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Главная > № 4 (2017) > Ахтямов

X-RAY AND MORPHOLOGICAL PARALLELS OF THE OSTEOREGENERATIVE PROCESS AFTER USING THE AGENT BASED ON ION LANTHANIDE ETIDRONATE

Akhtyamov I.F., Zhitlova E.A., Tsyplakov D.E., Boychuk S.V., Shakirova F.V., Korobeynikova D.A.

 Kazan State Medical University, Kazan State Academy of Veterinary Medicine, Kazan, Russia

Closure of defects and damages of bone tissue presents the quite longand multi-staged process. The search for osteogenesis-stimulating substances is the important task in the modern traumatology and orthopedics. This complex problem has not been solved with significant progress in use of osteoinductors and osteoconductors for recovery of bone defects [1-4]. Complex compounds including some mineral components (such as organic stimulators of osteoblast function) are the perspective measures for bone reparation activation. In this regard, one should mention a new patented agent of the group of nitrogen-free bisphosphonates (etidronate) including lanthanide-ions and calcium ions [5]. The etidronate is the disodium salt of 1-hydroxyethane 1,1-diphosphonic acid, which is the derivative of etidronic acid.
Etidronic acid (H4L) falls into the group of bisphosphonates and is used in medicine for prevention of excessive calcium discharge from the bones and pathologic calcification in soft tissues. Owing to high similarity with phosphates after addition of the complexes of lanthanide ions to hydroxyapatites forming the basis of bone tissue, they closely bind to the minerals and do not influence on the structure of hydroxyapatites. Lanthanides suppress the development of cells (osteoclasts) responsible for bone tissue resorption. This ability to imitate the functions of calcium ions allows modelling their behavior by means of lanthanide ions, as well as using lanthanides as the components for treating the bone tissue diseases.
It is considered that gadolinium (III) is a “paramagnetic probe”, which models the behavior of calcium in the biosystems in absence and in presence of etidronic acid regulating the calcium metabolism. It is important to match the behavior of these ions and identification of similarity and differences in chemical quality (stoichiometry and stability) of complex formation with etidronic acid. The unofficial name of the drug is Inrok.
The primary publications showed the potential ability to influence on the reparative processes in bone tissue [6, 7].
The objective of the study – to study the effect of the agent based on ion lanthanide etidronate and calcium on the process of osteoregeneration in the experimental model of the tibial defect.

MATERIALS AND METHODS

The experimental project was the agent with the following chemical composition and the ratio of the ingredients:
1. Etidronic acid monohydrate – 1.8
2. Calcium chloride dihydrate – 1.44
3. Gadolinium (III) nitrate hexahydrate – 0.30
4. Dysprosium (III) chloride hexahydrate – 0.038
5. Water for injections
6. Solution pH 7.3-7.8.

Description. The agent is the water suspension of white color with pearl shade.
The study included 36 rabbits (age of 6-10 months, body weight of 2,500-2,800 g). The experiment included the animals, which were kept in the conditions of the vivarium. The animal management and care corresponded to the requirements of GOST ISO 10993 (R). The reparative osteogenesis was estimated in the experimental model of a non-penetrating defect in the medial surface of the proximal tibial bone [8] with use of neuroleptanalgesia (Rometar 2 %: 0.15-0.2 ml/kg; Zoletil 100: 10-15 mg/kg). The surgical approach was made two cm lower than the femoral-tibial junction. The drill was used for making a hole in the single cortical layer of 3 mm. The wound was sutured with intermittent knotted sutures.
The republican ethical committee of Kazan State Medical University gave the permission for the study (the session protocol No.9, November 25, 2014).
The agent was introduced into the modelled defect of the tibial bone of the rabbit on the third and the fifth day after surgery. The dosage was 0.2 ml in the experimental group (n = 18). The comparison group did not receive the agent (n = 18).
The X-ray images for estimation of time course of changes and degrees of intensity of the reparative osteogenesis in the defect field were conducted in the end of the first, the second and the eighth weeks of the experiment using the device 9LU2*. The exposure time was 0.1 sec. with the distance of 70 cm and current of 20 mA. At the same time, the morphological analysis of the tissue in a perforative hole and of the boundaries of the defect was conducted. The histological material was fixed in 10 % neutral formaline, with further decalcification with a well-known technique [7, 9], dehydration and addition of paraffine. The histological sections of 5-7 µm thickness were made with the microtome Leica SM 2000R with hematoxylin-eosin and picrofuxine (van Gieson) staining. The morphometric method was used for quantitative estimation of the squares of the structures [10]. The calculation was in percentage to the general square of a histological section [11].
The statistical analysis was conducted with SPSS 13.0. The normalcy of distribution was estimated with Kolmogorov-Smirnov test. Student’s test was used for comparison of the values in two groups. The variance analysis was used for comparison of the values in three groups and more. The subsequent intergroup comparison was conducted with Student’s test with Bonferonni adjustment. The differences were statistically significant with p < 0.05. The data was presented as M ± m, where M – mean arithmetic, m – standard error of mean.
Along with X-ray and morphometric analyses, the osteoblast activity of the drug was performed with MC3T3-E1 Subclone 4 (ATCC® CRL-2593™). The control measure was the solution of melatonin (50 nm/l). The qualitative estimation of the compound was conducted according to the general protocol with use of the commercial set In Vitro Osteogenesis Assay Kit (Millipore, USA) (Cat. ECM810). MC3T3-E1 line cultivation was conducted in the complete cultural media alpha-MEM (Gibco, USA) up to achievement of cellular confluence with subsequent passaging in 24-basin flat-bottomed tray (Corning, USA). TC-20 automatic counter was used for calculation of cells in suspension (BioRad, USA). In vitro examination of osteoblast activity of the agent was performed with the common techniques using In Vitro Osteogenesis Assay Kit (Millipore, USA) and the cell line MC3T3-E1 from the American bank of ATCC cellular cultures. The set and the above-mentioned cell line have been used for in vitro estimation of osteoblast activity of substances for many years. Melatonin was in this set and was used by us (according to the protocol) as the positive control, i.e. the inductor of osteoblast activity in the examined cell line. Therefore, there were the cell cultures incubated with the various concentrations of the substance, and the cell cultures incubated with melatonin (the positive control) and without introduction of any substances (the negative control).
We have shown that the osteoblast activity of the compound increased the values of the positive control within the levels of 500 µm and 1 mM. Moreover, an evident dose-depending effect was identified (induction of osteoblast activity). The conclusion was made that one of the possible molecular mechanisms of action of the studied compound was its ability to increase the activity of osteoblasts in the injury site.
After achieving cellular confluence, the complete nutritional media on the basis of Alpha Minimum Essential Medium was replaced by Osteogenesis Induction Medium. After six days of cultivation of MC 2T3-E1 cell line, the cultural media was replaced by Osteogenesis Induction Medium with solution of the examined compound with levels of 1, 10, 100, 250, 500 and 1,000 µm. After completing six days of cultivation, the qualitative response to the osteoblast activity was estimated with alizarin. After completion of incubation, the basins were washed with d water (4 times), and optic microscopy was conducted.

RESULTS

The rabbits showed good tolerance to general anesthesia and surgery. Motion activity restored 30 minutes after surgery. The animals could eat 5 hours later. The postsurgical wounds healed with primary intention in all cases. Wound infection, allergic reactions and other complications were not identified.
The X-ray examination showed the perforative holes in the upper parts of the tibial bones with smooth and clear borders in both groups (Fig. 1a).

Figure 1. The X-ray image and photomicrography (x400) of the defect in the animal of the experimental group 7 days after surgery: a) a perforative hole of the shin bone; b) granulation tissue. The osteoblasts are marked with the arrows. Hematoxylin and eosine staining.

  

The microscopic analysis showed the unfilled defect in the experimental group at that stage of the experiment (15.9 ± 1.4 %, p = 0.003). Traumatic edema was absent at that stage, or it was insignificant. The vessels were extended and full-blooded. The blood clots were organized in the field of the defect. Granulation hematoma was present in each case at the background of hematoma organization. The square of granulation tissue was 70.6 ± 1.1 % (p = 0.02), in the comparison group – 53.6 ± 3.1 % (Fig. 1b). Osteoblasts were visualized along with new vessels and mesenchymal cell elements. Inflammatory response was only in individual cases, where insignificant macrophageal infiltration was present. Insignificant leukocytic necrotic masses were in two cases in the experimental group: 5.5 ± 0.8 % (p = 0.001). They had the lower square than in the comparison group (14.6 ± 1.4 %).
In the comparison group, the square of the unfilled perforative hole was 27.6 ± 2.1 %. Anatomically, the residual events of reactive processes (exudative inflammation) as consequences of trauma were evident. Infiltration by polymorphic nuclear leukocytes was present to a greater or lesser degree, followed by macrophageal infiltration.
In some cases in the comparison group, some necrotic changes with desolate cavities of osteocytes and calcification foci appeared. Totally, leukocytic necrotic masses occupied 14.6 ± 1.4 % of the square of the section. In most cases, the process of regeneration with proliferation of blood vessels and migration of fibroblasts, which were between vascular slings, began. Granulation tissue appeared. Its square was not more than 53.6 ± 3.1 % (the table). One case had the inflammatory process with extensive leukocytic necrotic masses. The signs of reparation were absent.

Table. The squares of structural components filling up the perforative hole at different time points of the experiment (the percent of total square of histological section, М ± m)

	7th day		14th  day		28th day		56th day
	Comparison group (n = 4)	Experimental group (n = 4)	Comparison group (n = 4)	Experimental group (n = 4)	Comparison group (n = 4)	Experimental group (n = 4)	Comparison group (n = 4)	Experimental group (n = 4)
Non-filled perforative hole	27.6 ± 2.1	15.9 ± 1.4*	10.4 ± 0.7	1.5 ± 0.7*	-	-	-	-
Leukocytic-necrotoc masses and blood clots	14.6 ± 1.4	5.5 ± 0.8*	2.2 ± 0.6	0.6 ± 0.6	-	-	-	-
Granulation tissue	53.6 ± 3.1	70.6 ± 1.1*	17.2 ± 2.1	6.7 ± 1.1*	1.5 ± 0.3	0.4 ± 0.2*	-	-
Connective tissue	4.1 ± 1.2	8.0 ± 0.9	48.2 ± 0.6	68.0 ± 2.5*	7.3 ± 1.1	3.6 ± 0.6	-	-
Cartilaginous tissue	-	-	16.1 ± 1.5	5.1 ± 1.3*	4.0 ± 0.9	0.6 ± 0.1	4.2 ± 0.2	0.4 ± 0.2
Membrane reticulated bone of beam structure	-	-	6.0 ± 1.0	18.2 ± 0.6*	86.0 ± 1.9	92.2 ± 0.9	7.1 ± 0.4	0.8 ± 0.1
Lamellar bone	-	-	-	-	1.2 ± 0.3	3.2 ± 0.4*	88.7 ± 0.6	98.8 ± 0.2*

Note: * – statistically significant differences in values between the groups.

In the end of the second week of the experiment, the square of the perforative defect decreased 2.65 times in the comparison group. Growth of collagen fibers and process of osteogenesis began in each case, where granulation tissue appeared at the previous stage. At the background of collagen homogenization, the bone rods developed, with loose areolar connective tissue filling the space between them. Transverse bridges appeared between some bone rods. Proliferating osteoblasts were found. Connective tissue took the general square of 48.2 ± 0.6 %. Necrotic masses (also in bone tissue) almost resolved.
The blood vessels grew into the Haversian canals on the edges of the defect. In some cases, bone formation happened by means of development of cartilaginous tissue, which was presented by small regions or extensive fields of connective tissue. The cartilage square was 16.1 ± 1.5 % for this time interval. Two rabbits had the persistent inflammatory processes with cellular infiltration and focal tissue necrosis, but their square was not high (2.2 ± 0.6 %). One case had some necrotic changes in the adjacent periosteal tissues.
At the same time, the main group showed the decreasing proportion of granulation tissue and the increasing volume of connective tissue: 6.7 ± 1.1 % (p = 0.004), 68 ± 2.5 % (p < 0.001) correspondingly. Three cases were associated with formation of rough fibrous tissue, which rods were connected to bone tissue of the defect’s borders. The square of rough fibrous bone tissue was 18.1 ± 0.6 % (p < 0.001). The defect had almost closed by that time, and its square was only 1.5 ± 0.7 % (p < 0.001). Formation of the cartilage was minimal and did not exceed 5.1 ± 1.3 % (p = 0.001). The inflammatory response was absent or minimal, with background reparative processes.
The bone defect of the tibial bone was almost closed in most cases in the end of the fourth week. The animals of the comparison group had the rough fibrous bone, with its square of 86.0 ± 1.9 %. The trabecules showed the partial resorption on the borders of the former hole. Lamellar bone formation began, but the volume of the bone was still insignificant (1.2 ± 0.3 %). When cartilaginous tissue formed during the process of the defect healing, its resolution, calcification and replacement by bone tissue happened. The signs of inflammatory response were absent. Some cases included the defect closing with presence of cartilaginous tissues on the borders (without ossification) and immature rough fibrous tissue bone.
At that period of observation in the experimental group, the perforative hole was replaced by the rough fibrous bone (92.2 ± 0.9 %) with diffuse calcification of the rods. The bone transformed to the lamellar bone 2.66 times more often than in the comparison group (Fig. 2b). The cartilaginous tissue was minimal (0.6 ± 0.1 %).

Figure 2. The X-ray image and photomicrography (x400) of the defect in the animal of the main group 28 days after surgery: a) a perforative hole of the shin bone; b) transformation of cartilaginous tissue into a rough fibrous bone. Hematoxylin and eosine staining.

  

TheX-ray examination showed the bone defect with non-uniform wideness of sclerosis zone (1-2 mm) and local hyperostosis in the animals in both groups. In the comparison group, the borders of the hole usually had the unsmooth internal contours. The experimental group showed some signs of periosteal response at the level of the defect’s boundaries (Fig. 2a).
In the end of the experiment (the eighth week of observation), most animals of the comparison group demonstrated the lamellar bone (88.7 % ± 0.6 % of the section). But some individual cases showed the fragments of rough fibrous bone (7.1 ± 0.4 %), the regions of resolution of cartilaginous tissue with ossification events and presence of necrosis foci and destruction of the cartilage and the bone. Generally, cartilaginous tissue remained only on 4.2 ± 0.2 % of the perforative hole.
The complete uncomplicated recovery of the bone defect was diagnosed in the experimental group at that time. The lamellar bone (the square of 98.8 ± 0.2 %, p < 0.001) appeared at the place of the perforative hole. It had the evolved system of Haversian canals and restored surrounding periosteal tissues.
The X-ray examination did not show any complete replacement of the bone defect in both groups at that time. The contours of the hole were unsmooth, with non-uniform wideness of sclerosis zone (1.5-3 mm) and local hyperostosis (0.5 mm). Therefore, objective estimation of the X-ray images did not identify any significant changes between the animals of the experimental and comparison groups.

CONCLUSION

1. Dosed administration of the agent with etidronate of lanthanide ions and calcium is efficient already in early stages of recovery of small bone defects. The feature of the agent (or its specificity) is the complex influence: 1) decreasing intensity of inflammation; 2) increasing reparation; 3) influence on osteogenesis, which is direct in most cases.
2. The efficiency of this compound can be based on increasing osteoblast activity of cellular elements in the region of the injury.
3. Scientific novelty of the studies consists in the fact of the first appearance of the injectable form of the agent based on etidronate of lanthanide ions and calcium with osteoinductive properties. The complex approach showed that introduction of the agent did not cause any acute inflammatory responses in tissues in the region of introduction. The morphometric (morphological) studies showed that the first seven days after initiation of the defect in the animals of the experimental group resulted in formation of granulation tissue with subsequent formation of reticulofibrous and lamellar bone.
4. On the basis of the received data of specific activity of the agent containing the lanthanide ions and calcium in the model of partial bone injury, one may make a judgment on the possible perspective of its clinical use.

Information about conflict of interests

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

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