Pilot study

A surgeon was trained using two sheep skulls: one refrigerated (with soft tissues still attached) and one dried (only hard tissues present). The surgeon was required to access the TMJ, perform a condylectomy, wax the two articular surfaces in situ, take an impression of the structures, obtain plaster models, and create 1 mm-thick silicone trays of the temporal and mandibular segments. These trays were used later in the study.

Aiming to draw comparisons with the acrylic components (mandibular and temporal) removed from the animals in the experimental period of 45 days (test samples), acrylic control components (C0) with the same preparation and dimension were created, but not exposed to the biological environment. Specific control samples were prepared for assessing the PMMA degree of conversion.

Sample allocation and study design

Sample size was calculated in accordance with the literature [21, 22]. Fourteen young adult (ten to fourteen months old) female Corriedale black sheep, weighing approximately 35 kg, were used in the present study. The sheep were divided into an experimental (E) and a control group (C) through unequal randomization (ratio 5:2). The animals in the experimental group (n = 10) underwent biconvex arthroplasty, and were further divided into two subgroups, E45 (n = 5) and E90 (n = 5), according to the interval between the surgical procedure and euthanasia (45 and 90 days, respectively). Control animals (C45, n = 2) underwent pre-, intra- and postoperative procedures, but surgery was limited to skin incision and subcutaneous and muscular dissection without approaching the joint capsule or bone. Variables were assessed before the surgery (T0), immediately after (T1) and at 45 or 90 postoperative days (T2). All surgical procedures were conducted by the same surgeon.

Pre-operative care and anaesthetic procedures

Pre-operative care involved transporting, weighing and housing the animals for 24 h at the Animal Experimental Unit, with a view to reducing stress. The animals were submitted to a 24-h fast from solid foods and a 12-h fast from liquids. Housing temperature was kept at 22 °C ± 2 °C, humidity was maintained at 60% ± 5 and the light/dark cycle was controlled by a timer (12:12, lights on at 7 h and off at 19 h).

The pre-anaesthetic medications meperidine (3 mg/kg) and midazolam (0.25 mg/kg) were administered intramuscularly (IM). After 15 min, the animals were contained and transported to the operating room. Cefazolin (22 mg/kg) was administered intravenously, as was the anaesthetic propofol (3–5 mg/kg). The animals were submitted to orotracheal intubation followed by mechanical ventilation and isoflurane anaesthesia.

Surgical procedures

A trichotomy and antisepsis were performed and surgical drapes were positioned. A preauricular incision (7 cm) and tissue dissection/detachment were performed to access the TMJ. A line was drawn from the mandibular condyle to the temporal bone to define the orientation of the jaw force axis (posterior-superior vector, 45o from the occlusal plane). This line was used to identify the future location of the contact point between the reconstructed segments, which allows for the jaw force vector to be directed downwards. The new mandibular condyle, which is articulated with the posterior surface of the glenoid cavity, guides the vector on a sagittal plane, directing it in an inferior to superior and anterior to posterior direction. An anterior–posterior osteotomy was performed in the condylar neck, 10 mm from the highest point of the condyle (Fig. 1A). Four and three bicortical perforations (1 mm) were made on the temporal surface (Fig. 1B) and condylar neck, respectively, for mechanical retention of the PMMA cement, since the material has no adhesive property to the bone. At this point, the animal was submitted to maxillomandibular immobilization. Auto-polymerized poly(methyl methacrylate) (Surgical Simplex P Bone Cement, Howmedica International Inc, Limerick, Ireland) was mixed according to the manufacturing company’s instructions and injected into the bone perforations. The temporal silicone tray (Fig. 1C) was also completely filled. As the material hardened, the tray was placed in position. During the heating phase, the area was constantly irrigated with distilled water. After the polymerization, the silicone tray was opened with a scalpel and removed. The same sequence was performed in the mandibular segment. When the reconstruction was completed (Fig. 1D), intermaxillary immobilization was removed. Retention of the alloplastic implants and maintenance of mandibular mobility were confirmed through manual examination of the mouth-opening amplitude and of lateral mandibular movements. The incision was closed using a simple interrupted suture with polyglactin 910 thread.

Fig. 1

TMJ reconstruction using the Puricelli biconvex arthroplasty technique. A Condylectomy; B Temporal bicortical perforations; C Silicone trays of the temporal and mandibular segments before the reconstruction; D Final reconstruction of the left TMJ

In the animals of the control group, a vertical preauricular 7-cm incision was made, followed by incision and divulsion of the masseteric musculature. In this group, the joint capsule was not ruptured, so the joint surfaces were not exposed and the procedures for joint reconstruction were not performed. The tissue planes were sutured with 4 × 0 polyglactin 910.

Postoperative management

Animals began to be fed immediately after recovering from the anaesthesia. After 48 h, the sheep were transferred to external housing, where they were identified by ear tags on both ears. A broad-spectrum antimicrobial agent (benzylpenicillin/streptomycin, 6 ml/100 kg) was administered IM following surgery, and once again after 72 h. Anti-inflammatory medication (ketoprofen, 2 mg/kg) was also administered daily for 48 h, while an analgesic (tramadol hydrochloride, 2 mg/kg) was given to the animals every 12 h for 48 h.

Experimental and control animals were returned to the Animal Experimental Unit after 45 or 90 days. After their body weight had been measured, mouth-opening and mandibular laterality amplitude were assessed under general anaesthesia. Following the evaluation of these parameters, the anaesthetized animals were euthanized using intravenous potassium chloride (1 ml/kg).

The reconstructed TMJ was surgically exposed post-mortem and the left temporomandibular joint was removed with a 6.0 cm (diameter) × 4.5 cm (depth) trephine drill. The samples were identified and stored in closed containers with 10% buffered formalin.

Histological analyses

After fixation, samples from E45 and E90 animals were decalcified in 5% nitric acid solution for approximately two weeks. Decalcification allowed the removal of prosthetic components from bone tissue without damage. Samples from the reconstruction capsule and pseudo-disc were collected from the main block, identified and submitted to routine histological processing before being embedded in paraffin. Slides were stained using hematoxylin–eosin (HE). Morphological descriptive analysis of the capsule and pseudo-disc samples (when present) in HE staining at 100 × magnification was performed using a CX41RF binocular microscope (Olympus Latin America Inc., Miami, FL, USA). Images of five selected fields from the most cellular areas of the samples under investigation were captured at 400 × magnification using a Qcolor 5, Coolet, RTV camera (Olympus Latin America Inc.) and Qcapture software version 2.81 (Quantitative Imaging Corporation Inc., Canada). All inflammatory cells were quantified using ImageJ for Windows version 3.0 software (NIH, USA) in the five fields and classified into neutrophils, eosinophils, lymphocytes, plasmocytes and macrophages. Data are presented as mean and standard deviation considering each animal as sample. A blinded, trained and calibrated examiner performed quantifications. For calibration purposes, 30 fields from six different samples were evaluated, and re-evaluated after five days, and quantifications were compared using the Intraclass Correlation Test (ICC) (α > 0.98).

PMMA degree of conversion

Vibrational Raman spectroscopy (Senterra, BrukerOptics, Germany) was performed at three random points in the areas corresponding to the contact point established between the temporal and condylar PMMA surfaces, the non-contact area between the components and the contact surface of the acrylic with the bone tissue, in order to evaluate the degree of conversion of the acrylic resin. The 100 mW and 785 nm diode laser was used for two seconds with 20 co-additions, totalling 40 s with 100% laser power, and a spectral resolution of 3–5 cm−1. Spectra were obtained between 400 and 1800 cm−1.

The percentage of unreacted carbon–carbon double bonds (%C = C) was determined by the ratio of the absorbances between the aliphatic carbon double bonds (peak at 1640 cm−1) and the internal standard in the monomer and the polymer. The absorbance of the carbonyl group (peak at 1720 cm−1) was used as an internal standard. The degree of conversion (DC) was determined by subtracting the %C = C of 100% (13). For this analysis, control specimens (n = 3) were made using a silicone matrix 2 mm in height and 3 mm in diameter. A spectrum was obtained soon after the manipulation of the material and insertion into the matrix to obtain the values referring to the peaks in the monomer. The same specimens were analysed immediately after the cure time (ImmC) and after seven days (7dC). The relative values obtained for the monomer were also used to calculate the degree of conversion of the components.

PMMA roughness assessment

An SJ-201 rugosimeter (Mitutoyo, Japan) was used to assess the components’ surface roughness. It contains a sensor which, when traversing the surface of the material, assigns values that define peaks and valleys present on this surface. The value assigned to the area of peaks and valleys was divided by the distance travelled by the straight-line sensor providing the roughness parameter in Ra in μm. The machine provides the average of three polls of 0.25 μm. Four measurements were performed randomly, by a blinded examiner, on each surface (with without wear and control C0) on the condylar and temporal samples.

PMMA scanning electron microscopy

The samples were metallized by a gold film using an ion deposition technique (sputter coater) due to the absence of electrical conduction of the acrylic material. Afterwards, they were stabilized in stubs and scanned with an electron microscope (SEM-FEG) (Inspect F50, FEI, Czech Republic). Images of the surfaces with or without wear, the acrylic contact area with the bone tissue and control C0 were obtained randomly, by a blinded examiner, with 30x, 100x, 200 × and 1000 × magnification. The energy of the electron beam was 20 kV.

PMMA wear assessment

Condylar and temporal acrylic components were digitized using a laser scanner (Tecnodrill, Digimill 3D, Brazil) with a conoscopic sensor (Optimet, ConoProbe 1000, Israel) and a 75mm lens and 0.05 mm resolution, creating a point cloud that was later transformed into a 3D mesh. Employing Geomagic Qualify software (Geomagic, USA), acrylic pieces removed from the animals were overlapped on control pieces (C0) – which represent the initial volume of the acrylic reconstruction – using manual and software alignment tools such as Best Fit Alignment. Image subtraction allowed observation of the linear wear (loss of height), in mm, by calculating the mean linear loss at three random points with the highest wear, as well as the loss of volumetric content of the material, in mm3, by calculating the volume difference from the wear plane in test samples in comparison to the control sample, during the postoperative experimental period of 45 days in both condylar and temporal samples. A blinded examiner performed the analyses.

PMMA microhardness assessment

The condylar and temporal acrylic components removed from the animals, as well as a control sample (C0), were positioned and fixed with the use of brown Godiva on resin blocks. The Knoop hardness of the acrylic resin was evaluated using an HMV-2 automatic micro durometer (Shimadzu, Japan) with a load of 25 g for ten seconds. Three random indentations were performed on the surfaces (with and without wear) of each specimen, 100 μm apart. The final microhardness value assigned to each surface (with or without wear and control) on the condylar and temporal samples was the arithmetic mean of the three measurements. A blinded examiner conducted the analyses.

Prosthetic stability

For analysis of macroscopic stability, the reconstructed TMJ was surgically exposed post-mortem for immediate evaluation of the retention of the acrylic structures. The presence of macroscopically observable movements of the prosthetic component in relation to the corresponding bone segment when the prosthesis was handled was taken to indicate a lack of prosthetic stability. The test was carried out with surgical instruments and manual pressure in lateral-medial directions, perpendicular to the direction of the force vector exerted by the reconstructed mandibular condyle on the reconstructed temporal surface. The examiner was blind to the time of death of the animals.

For radiographic evaluation, the left and right temporomandibular joints were removed post-mortem with a 6.0 cm (diameter) × 4.5 cm (depth) trephine drill and stored in enclosed containers with buffered formalin (10%). Within seven days following euthanasia, lateral X-ray examinations were used to examine the left TMJs.

The characterization of reconstructed mandibular and temporal structures as stable or unstable based on imaging examinations corroborated the results of the macroscopic analyses (T2). The experimenters were blind to the time of animal death.

Functional evaluation

Maximum amplitude of mouth-opening and bilateral mandibular lateral movements were measured in millimetres using an optical pachymeter at three different times (T0, T1 and T2). The edges of lower central incisors and the upper edentulous alveolar ridges were used as reference for the measurement of the maximum mouth-opening amplitude. Since sheep do not have upper anterior teeth, the upper and lower labial frenums were used to measure the maximum amplitude of lateral movements. Mouth-opening and lateral movements amplitude were assessed through the application of a standard force of 2.5 kg, which was measured using a portable electronic scale. The same blinded examiner assessed the animals following the induction of muscle relaxation by carrying out the same measurements after obtaining a 1.5 MAC (minimum alveolar concentration) under isoflurane sedation.

Body weight evaluation

Animals were weighed at the experimental unit, using a digital scale. The weight was recorded at two different times (T0 and T2). The examiner was blind to the animal group.

Statistical analysis

Data were analysed using SigmaPlot 12.0 (Systat Software Inc., EUA), except for prosthetic stability, and functional and body weight evaluation, which were assessed using SPSS. The normality of data distribution was determined using the Shapiro–Wilk test, and variables were expressed as mean, standard deviation and median. A T-test for independent samples was applied to compare experimental groups for the number of inflammatory cells at different experimental times, and to compare volumetric and height wear between the experimental and control groups. Changes in the mean value of variables over time were compared within groups through a repeated-measures analysis of variance (functional measurements) and through paired-sample Student’s t-tests (weight). Variables were compared between the test and control groups by calculating deltas (differences between mean values at different points in time) and comparing these values between the groups using a one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. A significance level of 5% was considered.