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Comparative study of the changes of the blood composition in rabbits utilizing coated and non-coated metal implants for osteosynthesisA. Yousef, I. Akhtyamov, F. Shakirova, L. Zubairova, E.Gatina, E. AlievNew MowasatHospital – Department of orthopedic surgery, 22077 P.O.BOX 6661Salmiya.Kuwait.Kazan State Medical University, Kazan, Butlirova St. 49, P.O.Box 420012 – Russia., Kazan State Veterinary academy. Kazan, Sibirsky Road-35, P.O.Box 420029- RussiaAddress correspondences to Dr. Yousef Ashraf.,MD,PhD- NEW MOWASAT HOSPITAL, 22066, P.O.Box 6661, Salmiya, Kuwait. Tel +96599060174 aismail@Running title: osteosynthesis with coated metallic implants.Declaration of conflict of interest: None.Authors :Yousef Ashraf., - NEW MOWASAT HOSPITAL, 22066, P.O.Box 6661, Salmiya, KuwaitTel +96599060174aismail@Akhtyamov Ildar., Kazan State Medical University, Kazan, Butlirova St. 49, P.O.Box 420012 – Russia.yalta60@mail.ruShakirova Faina., Kazan State Veterinary academy.Kazan, Sibirsky Road-35, P.O.Box 420029- Russiashakirova-fv@yandex.ruZubairovaLaily., Gatina Elmira., AlievElchinKazan State Medical University, Kazan, Butlirova St. 49, P.O.Box 420012 – Russia.yalta60@mail.ruAbstractThe authors conducted an experimental study on using implants (pins) coated with titanium and hafnium nitrideon blood composition of the rabbits before and after osteosynthesis. On the control group we did osteoclasis followed by ostesynthesis with metallic pins (non-coated). The study revealed that the reactive phase of healing after osteotomy and osteosynthesis was short-term, and issomehow less pronounced when using implants coated with titanium and hafnium nitride compared with implantation of non coated steel. In the experimental group, inflammatory markers such as CRP, ESR, and the relative increase in the granuloytes count, were normalized in a shorter time than in the control group. Post-operative inflammation, providing systemic and local regulation of humoral and cellular mechanisms involved in restoring the damaged bone is not accompanied by complications during implantation of implants coated with titanium and hafnium nitride. IntroductionThe use of medical implants increases with each decade due to an increase in life expectancy, changing of lifestyle and improvement in technology of production. [1].Mechanical and biological properties of these implants must be optimal for a fast and strong links with the surrounding tissue. The interaction between the device and the surrounding tissue is important as it determines the success or failure of implantation, in this regard;coatedimplants should provide surface hardness, corrosion resistance, low coefficient of friction and wear resistance.When developing new coating implants with improved functionality, we should take into account the biological tolerance [2]. Along with widely used Titanium Nitride coating, a technology of coating implants with Hafnium Nitride was developed, but a few researches were done for this type of coating.[3, 4, 9].The aim of the study: A comparativestudy of the changes in the composition of blood in experimental and control groups of animals when using implants coated withtitaniumand hafnium nitride.Materials and methods The experiment, content and care, all were done according to the requirements of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes "(1986). Studies have been conducted on 30 rabbits of both sexes, at the age of 6-7 months, weighing 2526.5 ± 74.4 grams.All the animals underwent open osteotomy of the tibia in the middle and lower thirds of the diaphysis with subsequent retrograde nailing. In the comparison group (15 rabbits ) a steel medical pins 12HI8H9T, dх2 mm, were used; in the experimental group (15 rabbits) we used medical steel pins 12HI8H9T,dх2 mm; coated biologically with inert titanium andhafnium nitride.The coating process consistsof ion-plasma coating with electron arc evaporation (EAE). As the coating thickness within 10 ? 3 mμ, then it can be classified as a nano-coating.Blood collection was performed by venipuncture before surgery and on the 1st, 5th 10th, 20th, 30th and 60th day after surgery, determining the level of C - reactive protein (CRP) using a semi-quantitative test based on latex – agglutination, company ?HUMAN? (Germany). Each animal on the stages of the study (prior to surgery, on the 1st, 10th, 20th, 30th, 60th day) was assessed: erythrocyte sedimentation rate (ESR), white blood count (WBCs) with leukocyte formula, hemoglobin (Hb) and red blood cells (RBCs) by standard methods.Statistical processing was performed using thepackageSPSSv.13.ResultsComparative analysis of the research results regarding systemic response of Medical steel implants (control group) andbioinertimplantscoatedwith titanium andhafnium nitride (experimental group) revealedraised CRP levels in both groups, starting from the first day, reaching a significant differencefrompreoperativevalues onfifthday (Table 1).The CRP of the control group exceededthe initial levelby 9.6 times (p=0.001), as in the experimental groupwaslowerandtheCRPlevelexceededthe pre-operative value only by 4.9times (p=0.004).CRP dynamicsalsodiffered: on the 10th day after the intervention, the rate of CRP was decreased, morepronouncedin the experimental group compared with the control group, by 3.3and 4times, respectively.Slight increasingrate wasmaintained in the control group up to 30th dayandby theday 60, amarkedsignificant decrease was noticed (p= 0.003), whilethe absolute values?ofCRPremainedabovebaselineby 1,3 times. In the experimental group, starting day 20, a dynamicdecrease in CRP rate was noticed, indicating a less pronouncedreactionof the rabbitsto the use of implants coatedwith titanium and hafnium nitride.In General, the revealed dynamics of CRP levels is consistent with the data of other authors, also noticed increased CRP levels in the early post-operative period. (Figure 1) [5,6].Erythrocyte sedimentation rate (ESR) is increased on the next day after surgery in both groups. In the comparison group, this figure was significantly higher than its pre-op level by 3.7 times (p = 0.024), and in the experimental group by 4.7 times (p = 0.022). In the subsequent periods the value of ESR gradually decreased and on the 10th day in both groups exceeded the original data only by 2.3 times. On the 30th day, ESR rate of experimental group was down to 1.7 times, while the comparison group did not change.By the end of the period of observation, ESR rate in both groups was compared with baseline values prior to the intervention.Minor variations of hemoglobin concentration in peripheral blood in animals of both groups throughout the experiment were within the physiological norms for this type of animal. We also did not observe statistically significant changes of RBCs during the experiment; meaning, surgery and implants have not been accompanied by the development of anemia (table. 2).Practically the entire match of dynamics of CRP levels and ESR after osteotomy and osteosynthesis allows considering the pathogenitic relationship, since it is known that desproteinemia, growing by increasing the level of globulin, accompanied by an acceleration of ESR. (Figure 2)The WBCs in the peripheral blood of experimental animals during the whole experiment did not change significantly; slight variations do not go beyond the physiological norm. At the same time, there has been a change in the composition of white blood cells.It was noticed a rise in banded neutrophilscounton the first day in thecontrol group 2.2 times, and in the experimental reliably in 2.4 times (p = 0.024) in relation to the initial indicators. Later, during the experiment, neutrophils were still rising up, while in the experimental group and by the 10th day, the value of neutrophils was almost the same as in pre op stage.The segmented neutrophils count in both groups was increased significantly (p = 0.001) in 1.7 times in thefirst 24 hours. In subsequent periods the figures in both groups steadily decreased: in the 10th day: control group - 1.4 times (p = 0.028), in experimental group - 1.8 times (p = 0.001); in the 30th day: control group - 1.6 times (p = 0.003), experimental group - 1.7 times (p = 0.001); and in the 60th day: experimental group - 2.3 times (p = 0.001), and in the control group - 1.8 times (p = 0.013).The lymphocytes count in the first 24 hours was significantly below the pre op values, in the control group for 31% (p = 0.001) in the experimental group - 36% (p = 0.001). Later on, indicators significantly increased compared to the first days after surgery. In the control group at the 10th day the rise was 22% (p = 0.021), 30th day- 25% (p = 0.019), 60th day increase up to 27% (p = 0.042). In the experimental group in the 10thday -36% (p = 0.001), 30th day- 35% (p = 0.001), and the 60thday-42% (p = 0.001)respectively.Numberofmonocytes inthecontrolgroup wasreduced bythe 10thdayaftersurgery. Subsequent growth restored on the initial level by the 30thday.In the experimental group showed a significant and sustained reduction in the number of monocytes from the day 1 to the day30(p =0.021).Osteotomy and the introduction of foreign material into the bone tissue is accompanied by multiple body response, including reactive inflammatory phase and the development of granulation, which takes days; reparative phase, lasting for weeks and bone remodeling phase lasting up to several months or years [7]. We observed changes in the composition of leukocytes, which showed relatively rise in granulocytefraction in the first days after surgery, alternating with the relative increase in mononuclear cell that reflects the change in the dynamics of acute phase inflammatory response (Table 3).A separate discussion in our view deserves a decrease of monocytes counts in the experimental group during the first month of observation.One explanation may be the mobilization of monocyte/macrophage lineage from the blood stream to the bone remodeling andreparation's area, as they considered as predecessors of osteoclasts [8]. (Fig 3)There has been no certain change in eosinophils and basophilscount during the experiment that may be indirect evidence of absence of hyper Eric reaction on the material implants. (Fig 4)Minor variations of hemoglobinconcentration in peripheral blood in animals of both groups throughout the experiment were within the physiological norms for this type of animal. We also did not observe statistically significant changes of RBCs during the experiment; meaning, surgery and implants have not been accompanied by the development of anemia (table. 2).Conclusion: The experiment showed, that after osteotomy and osteosynthesis, the reactive phase was short-lived, and is somewhat less pronounced when using implants of medical steel coated with biologically inert Nitride titanium and hafnium compared with implantation of non-coating steel. In the experimental group of animals,inflammatory markers such as CRP, ESR, and the relative increase in the number of granulocytes in the blood, normalized in a shorter time than in the control group. In our experiments we did not reveal pathological changes of the eosinophilic and basophylic granulocytes and also red blood cells. That means, post-operative inflammation, causing systemic and local regulation of humoral and cellular mechanisms involved in restoring the damaged bone is not accompanied by complications during implantation steel coated with nitride titanium and hafnium.References: BarrèreF.,MahmoodT.A., deGrootK., VanBlitterswijkC.A. Advancedbiomaterialsforskeletaltissueregeneration: Instructiveandsmartfunctions. Mater. Sci. Eng. Rep. - 2008, - 59, - P. 38–71.Anderson, J.M., Rodriguez, A., Chang, D.T. Foreign body reaction to biomaterials. Semin.Immunol. - 2008, - 20, -Р. 86–100.Chen Y., Laha T., Balani K., Agarwal A. Nanomechanical properties of hafnium nitride coating // ScriptaMaterialia, 2008, 58. - Р. 1121–1124. Abdulin I.S., Mironov M.M., Garipova G.I. Resistant bacterial and biological coating for medical implants and instruments.// Med. Technika 2004-N4, P 20-22.Orrego L. M., Pérez C. M., Pérez Y. M., Cheyre E. J., Mardones P. R. Plasma C reactive protein in elective orthopedic surgery // Rev Med Chil. - 2005, - Vo1.33 (№11): -Р. 1341-1348; Lee D.Y., Cho T.J., Lee H.R., Park M.S., Yoo W.J., Chung C.Y., Choi I.H. Distraction osteogenesis induces endothelial progenitor cell mobilization without inflammatory response in man // Bone. - 2010, - Mar; 46 (3): - Р.673-679. EinhornT.A.. One of Nature's Best Kept Secrets // Journal of Bone and Mineral Research – 1998.– Vol. 13, Issue 1, P. 10–12.Roodman G.D.: Cell biology of the osteoclast. ExpHemato– 1999.– Vol. l27 : P.1229– 1241.Akhtyamov I.F., Shakirova F.V., Gatina E.B., Zubairova L.D. and others. //Vestniktravmatologii and orthopedii named by N.N. Priopov 2013, N2.- P 60-64.Figure legendsFigure -1 The dynamics of CRP during the period of experimentFigure 2. Dynamics of ESR during the experiment Figure 3.dynamics of monocytes from day 1 to day 60Figure- 4.dynamicseosinophils and basophilsTable 1.Change of serum CRP concentration (mg/l).daysgroupPre Op.Day 1Day 5Day 10Day 20Day 30Day 60Control group17,6±3,1128,0±14,6***169,6±24,6***72,0±14,1###74,0±21,2##84,6±20,4#41,4±14,8##Experimental group22,9±6,594,4±17,1*113,1±25,9**59,6±12,770,8±22,650,4±12,516,8±2,9* Significant differences with the pre op values of p 0,05, **р 0,01,***р 0,001.#-significant differences on day 5-р 0,05, ##,р<0,01, ###,р<0,001.Table 2.Blood morphological parametersRBCs(1012/L)Hb (gr/%)ESR(mm/h)WBCs (109/L)Norma5 – 8 10,0 – 17,0 5 – 12,5 GroupExperimentalcontrolExperimentalControlExperimentalcontrolexperimentalcontrolPre Op4,30±0,154,77±0,1612,1±0,412,9±0,31,16±0,11,1±0,115,15±0,304,66±0,27Day 14,13±0,214,22±0,1611,5±0,611,5±0,65,7±1,37*4,06±0,91*5,87±0,495,44±0,46Day 104,43±0,404,41±0,2011,7±0,611,5±0,32,84±1,372,56±0,725,69±0,566,26±0,78Day 304,56±0,274,43±0,1410,4±0,911,0±0,81,65±0,42,55±0,885,05±0,714,91±0,40Day 603,46±0,484,17±0,1912,2±0,811,8±0,81,3±0,30,9±0,16,16±0,657,30±0,81* Significant differences with the pre op values p0,05 **р0,01 ***р0,001Table 3.Indicators of leukocyte formulaNorma2-10%20-75%30-85%1-4%1-4%1-7%groupExperimentalControlExperimentalContRolExperimentalcontrolExperimentalControlExperimentalControlExperimentalControlPre op3,3±0,72,7±0,426,7±2,023,9±2,565,6±2,469,7±2,71,7±0,31,3±0,31,6±0,51,3±0,31,1±0,41,0±0,3Day 18,0±1,0*6,1±1,144,1±2,2***40,6±2,5***43,5±2,5***48,7±2,8***1,2±0,21,2±0,22,2±0,32,7±0,41,1±0,20,7±0,3Day 105,3±1,25,1±1,224,2±1,9~~~29,3±2,9~67,0±2,1~~~62,4±3,3~0,7±0,2**0,6±0,21,7±0,41,8±0,41,2±0,40,9±0,4Day 307,8±1,57,3±2,024,0±2,5~~~24,8±3,3~~66,0±3,9~~~64,2±4,4~~0,6±0,2*1,3±0,31,2±0,21,4±0,30,4±0,21,0±0,3Day 604,0±1,57,0±2,018,4±3,6~~~23,2±2,5~~74,4±4,2~~~66,6±3,3~1,2±0,41,6±0,42,0±0,41,2±0,50,0±0,00,4±0,2~-significant differences of the values in the first days р0, 05~~р<0,01 ~~~р<0,001*-significant differences with the pre op valuesp0,05 **р0,01 ***р0,001Figure 1344170153670Figure 2Figure 3Figure 4 ................
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