The influence of “Efial” medicine on the chondrocytes functional state

Abstract

Renewal of articular cartilage is a topical issue of modern orthopedics. High frequency of injuries, complexity of clinical diagnosis and subsequent treatment, and also the delay in recovery lead to the development of osteoarthritis, and in some cases, to disability. Articular cartilage belongs to the highly specialized tissues, which is characterized by the lack of blood supply, the low number of cell elements that are placed in the matrix, include collagen, proteoglycans, non-collagenous proteins and water. For the treatment of articular cartilage lesions the medicine which are tissue specific promoters of regeneration are used. The ability of most reparants to stimulate cartilage regeneration combines with other effects, such as: anti-inflammatory, antioxidant and antibacterial. The purpose of administration of these medicines is to stimulate regeneration of tissue in the area of injury.

The aim of research was to investigate the effect of “Efial” medicine on functional state of chondrocytes in cultivation conditions.

Materials and methods. The chondrocytes were obtained from articular cartilage of rats by enzymatic disaggregation. In all experiments the seeding concentration of chondrocytes was 1.2 x 10⁴ cells/cm².The "Efial" medicine in concentration of peptides of 0.137 mg/ml was used. Investigated concentration range was 70; 7.6; 1.5; 0.15µg/ml and 75; 15; 1.5 ng/ml. The medicine was added to the cell culture medium when seeding and on the 3rd cultivation day. The control (comparison group) was the cultures of chondrocytes which were cultivated under the same conditions without medicine addition. Functional state of chondrocytes under interaction with investigated "Efial" medicine was evaluated by the presence of glycosaminoglycans after Toluidine blue staining (Fluka, Germany) and collagen type II (1:200 and FITC-conjugate, Sigma -Aldrich, USA). For statistical study ANOVA and t-Student tests were used with application of Microsoft Excel Programm and Statistica 8.

Results. Adding the medicine at concentrations from 70 µg/ml to 1.5 µg/ml in a nutrient medium at plating and the on 3-rd cultivation day resulted in a reduction of glycosaminoglycans (GAG) and collagen type II content compared to the control (without medicine). Using the test medicine at concentrations from 0.15 µg/ml to 1.5 ng/ml caused the increase of GAG by 17.1±2.3 % (in conditions of adding at seeding) and by 19.4±2.5 (in conditions of adding on 3-rd cultivation day), and the increase in collagen type II content in the chondrocyte culture by 10.3±1.7 % (in conditions of adding on 3-rd cultivation day) and without effect in conditions of adding when seeding (0.1-1.7 x10⁵ cells) versus the control. It should be noted that the use of the studied medicine on the 3-rd day of cultivation led to more pronounced increasing of GAG content in chondrocyte culture compared with the addition of the medicine at plating (in cases of medicine concentrations 7.6 µg/ml - in 2.6 times and 1.5 µg/ml - in 3.3 times). Similar results were obtained in the study of collagen type II content. Namely, in case of medicine concentration of 1.5 µg/ml the investigated parameter increased in 1.5 times, 0.15 µg/ml – in 2.7 times, 75ng/ml - in 2.1 times. This effect of the medicine may be connected with the fact that at the time of medicine addition (on the 3-rd cultivation day) adhesion processes are completed in cells and stimulation of proliferation occurs and as a result the activation of biosynthetic processes in chondrocytes takes plase.

Conclusion. JSC "Farmak" designed the "Efial" medicine as a spray based on concentrate of deproteinised dermal layer of pig skin and phosphatidylcholine from soybeans to heal wounds of various etiologies. The results of influence of the "Efial" medicine on functional state of chondrocytes is the basis for further study of this medicine efficiency to treat a damaged articular cartilage.