| کلیدواژههای انگلیسی مقاله |
Autogenous bone graft, Osteoblast, Extracorporeal preservation, Osteogeneic cells, Histomorphometrical analysis, IntroductionAutogenous bone graft is considered the best method and gold standard for bone defect reconstruction, as it provides three basic factors including osteoinductive molecules, a proper scaffold, and osteogenic cells. It is crucial to keep graft osteogenic cells viable because they are responsible for new bone formation after transplantation [ 1, ]. Although it is highly recommended to implant the autogenous grafts immediately after harvest, it may not be possible. As a result, the bone graft must be stored for a certain amount of time extracorporeally [ 2, , 3, ]. Several storage media have been introduced for graft preservation including normal saline solution, Ringer&,rsquo s lactate solution, distilled water, different culture media [ 4, ], and autologous blood derived products including whole blood, plasma, serum, platelet-rich-plasma (PRP) [ 5, ], platelet-poor-plasma (PPP) [ 6, ], and so on.Normal saline solution and Ringer&,rsquo s lactate solution are the most common infusion fluids in operation room settings. Normal saline solution is also the most common solution used in surgeries as coolant and irrigation [ 4, ]. Most surgeons also consider whole blood a proper medium for bone graft maintenance and it can easily be obtained during surgery [ 5, ]. Although air exposure is considered the most inappropriate medium for bone graft maintenance, in clinical practice the grafts are often stored in dry environments [ 7, ].Rocha et al. [ 8, ] studied the effects of preserving bone grafts for 30 minutes in different storage media including normal saline solution, PPP, and dry environment in comparison with a control group, which was implanted immediately. The results showed more empty lacunae in the dry group when compared with the control group, the saline group, or the PPP group. No significant difference was reported in the count of empty lacunae between the saline group, the PPP group, and the control group.Another factor is preservation temperature. There are three temperature ranges usually possible in operation rooms, during surgeries. These include cold preservation (2-8&,deg c), room temperature (18-24&,deg c), and incubator (37&,deg c). Antonenas et al. [ 9, ] preserved blood stem cells in room temperature and refrigerator for 24 hours, 48 hours, and 72 hours. His study showed a great loss of viable cells in the grafts stored in room temperature (21.9%) compared to those stored in refrigerator (9.4%). Finally, the time interlude that a graft is preserved extracorporeally, can affect the quality of the bone graft. Williams et al. [ 10, ] stored canine femoral condyles at 4&,deg c for 14, 21, and 28 days. His study showed &,gt 95% cell viability at 14 days, 75-98% at 21 days, and 65-90% at 28 days of preservation. In this study, we have assessed the effects of three major factors on regenerative potential of autogenous cancellous bone grafts.Material and MethodA one-year-old healthy brown female goat (Capraaegagrus hircus) weighing 28 kg was chosen as the animal model. This study was done according to International animal rights with adherence to the animal experiment rules assigned by Shiraz University of Medical Sciences. Before the surgery, 30cc of blood was obtained from right jugular vein under aseptic condition. The blood was mixed with 6cc of sodium citrate 3.8%, as anticoagulant. The blood was transferred to previously sterilized and labeled laboratory tubes assigned for blood as storage medium. Other tubes contained sterile normal saline solution Ringer&,rsquo s lactate solution, or they were empty. The animal was anesthetized by Ketamine (25mg/ kg) and midazolam (0.2mg/kg) intramuscularly. For local anesthesia, 3.8 ml of lidocaine with epinephrine was injected in surgical site. A single dose of Pen &,amp amp Strep (2.5mg/kg) was injected intravenously in right jugular vein. The animal was placed on surgery table in sitting position. Surgery was conducted under aseptic condition. The hair on right iliac crest area was shaved and antisepsis was done with aqueous solution of povidone. Animal was covered with sterile drapes and the surgical site was exposed through a perforated drape.A full-thickness 5cm long incision was placed over right anterior iliac crest with a # 15 blade. Using periosteal elevator, medial and lateral subperiosteal dissection was performed to expose the lateral surface of the iliac crest completely. A 3cm&,times 3cm cortical window was removed by chisel and osteotome to gain access to the cancellous bone. Cancellous blocks were removed by curette and chisel. Blocks were cut by a fine scissor to 180 pieces the mean volume was 4mm&,times 4mm&,times 4mm. each bone graft sample was transferred to a tube containing storage media.Copious irrigation of the surgical site with normal saline was done. No active bleeding was noticed. Periosteum, muscle, and fascia were separately sutured with vicryl 4-0. The skin was sutured with Nylon 3-0 and dressing was applied. For studying the effects of three major factors on regenerative potential of autogenous cancellous bone grafts, we used histomorphometrical analysis to evaluate osteoblast count in graft volume unit. These factors included,1. Different storage media including normal saline solution, Ringer&apos,s lactate solution, autologous blood, and dry environment2. Different storage temperature including cold preservation (2-8&,deg c), room temperature (18-24&,deg c), and incubator (37&,deg c)3. The time interval that the bone grafts were preserved, 2 hours, 4 hours and 12 hoursThe tubes containing specimen and storage medium were distributed evenly in three categories. Each category included 60 tubes and represented storage temperature, including cold preservation, room temperature, and the incubator. Among the 60 tubes in each category, 15 contained normal saline solution, 15 contained Ringer&,rsquo s lactate solution, 15 contained autologous blood with anticoagulant and 15 tubes did not contain any medium representing the dry environment. Two hours after the harvest time, from each temperature category and storage medium groups, five tubes were selected randomly and the specimens were immediately immersed in 10% formaldehyde solution for 48 hours. Same procedure was done after 4 hours and 12 hours of bone graft preservation. Bone graft decalcification was performed by EDTA 4.13%, dehydration by ethanol 95% and embedded in paraffin. We used oriented method in order to obtain isotropic uniform random (IUR) sections. Paraffinized bone grafts were semi-serially sectioned in 20&,micro m thickness (H&,amp amp E staining) using a microtome.A video-microscopy system consisting of a microscope (Nikon, E-200, Japan) linked to a video camera (SONY, SSC Dc 18P, Japan), a P4 PC computer and a LG monitor (795 FT plus) was utilized for analysis.The number of osteoblasts was counted with an optical dissector design for 20&,micro m thick sections. Through this method, the count of osteoblasts in a volume unit of the bone specimen was determined. An unbiased counting frame was superimposed on the image of bone graft section on monitor on average 70-100 microscopic fields, which were selected for every bone graft sample. The counting frame had two borders of inclusion and two borders of exclusion. The initial field was selected randomly out of the sample section the remaining fields were selected by moving the microscope stage using microscope stage along X- and Y-axis in equal intervals. An oil immersion lens with &,times 100 magnification was used. To assess the numerical density of osteoblasts, the focus area was also moved on Z-axis. Through traveling on Z-axis and using magnification of 60&,times , a microcater (Hidenhain MT-12, Germany) which calculated the Z-axis movements was employed. Each plane thickness is 5&,micro m, but the first nuclei that came into focus, were excluded. Within the next 5&,micro m of traveling on Z-axis (height) any nucleolus which came into maximal focus was counted, if located within the counting frame or in contact with the inclusion border and did not touch the exclusion border or the frame. Numerical density of osteoblasts was calculated by the following formula [ 11, ], N v = &,#425 Q (&,#425 p&,times a(f)&,times h) In this equation, &,#425 Q represents the sum of the counted osteoblasts for each sample, a(f) represents the frame area and &,#425 p represents the total number of fields in X- and Y-axis on which osteoblasts were counted.Kruskal-Wallis non-parametric test was used for data analysis and a p Value &,lt 0.05 was considered statistically significant. SPSS statistical software (version 15) was utilized for statistical analysis.ResultsStorage Media ComparisonFour groups of storage media were compared with each other sorted and layered by similar time interval and storage temperature, each group contained five specimens. Table 1, shows different storage media comparisons in different preservation temperatures and times.1. Comparison of four storage media groups, which preserved bone grafts for 2 hours in cold temperature, showed that the highest count was noted in samples stored in autologous blood, followed by dry environment, Ringer&,rsquo s lactate solution, and finally normal saline solution respectively (p&,lt 0.05).2. Comparison of four storage media groups which preserved bone grafts for 4 hours in cold temperature showed the highest count was noted in samples stored in normal saline solution, followed by autologous blood, dry environment and finally Ringer&,rsquo s lactate solution respectively (p&,lt 0.05). 3. Comparison of four storage media groups which preserved bone grafts for 12 hours in cold temperature showed that the highest count was noted in samples stored in dry environment, followed by Ringer&,rsquo s lactate solution, normal saline solution, and autologous blood, respectively (p&,lt 0.05).4. Comparison of four storage media groups, which preserved bone grafts for 2 hours in room temperature, showed the highest count was noted in samples stored in autologous blood, followed by ringer lactate or normal saline solutions and finally dry environment respectively (p&,lt 0.05). No significant results were shown between samples stored in normal saline solution and Ringer&,rsquo s lactate solution (p&,gt 0.05). Solution (I)Solution (J)2 hours4 hours12 hoursMean Difference (I-J)Sig.Mean Difference (I-J)Sig.Mean Difference (I-J)Sig.Cold temperatureSalineRinger-3955.92140*,.00025196.30000*,.000-24263.0960*,.000Blood-59558.8714*,.00011199.06000*,.0006843.42000*,.000Dry-43916.7214*,.00017637.41000*,.000-43860.2200*,.000RingerSaline3955.92140*,.000-25196.3000*,.00024263.09600*,.000Blood-55602.9500*,.000-13997.2400*,.00031106.51600*,.000Dry-39960.8000*,.000-7558.89000*,.000-19597.1240*,.000BloodSaline59558.87140*,.000-11199.0600*,.000-6843.42000*,.000Ringer55602.95000*,.00013997.24000*,.000-31106.5160*,.000Dry15642.15000*,.0006438.35000*,.000-50703.6400*,.000DrySaline43916.72140*,.000-17637.4100*,.00043860.22000*,.000Ringer39960.80000*,.0007558.89000*,.00019597.12400*,.000Blood-15642.1500*,.000-6438.35000*,.00050703.64000*,.000Room temperatureSalineRinger-.000201.000.000601.000-.053201.000Blood-50540.8204*,.000-70851.9100*,.000-49492.7368*,.000Dry3804.14960*,.000-11804.9298*,.000-59024.6604*,.000RingerSaline.000201.000-.000601.000.053201.000Blood-50540.8202*,.000-70851.9106*,.000-49492.6836*,.000Dry3804.14980*,.000-11804.9304*,.000-59024.6072*,.000BloodSaline50540.82040*,.00070851.91000*,.00049492.73680*,.000Ringer50540.82020*,.00070851.91060*,.00049492.68360*,.000Dry54344.97000*,.00059046.98020*,.000-9531.92360*,.000DrySaline-3804.14960*,.00011804.92980*,.00059024.66040*,.000Ringer-3804.14980*,.00011804.93040*,.00059024.60720*,.000Blood-54344.9700*,.000-59046.9802*,.0009531.92360*,.000IncubatorSalineRinger-11198.3490*,.00015747.68000*,.00033361.77040*,.000Blood-16797.5278*,.0001283.14220.05629862.28000*,.000Dry-28891.7600*,.0006415.72040*,.0001866.40060*,.004RingerSaline11198.34900*,.000-15747.6800*,.000-33361.7704*,.000Blood-5599.17878*,.000-14464.5378*,.000-3499.49040*,.000Dry-17693.4110*,.000-9331.95960*,.000-31495.3698*,.000BloodSaline16797.52778*,.000-1283.14220.056-29862.2800*,.000Ringer5599.17878*,.00014464.53780*,.0003499.49040*,.000Dry-12094.2322*,.0005132.57820*,.000-27995.8794*,.000DrySaline28891.76000*,.000-6415.72040*,.000-1866.40060*,.004Ringer17693.41100*,.0009331.95960*,.00031495.36980*,.000Blood12094.23222*,.000-5132.57820*,.00027995.87940*,.000*Statistically meaningful difference(p &,lt 0.05) |