FEATURES OF FORMATION OF BONE CALLUS AT THE BACKGROUND OF CONTACT LOW-FREQUENCY ULTRASOUND IN THE CLINIC AND IN THE EXPERIMENT

FEATURES OF FORMATION OF BONE CALLUS AT THE BACKGROUND OF CONTACT LOW-FREQUENCY ULTRASOUND IN THE CLINIC AND IN THE EXPERIMENT

Reznik L.B., Rozhkov K.Yu., Dzyuba G.G., Zavodilenko K.V.

Omsk State Medical University, Medical and Sanitary Unit No.4, Omsk, Russia

 The modern technologies and approaches to treatment of patients with fractures of long bones prevent the disorders of reparative regeneration of bone tissue in some cases [1]. However the presence of an opened fracture, which always predisposes to regeneration disorders, significantly limits the choice of methods for stimulation of osteogenesis. The use of the external fixation device (the gold standard of treatment of opened fractures of long bones) does not solve all problems [2]. Osteogenesis stimulation is required in some cases [1-3].
One of the ways of osteogenesis stimulation is low frequency ultrasound. The positive influence of ultrasound on bone tissue regeneration in conservative [3-6] and surgical [7, 8] treatment of fractures of long bones has been proved. Some mechanisms of ultrasonic impaction have been studied: neoangiogenesis activation [9, 10], growth factors [11, 12], influence on osteoblasts and osteoclasts [13, 14]. Besides influence on bone tissue, low frequency ultrasound makes the positive influence on soft tissue regeneration [15-17] that is especially important for treatment of opened fractures. The feature of ultrasonic waves is their weakening when passing through soft tissues [18]. So, the maximal efficiency is observed in direct influence on a bone [14, 19]. The international literature describes the use of low frequency ultrasound for osteogenesis stimulation, determines the mechanisms, but does not describe any data of features of callus formation under ultrasonic impact, particularly, in direct impact on a bone.
Objective – to study the features of formation of the bone regenerate in the experiment in and the clinical course for estimation of efficiency of influence of contact low-frequency ultrasound impact on union of opened fractures of long bones in conditions of extrafocal transosseous osteosynthesis.

MATERIALS AND METHODS

The study included the experiment with animals and the clinical examination.
The experimental examination included 16 outbred pubertal dogs (body mass of 24.2 ± 3.2 kg). The study was approved by the ethical committee of Omsk State Medical University. The animal management and the experimental study corresponded to the order of Health Ministry of USSR (August 12, 1977, No.755) and the claim of European Convention for the Protection of Experimental Animals (Strasbourg, 1986).
The animals were distributed into two groups (8 animals in each group): the main group and the comparison group. All animals received the modelling of an opened fracture of humerus diaphysis in the middle one-third and with 2 mm bone tissue defect. The fracture modelling was performed from the longitudinal lineal incision along the anterior surface of the shoulder with use of the cutter. Osteosynthesis was conducted with the monolateral rod device with 4 cannulated nail-screws (two in each fragment) which were fixed on the plate. The outside nail-screws were fixed in two cortical layers of the bone, and the nail-screws near the fracture – in one cortical layer. 2 mm diastasis between the fragments was observed after osteosynthesis. The surgical intervention was conducted under intravenous general anesthesia: 1 ml of xylazine hydrochloride per 10 kg of body mass, 1 ml/h tiletamine hydrochloride per 10 kg of body mass. The animals of the main group received the contact influence of low frequency ultrasound with introduction of the wave guide through the perforated hole of the cannulated nail-screw into the cavity of the intramedullary canal until reaching the contralateral cortical layer [20]. The impaction was performed with Tonzilor M device with the ultrasound parameters of 26.6 kHz and 50 W. The radiologic examination of the humerus with Radrex (Toshiba, Japan) device was carried out on 41st day. The experiment was completed on the day 42 and corresponded to the euthanasia standards (Zoletil 10 ml, i.v., by stream infusion, after narcosis with KCl 20 % - 20 ml, i.v., by stream infusion). The histological examination of the bone regenerate with hematoxylin and eosine was conducted. The results of the treatment were conducted from the moment of appearance of support to the operated extremity till disappearance of abnormal mobility after removal of the bar of the external fixation device.
The clinical study included 24 patients of young age according to WHO classification (age of 25-44) with opened fractures of the leg of 42A2-3; В2-3 (АО, ASIF) 2-3А types according to Gustilio and Anderson within 24 hours after trauma. With the random numbers table generated in Statistica 6.1, the patients were divided into two equal groups – the main group and the comparison group (12 patients in each group). The groups were similar according to gender and age. All patients received osteosynthesis of the leg with the external fixation device. The cannulated nail-screw was introduced into the distal end of the proximal fragment over the distance about 25 mm after achievement of reposition. The nail-screw was fixed to the ring of the device with use of the bracket. The patients of the main group received low frequency ultrasonic therapy with the technique similar to the animal experiment from the 2nd day after surgery, 1 time per 3 days [20]. MSCT was conducted with GE 750 HD Discovery in the mode of dual energetic scanning with assessment of callus density (Hounsfield units) and calcium level, with use of software in the mode of GSI in mg/cm3. The device was dismounted if positive clinical trends were found.
The analysis of results with Shapiro-Wilk method showed the distribution, which differed from the normal one. Therefore, the techniques of non-parametrical statistics were used for estimation of the results: median, interquartile range, Mann-Whitney test. The binary signs were compared with Fisher’s test and frequency analysis method. The calculations were made with Statistica 6.1 (the license No. BXXR904E306823FAN10). The ethical committee of Omsk State Medical University approved the study. All patients gave their written consent for participation in the study and analysis of personal data.

RESULTS AND DISCUSSION

Experimental study

The animals demonstrated the disappearance of abnormal mobility on the day 33 [33; 31] in the main group and on the day 39 [40; 39] in the comparison group (p = 0.0009). Micromobility was in 4 animals. On the day 41 of the experiment, the analysis of X-ray images gave the following results: all animals in the main group achieved the union (the line fracture was not visualized, there was sufficient periosteal callus) (Fig. 1). The comparison group: 5 animals did not show any signs of consolidation according to X-ray examination – the fracture line was visualized, periosteal callus was weak (Fig. 2); 3 animals showed weak consolidation (the fracture line visualization, a periosteal response) (p = 0.0035).

Figure 1. X-ray of the operated limb (main group) on 41st day: 1 – external fixation; 2 – callus



Figure 2. X-ray of the operated limb (comparison group) on the 41st day: 1 – fracture line; 2 – external fixation

The histological characteristics of the bone regenerate were as described below. In the main group of the experimental animals, the regenerate was presented by the formed bone rods with mature structure and dense homogenous matrix and non-active osteoblasts (Fig. 3). The morphological picture was homogenous along the whole regenerate. The experimental animals of the comparison group showed the regenerate on the basis of cartilaginous tissue with unconsolidated matrix and inhomogeneous density. The spaces between the rods were formed and filled with loose fibrous tissue with thick-wall new vessels (Fig. 4). The more distance from the periosteum, the less mature regenerate: near the periosteum, the forming bone rods are more pronounced, in the depth – cartilaginous tissue.

Figure 3. Regenerate on the 42nd day, the main group, staining with hematoxylin and eosin, an increase of 200: 1 – inactive osteoblast; 2 – formed bone beams; 3 – homogeneous matrix



Figure 4. Regenerate on the 42nd day, comparison group, staining with hematoxylin and eosin, an increase of 200: 1 – forming inter-beam spaces; 2 – periosteum; 3 – inhomogeneous matrix; 4 – proliferating osteoblast

The clinical study

According to MSCT data, the callus density was 686 HU [702; 595] in the main group, 465 [494; 424] (p = 0.0003) HU in the comparison group on the 12th week after the surgery. The level of calcium in the unit of volume was 344 mg/cm3 [387; 300] in the main group, and 198 mg/cm3 [214; 187] in the comparison group (p = 0.0003). The callus was even and had the equal density along the whole length in the main group. The comparison group showed the higher density in the proximal and distal part of the oblique fracture in the sagittal images as compared to the central part of a fracture (Fig. 5).

Figure 5. Callus in patients on the 12th week after surgery: 1 – the main group; 2 – comparison group

The device was dismounted after 14weeks [13; 14] in the main group and after 16 weeks [17; 16] in the comparison group (p = 0.004).
Therefore, the offered technique of formation of an opened fracture in the experiment (with a bone tissue defect) and the chosen technique of osteosynthesis (the statistical model, without possibility of compression) are unfavorable for consolidation. Without additional stimulation, the comparison group did not show any clinical, radiological or histological signs of consolidation. The callus is formed on the basis of cartilaginous tissue with initiation of processes of enchondral osteogenesis. The process of enchondral osteogenesis is more pronounced in the periosteal region, and in the depth, the regenerate is mainly the cartilaginous tissue. In the main group, despite of factors unfavorable for consolidation, all animals showed complete consolidation confirmed by histological and radiological examination. Enchondral osteogenesis was completed, and the regenerate with bone tissue structure formed. The beginning of enchondral osteogenesis is associated with vessels budding into cartilaginous tissue of a forming regenerate [6]. The improvement in microcirculation in the injury site is the mechanism explaining the stimulating effect of ultrasound [6, 10].
The clinical findings show that ultrasonic impaction improves the fracture union in presence of the factors predisposing to disordered course of the reparative process. Low frequency ultrasound stimulates the callus with high optical density and high level of calcium, but such trends are not observed in patients without ultrasonic impaction. It can be estimated as the radiological equivalent of enchondral osteogenesis – the higher level of calcium is determined by maturity of bone tissue of the regenerate. The mechanism can be associated with improvement in microcirculation in the injury site (earlier formation of callus in the region of disordered vascularization after a fracture) and is explained by early ingrowth of vessels into the injury site. Moreover, the high volume of callus is explained by creation of micromobility in the injury site under influence of low frequency ultrasound. 

CONCLUSION

1. The experimental contact low frequency ultrasonic impaction provides the formation of clinically and structurally mature callus in presence of the factors predisposing to disordered consolidation (diastasis between fragments, absence of compression between fragments).
2. In conditions of extrafocal transosseous osteosynthesis, contact low frequency ultrasound provides 1.14-fold decrease in union time by means of callus with 1,43-fold higher density, with 1.76 times higher level of calcium per the unit of volume. 

Information on financing and conflict of interests

The study was conducted without sponsorship.
The authors declare the absence of any clear or potential conflicts of interests relating to publication of the present article.

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