage of occlusal forces on both sides of the dental arch. The total force of the combined left and right arch halves is shown in the Force vs. Time graph in the black line.

In the 3D graphics, registered contacts are displayed as different colour and height columns that quantify the intensity of the forces generated in the occlusion.

The 2D graphics show the halves of the right and left arch, which are outlined in green for the left side and red for the right, they also show the percentages of force of each dental arch, and by sector (Figure 2).

Figure 2: Force vs. Time Graph

A: Represents the start of occlusal contact.

B: Displays when all teeth go into intercuspation during closing.

A.B.: Time elapsed between initial tooth contact and maximum intercuspation. This is represented as occlusion time (O.T.) and should ideally be below 0.2 s;

B.C.: Is the time elapsed when teeth begin intercuspation during closing.

C: Represents the beginning of disclusion.

CD: Represents disclusion time (DT). During the tour, the D.T. should be less than 0.4 s; Longer D.T. indicates more occlusal surface friction contacts during the excursion ^{(180) (181) (182) (183)}.

Fuerza Máxima Total=Total maximum forcé.

The occlusion time is defined as the duration from the initial contact of the tooth until it reaches the maximum intercuspation, which ideally should be less than 0.2 seconds. Longer times indicate greater interference and premature contacts during closing.

The subsequent disclusion time (D.T.) was first described by Kerstein and Wright as the required elapsed time for all posterior molar and premolar teeth to separate bilaterally from each other during a single excursion movement made in one direction (right, left or protrusive). Excursion recordings using the T-Scan III are used to measure the duration of the subsequent disclusion time and the existence of friction on the posterior occlusal surface during excursion movements [13].

The data were processed with the statistical programme SPSS Statistics 22. 0 (IBM, Armonk, NY, USA). The comparison between the occlusion and disclusion times that occur before and after the installation of a stabilisation splint and the percentages of bite force was performed by the t Student sample test for repeated measurements. The Wilcoxon or Mann-Whitney U tests were used when the assumption of normality was not met (Figures 3 and 4).

Figure 3: Occlusion and disclusion times that occur after the installation of a stabilisation splint.

Con/sin férula=With/without splint

O.T Oclusión/Disoclusión=T. Occlusion/Disclusion

LDT=RLT (Right Laterality Time).

LIZT=LLT (Left Laterality Time)

PTT=PT (Protrusion Time).

Figure 4: Porcentage de fuerza de mordida en máxima intercuspidación – sin férula/con férula

Percentage of bite forcé in maximum intercuspation – without splint/ with splint.

Porcentage lado derecho/izquierdo de la arcada sin férula/con férula

Percentage right/left side of the dental arch without splint/with splint.

Porcentage sector anterior/posterior de la arcada sin férula/con férula

Percentage anterior/posterior sector of the dental arch without splint/with splint.

Results

The reproducibility of the study was analysed using the interclass correlation coefficient and sensitivity of T-Scan III. Although statistical significance was not found in all records, a pattern was observed in all of them.

The results in terms of the occlusion time at maximum intercuspation, recorded before and after the installation of the stabilisation splint, were an average value of 0.18″ and the occlusion time after the installation of the splint decreased to 0.14″, this difference was statistically significant, p < 0.05.

The Wilcoxon test was used to assess the results of disclusion times at maximum intercuspation. Records without splints have an average value of 0.26″. There are no significant differences between the two records.

Disclusion time analysis in right laterality showed statistical significance. The average of the records without splints was 1.07″, well above the 0.40″ admitted as normal and the splint records showed a decrease in disclusion time to 0.54″.

Upon analysis of the disclusion time in the left laterality movement, a statistically significant difference is observed. We note that the disclusion time decreases from 0.98″ to an average of 0.57″ in records taken with stabilisation splints.

When analysing the moving protrusion records, we observe that the average disclusion times before installing the splint is 0.90″ and that this time decreases with the splint to 0.64″. This data has statistical significance (Table 1).

Table 1: Occlusion and disclusion times that occur after the installation of a stabilisation splint.

Variables		Average	C.I. at 95%		Mdn	SD	p
Dependent	Independent		L.L.	U.L.
Occlusion time at maximum intercuspation	Without splint	0.18	0.16	0.21	0.17	0.07	<0.001 (t)
	With splint	0.14	0.12	0.15	0.14	0.05
Disclusion time at maximum intercuspation	Without splint	0.26	0.20	0.31	0.18	0.18	0.554
	With splint	0.22	0.19	0.25	0.20	0.11
Right laterality occlusion time	Without splint	0.19	0.16	0.23	0.19	0.11	0.044
	With splint	0.16	0.14	0.18	0.17	0.06
Right laterality disclusion time	Without splint	1.07	0.93	1.20	1.08	0.43	<0.001
	With splint	0.54	0.48	0.61	0.43	0.22
Left laterality occlusion time	Without splint	0.17	0.15	0.20	0.17	0.08	0.043
	With splint	0.15	0.13	0.16	0.15	0.05
Left laterality disclusion time	Without splint	0.98	0.87	1.09	0.94	0.35	<0.001
	With splint	0.57	0.49	0.64	0.49	0.24
Occlusion time in protrusion	Without splint	0.21	0.18	0.24	0.20	0.10	0.067
	With splint	0.18	0.16	0.20	0.19	0.06
Disclusion time in protrusion	Without splint	0.90	0.79	1.01	0.91	0.36	<0.001 (t)
	With splint	0.64	0.55	0.73	0.62	0.28
Wilcoxon signed-rank test; t, Student´s T-Test (paired samples); C.I, Confidence interval; L.L., lower limit; U.L., upper limit; S.D., standard deviation, Mdn, median. Sample Size 42 patients.

Record as a percentage of forces that occur in each dental arch, right and left. The Student’s T-Test was used for statistical analysis for repeated samples. It is observed that at maximum intercuspation, the average percentage for the right dental arch recorded without stabilisation splint was 53.25% and for the left, it was 46.76%. With the stabilisation splint, the average values were 51.22% for the right dental arch and 48.80% for the left. It is noted that in stabilisation splint registers, there is a tendency to balance between the two dental arches (Table 2).

Table 2: Percentages of forces that occur in the right and left dental arches before and after the installation of a stabilisation splint.

Variables		Average	C. I. at 95%		Mdn	SD	p
Dependent	Independent		L.L.	U.L.
Percentage right side of the dental arch	Without splint	53.25	49.22	57.27	53.60	12.91	0.067
Percentage left side of the arcade		46.76	42.74	50.78	46.40	12.91
Percentage right side of the dental arch	With splint	51.22	48.62	53.81	50.95	8.32	0.400
Percentage left side of the dental arch		48.80	46.19	51.41	49.05	8.37
Wilcoxon signed-rank test; t, Student´s T-Test (paired samples); C.I., Confidence interval; L.L., lower limit; U.L., upper limit; S.D., standard deviation; Mdn, median.

Record as a Percentage of Forces Produced in the Premolar and Molar Sector

For statistical analysis, the Student’s T-test for repeated samples and the Wilcoxon signed-range test were used. The average value of registers taken without stabilisation splints for the premolar sector was 40.27% and for the posterior molar sector of 60.09%. When registers are taken with the splint, the average was 52.26% in the premolar sector and 47.71% in the posterior molar sector. It is noted that records taken with stabilisation splints tend to balance both sectors (Table 3).

Table 3: Comparison of forces produced in the anterior and posterior sector before and after the installation of the stabilisation splint.

Variables		Average	C.I. al 95%		Mdn	SD	p*
Dependent	Independent		L. L.	U.L.
Anterior sector percentage (premolars)	Without splint	40.27	34.27	46.27	38.65	19.26	0.002
Posterior sector percentage (molars)		60.09	54.25	65.93	61.35	18.75
Anterior sector percentage (premolars)	With splint	52.26	47.97	56.55	53.70	13.75	0.149
Posterior sector percentages (molars)		47.71	43.42	51.99	46.00	13.76
*Wilcoxon signed-rank test; C.I, Confidence interval; L.L., lower limit; U.L., upper limit; S.D., standard deviation; Mdn, median.

Analysis of the results without stabilisation splint reveals there is a higher percentage of force at the molar than at the premolar level. With the splint, however, there is a change in the ratio of the force percentages, so that the loads are advanced, increasing the percentage of force at the premolar level, so much so that the values are inverted.

Discussion

At the start of this study, the reproducibility and sensitivity of the T-Scan system have been considered, using the interclass correlation coefficient. While no statistical significance was found in all records, a pattern is observed.

Several studies have assessed the ability of the T-Scan III system to detect the number and location of occlusal contacts and conclude that its operation is correct, particularly when used in conjunction with traditional methods [14,15].

Throckmorton [14] and Cerna [15] studied the validity and reliability of T-Scan III sensors under laboratory conditions, noting that reliability was high when used in consecutive measurements. Silva (2014) confirmed the high reliability of the sensor in consecutive measurements, as in our study [16].

Kerstein [17] related the size of the marks and the occlusal force with strips of articulating paper, he considered that reliability was only 21% and that to increase it, higher forces were needed [17]. Koos [9] assessed the accuracy and reproducibility of the T-Scan III to calculate the normal distribution of relative force in the dental arch and found that the difference between measurements and actual values were less than 2%. The method can, therefore, be considered sufficiently accurate and reliable [9].

Kerstein et al. have considered that, according to the available evidence, the computerised occlusal analysis system is the only occlusal indicator that displays the ability to quantify the force and time of occlusion in real-time from initial MIP contact to disclusion. The accuracy of displaying occlusal contacts makes this system a better occlusal indicator than other available conventional non-digital materials. Recordings of T-Scan’s occlusal analysis, if successful, can provide an objective view of the occlusion and its discrepancies. Subsequent publications have reported that T-Scan III shows good reproducibility of surface measurements and register of relative forces [18]. It is, therefore, endorsed as a good method of study, and even more so for this study, in which repeated measurements were made, with and without a splint.

The T-Scan III system is useful for calibrating occlusal contacts. Occlusal contact between the teeth is considered to exist when the interocclusal distance between the occlusion areas is less than 50 μm; while contacts close to occlusion occur when the distance is between 50 and 350 μm

For proper function, occlusal contacts must be synchronised with the stomatognathic system [19].

A wide variety of occlusal analysers have been used to record occlusal relationships between dental arches. In dental practice, the role of articulating paper has been the most commonly used diagnostic tool to identify the points of contact between the teeth of both arches. Paper can easily highlight or mark contact, but it cannot accurately quantify its intensity or measure the magnitude of the occlusal forces generated. It is the size of the marked area on the articulating paper that is used as an indicator of the occlusal load intensity [20].

Authors such as Saad, Weiner and Ehrenberg, Millstein, consider the interpretation of paper marks to be subjective and therefore inaccurate since similar occlusal loads correspond to marks of different intensity. The T-SCAN III computerised occlusal analysis system largely avoids subjectivity in the interpretation of the articular role [21,22].

T-Scan III occlusal analysis technology, while not being able to measure absolute bite force, does provide occlusal force times and variability, which can be quantified from the first point of contact to MIP when the subject bites on the occlusal sensor. The sensor thickness is 100 μm (0.1 mm), which is compressed up to 60 μm under the occlusion force. Due to the high compression capacity of the sensor, it provides bilateral contact during jaw movement.

The T Scan system allows accurate assessment of occlusion and disclusion times. The occlusion time, which runs from the first contact during closing to full intercuspation (Koos, 2010), is mostly conditioned by dental morphology. Furthermore, the canines and first premolars play a crucial role in leading teeth to contact [9].

The posterior disclusion time (D.T.) was defined by Kerstein and Wright as the elapsed time for all molar and premolar posterior teeth to separate from each other during a single excursive movement performed in one direction; right, left, or protrusive.

Several studies have shown that with treatments that minimise disclusion time to less than 0.5 s per excursion, it is possible to reduce pain and dysfunctional muscle symptoms quickly, both in frequency and intensity. This improvement was maintained during the observation time of the studies [13].

In this study, it has been confirmed in records taken with stabilisation splints in right/left laterality and protrusion movement that disclusion times decrease, and pain also decreased in most subjects.

In our study, patients showed decreased pain on the VAS scale when evaluated one month after the installation of the stabilisation splint.

Kerstein (2016) also related disclusion time to symptomatology, proving that a reduction of fewer than 0.4 seconds per excursion is effective in reducing symptoms of myofascial pain [23].

Modifying an occlusal scheme by shortening disclusion time can be achieved through the occlusal adjustment process known as Immediate Complete Anterior Guidance Development (ICAGD). In the study that developed this method, significant reductions in myofascial pain muscle symptoms were found, which began even after the first day of treatment. When comparing the right and left excursive disclusion times of 100 pre- and post-treatment patients with ICAGD, several conclusions were reached. First, that time of disclusion in excursive movements, if prolonged can result in symptoms of myofascial pain. Reducing disclusion times in left and right laterality movements below 0.4 seconds per excursion decreases symptoms of myofascial pain. When patients with myofascial pain are treated using the ICAGD technique, which uses T-Scan III computerised occlusal analysis to measure the correctness of the outcome of ICAGD treatment, symptomatology will improve rapidly.

Thus, treatment based on reducing disclusion time by applying the ICAGD method is a new therapeutic possibility for patients with dysfunction and myofascial pain. After the installation of the stabilisation splint, disclusion times are shortened, creating a new occlusal scheme, and establishing new guides for excursive movements [24].

It has been shown that the longer the back teeth are in occlusion during a jaw excursion, the greater the friction that occurs. This leads to temporal and masseter muscle hyperactivity, contributing to the occlusal pain and muscle symptomatology of TMDs. High disclusion times of more than 1.39 seconds significantly increase contractile muscle activity [25]. When these disclusion times are reduced, below 0.5 seconds, (<0.5″) contractility is significantly reduced, reaching values close to those of the resting state. It has been observed that with a coronoplasty, disclusion time can be shortened by 0.5 seconds per excursion.

In our study, coinciding with these observations, disclusion times decreased considerably in the different excursive movements with the splint.

Finally, the T-Scan system provides data relevant to the assessment of the distribution of force percentages in the different sectors of the dental arches. Misirlioglu et al. have suggested that T-Scan is a successful diagnostic device for detecting premature contacts and excessive occlusal forces [26].

T-SCAN III analyses the order of occlusal contacts while simultaneously measuring the percentage changes in the force of those same contacts, from the moment the teeth begin to make occlusal contact to central occlusion intercuspation. It shows abnormal forces that lead to trauma or pain in each tooth in the dental arch. This helps to balance forces on both sides of dentition [27].

Kerstein RB et al. consider the T-SCAN III system to be a highly accurate technique for studying and analysing occlusal relationships.

KERSTEIN et al., by synchronising computerised occlusal system data with electromyography, observed that it was possible to detect muscle dysfunction through the centre of force patterns and timing of disclusion. They also determined that the T-Scan III system can provide an accurate and definitive diagnosis of occlusal force balance and chewing muscle function for the clinician and is a comprehensive educational tool for the patient undergoing occlusal balance procedures [28,29].

Treatment with stabilisation splints is most commonly indicated for any of the following; TMDs, bruxism, and myofascial pain.

Scientific and health organisations in the United States; the National Institutes of Health-National Oral Health Information Clearing House (NIH-NOHIC), the American Academy of Oral Pain (AAOP), the American Association Of Oral and Maxillofacial Surgeons and the American Academy of Craniofacial Pain (AACFP), recommend short-term stabilisation splint therapy without occlusal changes as a treatment for TMD (Clark and Minakuchi, 2006).

Montgomery considers the T-Scan III system an element of routine analysis of occlusal physiology and its relationship to adjacent oral musculature. He concludes that T-Scan III is a precise method that, in a computerised manner, records occlusal contacts, generating a dynamic video that allows identification of the percentage of occlusal force per tooth, dental arch and quadrant. This procedure enables occlusal adjustment to be performed with greater precision, allowing functionally balanced restorative treatments, facilitating muscle activity and balancing periodontal support [30].

Ferrario et al. compare electromyography records in patients with and without stabilisation splints and find a significant decrease in the activity of the masseter and temporal muscles in the records of maximum intercuspation with the splint. They also observe a greater balance between both right and left sides, and an increase in masseter muscle activity compared to the anterior temporal muscles [31].

Thus, the results of this T-scan III study show that the installation of a stabilisation splint promotes balance in the distribution of forces. The splint tends towards symmetry between both sides of the dental arch, which is also confirmed by electromyography. On the other hand, the loads are redistributed, increasing the percentages of occlusal force over the premolar zone, relative to the rear area of molars. These results in an increase in masseter muscle activity compared to the previous temporal one, favouring balance in the joint activity of the muscles.

In conclusion, we can establish that the disclusion times in the excursive movements of left/right and protrusive laterality are reduced after the installation of the stabilisation splint. It is also observed that the distribution of forces between the two dental arches tend to be balanced after the installation of the splint. As for the percentages of forces at the anterior (premolar) and posterior level (molars), it is observed that there is a very clear difference, so the percentages of force between the two sectors tend to be balanced.

Given the results of this study, it can be said that the T-Scan III system provides useful information for the adjustment of the stabilisation splint in each patient, looking for guides with disclusion times of less than 0.4 seconds and a balance in the distribution of force percentages, providing orthopedic stability to the chewing apparatus.

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