Many different surgical and non-surgical techniques are used for the treatment of temporomandibular joint (TMJ) hypermobility. One of these methods is autologous blood injection into the TMJ. The fate of the autologous blood used for treatment of recurring condylar dislocation is still not completely understood. The authors used 12 pigs ( Sus scrota f. domestica ) as a model species for autologous blood delivery into the TMJ. Blood injection was followed by histopathological analysis at different times after treatment (1 h, 1, 2 and 4 weeks). Samples were examined by magnetic resonance imaging, macroscopic and histological methods. The deposition of the remaining blood was observed in the form of clots in the distal parts of the upper joint cavity 1 h and 1 week after treatment. 2 weeks after treatment, small blood clots were still apparent in the distal part of the upper joint cavity. 4 weeks after surgery, no remnants of blood, changes or adhesions were apparent inside the TMJ. No morphological or histological changes were observed in the TMJ after the injection of autologous blood suggesting another mechanism is involved in the hypermobility treatment.
The temporomandibular joint (TMJ) provides the junction between the jaw (mandibular condyle) and the neurocranium (temporal bone). Condyle dislocation (or hypermobility) of TMJ is one of the most frequent TMJ disorders in humans. In the case of hypermobility, the condyle reaches a position in front of the articular tubercle at wide mouth opening, which can be caused by abnormalities in the shape of the joints, by ligament looseness or by reduced muscle tension.
The treatment of TMJ hypermobility includes surgical or non-surgical approaches. Surgical procedures can be divided into two categories; those that limit the range of condylar movement, and those that remove the blocking factor that prevents the condyle from returning. Non-surgical treatment includes a soft diet, pharmacotherapy, physical therapy, stress reduction, movement limitation and occlusal splint therapy. Joint movement reduction can be caused by the injection of different substances such as autologous blood or sclerosing solutions into the upper joint cavity. Although there are many clinical studies about a high success rate of autologous blood injection into TMJ, the effect and the detailed mechanism of this therapy are not well understood.
It has been proposed that autologous blood in the TMJ may result in joint degeneration and/or formation of adhesions inside the joint. There is a lack of studies examining in detail the mechanisms of the effect of autologous blood injection into the TMJ. This type of study can only be carried out using a suitable animal model (human-like size, with similar structure and motion of TMJ) paired with subsequent histological evaluations.
Several studies have been performed to understand the joint pathology and different approaches have been tested for their treatment. A sheep model was used for the evaluation of histopathological changes after intracapsular condylar fracture, the fate of auricular cartilage graft in the surgical treatment of TMJ ankylosis, intraarticular scarring and ankylosis management. Blood was injected into the rabbit TMJ. The rabbit and sheep condyles are adapted to a herbivorous diet so are more rounded than those of humans, which causes greater mobility in the transverse plane and limited mouth opening.
In contrast, pigs are omnivorous like humans and therefore the structure of their TMJ resembles that of humans. Their diarthrodial synovial TMJ consists of an articular pit, articular disc and articular condyle surrounded by a ligamentous capsule. The articular disc, as in humans, divides the joint into two compartments described as two articulations: the meniscotemporal (suprameniscal) joint permitting translational movements, and the condylomeniscal (inframeniscal) joint, which permits rotational movements. The disc has a biconcave shape; the fossa is shallow, and the condyle is elliptic. The masticator muscle arrangement is similar to that in humans, therefore moderate translation movements are allowed in all planes; the major movement is provided by rotation of the joint condyle.
Based on these morphological similarities, the authors selected the pig as a model organism for this study, which aims to confirm or disprove the hypothesis that aseptic inflammation and subsequent formation of lesions and adhesions are responsible for the therapeutic effect of autologous blood used for TMJ hypermobility treatment.
Materials and methods
12 pigs ( Sus scrofa f. domestica ) aged 2 years were obtained from the breeding unit of the Institute of Animal Physiology and Genetics, Academy of Science of the Czech Republic in Libechov. Animals were divided into four groups of three animals, none of which had any previous history of TMJ hypermobility. The first group was killed 1 h after treatment, the second group 1 week, the third group 2 weeks and the fourth group 4 weeks after autologous blood injection. The animals were housed in separate breeding boxes under conventional conditions and provided with water and food ad libitum. The experimental procedure was approved by the Animal Research Committee of IAPG CAS, v.v.i. (Nr. 67985904).
All surgical procedures were performed in the aseptic conditions of an operating theatre with disinfectant applied over the operating field. All animals were premedicated with ketamine (22 mg/kg) and atropine (0.04 mg/kg) and anaesthetized with thiopental (15 mg/kg) prior to intubation. Anaesthesia was maintained with inhaled isoflurane (1.5%). The animals were mechanically ventilated with an initial tidal volume of 10 ml/kg and a respiratory rate of 15 breaths per min. The tidal volume was adjusted to maintain an arterial PaCO 2 of 35–40 mmHg during the experiment. Hydration was maintained using lactated Ringer’s solution delivered through a cannulated dorsal auricular vein. Body temperature was maintained at 38.0–39.0 °C using a circulating hot water heating pad. Both heart rate and oxygen saturation levels were monitored throughout all surgical procedures.
The lateral approach was carried out from the lateral side of the articular capsule. A small incision was made above the lateral part of the left condyle, approximately 1 cm below the external auditory meatus during a wide mandible opening. The first 20-gauge needle was inserted towards the posterior aspect of the condyle in the posterior part of the superior joint cavity in the anterior-medial direction, before being withdrawn slightly (about 1 mm) to prevent subchondral application and saline solution was injected. During the saline solution application, no protrusive movement of the mandible was observed and the correct position of the needle therefore had to be checked by arthrocentesis with saline solution. The second 20-gauge needle was inserted approximately 0.5–1 cm before the first needle at the same horizontal level but in the posterior-medial direction. After this procedure, successful arthrocentesis with saline solution was performed in all cases. The next step was collecting blood from the jugular vein and injecting 1.0 ml of this blood into the superior joint cavity and 0.5 ml around the articular capsule, followed by wound suture ( Fig. 1 ). The left TMJ served as the experimental joint for blood application and the right TMJ was left without treatment as the control tissue. As it would be difficult to maintain pigs in a sterile environment after the procedure, antibiotics (amoxicillin Bioventa 15% inj. ad us.vet, 15 ml/kg and day, divided into two doses) were administered to prevent infection.
The experimental pigs were killed by intravenous injection of thiopental at four different time intervals after successful autologous blood delivery (1 h, 1, 2 and 4 weeks after treatment). The whole heads were placed on ice and immediately transported for examination by 3T nuclear magnetic resonance imaging (MRI; Siemens Ltd., Siemens Magneton Trio 3T) at the Institute of Clinical and Experimental Medicine (Prague, Czech Republic). After this analysis, both TMJs were dissected, fixed in 10% paraformaldehyde and examined using a stereoscopic microscope (Leica, Germany). After 10 days in paraformaldehyde, decalcification was performed in Livrea’s solution (4% HNO 3 , 0.15% CrO 3 ) for approximately 1 month, after which the specimens were embedded in paraffin, cut into 5 μm sagittal histological sections, and split over four parallel slides. Haematoxylin–eosin (HE) was used as the primary staining for the histological analysis, elastic fibres were visualized by Orcein, reticular fibres were stained with Gömori, and Van Gieson staining was used for the detection of collagen fibres.
In the samples taken 1 h and 1 week after treatment, macroscopic examination revealed deposition of the remaining blood in the form of clots in distal parts of the upper joint cavity. No alterations on the articular surface were observed. 2 weeks after treatment, small blood clots were still apparent in the distal part of the upper joint cavity. 4 weeks after surgery, no remnants of blood, changes or adhesions were apparent inside the TMJ ( Fig. 2 ).
In MRI, the injection injury caused by the needle was visible in the articular disc and surface of the temporal bone 1 h after surgery. There were no apparent morphological changes in the nuclear magnetic resonance (NMR) images when comparing the control and experimental joints ( Fig. 3 ).
Regarding histological analysis, no inflammatory or non-inflammatory morphological lesions were observed at any time after the treatment. The superior and inferior articular spaces showed no sign of joint exudation. The surface of the synovium was covered with small finger-like projections (villi) with no morphological lesions. The fibrous articular discs (menisci) formed fibrocartilage pads containing bundles of elastic and collagen fibres between opposing surfaces of the joint. Fibroblasts were rare and were dispersed among fibres. Articular cartilage covering the condyle of the mandible and temporal bone was formed of hyaline cartilage with no apparent erosion or pathological defects ( Fig. 4 ). The middle and deep layers of cartilage were organized into columns of chondrocytes with normal appearance.