| ObjectivesAllograft bone transplantation takes advantage of abundant resource, plenty of sizes and shapes and having activity of biology in bone repairing, competed with other materials. However, allografts in bone bank still have immunogenicity, and their ability of osteogenesis have been damaged partly in the process of manufacture. As a result, Pei Guo-xian, Lu Hai-bo impragant cyclosprin to the deproteined and defatted bone and use low temperature plasm to sterilize the materials to manufacture cyclosporine-impragnated allograft bone(CAB). And they obtained an invention patent of China (A new kind of allograft and the manufacture process,200610122058) because of this invention. The objectices of this study are:(1) study in vitro drug release characteristic of cyclosporine-impregnated allograft bone to observe whether the drug loading pattern and drug capacity are reasonable, (2) compete CAB with freeze-dried allograft bone(FDAB) in terms of repairing bone defects and transplantation immunology. MethodsFirst part In vitro drug release characteristic of cyclosporin-impregnated allograft bone1 The iliums of 4 New Zealand rabbits were obtained under condition of sterilization after they were sacrificed. To make CAB in accordance with documents: infuse cancellous bones in 30% hygrogen peroxide to deprotein for 24 h. Then took chlorlform, methanol and water 20 ml respectively as extractant to defat the bones at 65℃. Then every bone was processed respectively. Infused deproteined and defatted bone in 0.6 ml 95% ethanol solution, then after the process of vibratiing at room temperature for 90 min, centrifugating at 5000 r/min for 20 min and unwater at 37℃for 12 h, the bone was weighted and the weight is expressed as M1. Infused the bone in 0.6 ml cyclosporine ethanol solution (the destiny of cyclosporine in 95% ethanol solution is 0.5 g/L), then after the process of vibratiing at room temperature for 90 min, centrifugating at 5000 r/min for 20 min and unwater at temperature of 37℃for 12 h, the bone was weighted again and the weight was expressed as M2. Drug capacity in CAB can be caculated by M2 minus M1, an anvenge of 0.0998 g.2 To observe the defatted and deproteined bones and CAB under scanning electron microscope.3 The CAB was incubated in 300 ml of physiological saline containing 0.2% sodium dodecyl sulphate at 37℃±5℃with an oscillating of 70 r/min. At predetermined time intervals,2 ml of the medium was sampled and analyzed by HPLC, then the cumulative amount of drug released was calculated and plotted versus time. Chromatographic condition:chromatographic column (Agilent C18,4.6 mm x250 mm,5μm), mobile phase:methanol:water(85:15), flow rate:1.0 mL/min, detection UV:210 nm, clumn temperature:55℃, sample injection volume:20μl.Second part Transplantation immunology of cyclosporine-impregnated allograft bone versus freeze-dried allograft bone:a comparative study1 Sixty Newsland rabbits were divided into three groups with 20 in each group: bone donor group, study group, control group. Forty iliums of New Zealand rabbits in donor group were obtained under condition of sterilization after they were sacrificed. Twenty iliums were used to make CAB in accordance with documents:infused cancellous bones in 30% hygrogen peroxide to deproteined for 24 h. Then took chlorlform, methanol and water 20 ml respectively as extractant to defat the bone at temperature of 65℃. Infused the deproteined and defatted bones in 0.5 g/L cyclosporine ethanol solution, then after the process of vibratiing at room temperature for 90 min, centrifugating at 5000 r/min for 20 min and unwater at temperature of 37℃for 12 h. Then after cacuum packaging respectively and been sterilized by low temperature plasm, CAB were made. The other twenty iliums were used to make FDAB in accordance with documents:the bones were stored at 4℃for 30 min, then at-20℃for 12 h, at-70℃for 7 d and freeze-dried for 24 h. Then after cacuum packaging respectively and been sterilized by irradiation of Coy(25 kGy,17 h), FDAB were made. Under condition of sterilization 10 mm bone in the middle of right radius of rabbit was intercepted. Study group and control group were repaired by CAB and FDAB respectively. Sutured and packed the wound. Prevent infection with penicillin for 3 d.2 Respectively 1.5 ml vein blood of 5 rabbits in each group was colleced at 2,4,8,12 week after operation, then the animals were sacrificed and the bone at the defect site of right radius were dissected. Blood and bone specimen were stored at-80℃.1 ml of blood specimen was centrifuged at 5000 r/min for 3 min, then 100μl of supernatant was collected to test IL-2 and TNF-a OD value in accordance with kit directions.100 mg bone was collected from proximal, distal and middle part of the bone specimen respectively. Added 3 ml PBS to 300 mg bone then after homogenatding the bone homogenation was made.1 ml of homogenation was collected and centrifuged at 5000 r/min for 3 min, then 100μl of supernatant was collected to test IL-2 and TNF-a OD value in accordance with kit directions. According to the kit directions:for IL-2 the smaller the OD value is, the bigger the density is, but for TNF-a the smaller the OD value is, the smaller the density is.Third part Effects of cyclosporine-impregnaged versus freezed-dried bone allografts in repairing bone defects:a coparative study1 Forty five Newsland rabbits were divided into three groups with 15 in each group:bone donor group, study group, control group. Tirty iliums of New Zealand rabbits in donor group were obtained under condition of sterilization after they were sacrificed. Fifteen iliums were used to make CAB in accordance with documents: infused cancellous bones in 30% hygrogen peroxide to deproteined for 24 h. Then took chlorlform, methanol and water 20 ml respectively as extractant to defat the bone at temperature of 65℃. Infused the deproteined and defatted bones in 0.5 g/L cyclosporine ethanol solution, then after the process of vibratiing at room temperature for 90 min, centrifugating at 5000 r/min for 20 min and unwater at temperature of 37℃for 12 h. Then after cacuum packaging respectively and been sterilized by low temperature plasm, CAB were made. The other fifteen iliums were used to make FDAB in accordance with documents:the bones were stored at 4℃for 30 min, then at-20℃for 12 h, at-70℃for 7 d and freeze-dried for 24 h. Then after cacuum packaging respectively and been sterilized by irradiation of 60Co y(25 kGy,17 h), FDAB were made. Under condition of sterilization 10 mm bone in the middle of right radius of rabbit was intercepted. Study group and control group were repaired by CAB and FDAB respectively. Sutured and packed the wound. Prevent infection with penicillin for 3 d.2 At postoperative 1,3,6 month 5 animals in each group were sacrificed respectively, then X-ray examinations were carried out and were scored by Lanej-Sandhu standerd. After gross observation the bone at the defect site of right radius was dissected, fixed in 10% formol, demineralized in 20% formic acid, dehydrated in ethanol, then embedded, sliced, stained by Masson method and observed under microscope.ResultsFirst part In vitro drug release characteristic of cyclosporin-impregnated allograft bone1 Scaning electron microscope observation:the deproteined and defatted bone was made of plenty of bone trabeculae which was one kind of stereo network structure. Collagen fiber on the surface of bone trabeculae couold be observed at high magnification. The surface of the CAB and the bone trabecula of its inner part were coated by cyclosprin in the form of crystallization.2 Drug releasing:The speed of drug release in the first three days was more fast, but it would slow down and turned to be stable later. And the drug releasing could last more than three months.Second part Transplantation immunology of cyclosporine-impregnated allograft bone versus freeze-dried allograft bone:a comparative study1 OD value of IL-2 in blood analysis:There are no significant difference between two groups (F=0.001, P=0.970), and among different time points (F=0.058, P=0.764). And interaction does not exist(F=0.493, P=0.690). Look at the time points, there is no significant difference between two groups at postoperative week 2,4,8,12 (t value is 0.998,0.474,0.287,0.590 respectively, P value is 0.362,0.648,0.782, 0.572 correspondly). Look at the groups, statistic difference does not exist among different time points both in two groups(F value is 0.843,0.134 respectively, P value is 0.495,0.938 correspondly). 2 OD value of IL-2 in bone at the transplant site analysis:Significant differences exist both between two groups and among different time points(F value is 12.128, 3.985 respectively, P value is 0.001,0.016 correspondly). The interaction exist between groups and different time points (F=3.859, P=0.018). Look at the time points, OD value of study group are bigger than control group at postoperative week 2 and 4(t value is 3.858,2.990 respectively, P value is 0.005,0.017 correspondly), and there are no statistic difference between two groups both at 8 and 12 weeks after operation(t value is 0.332,0.025 respectively, P value is 0.748,0.981 correspondly). Look at the two groups, there is no significant difference among different time points in study group(F=0.045, P=0.987). But satistic difference exist in control group(F=8.896, P=0.001), post analysis were down by LSD method:OD value of postoperative week 2 is bigger than the other time points(P value is 0.028,0.001, 0.000 respectively), and there is no statistic difference among 4,8,12 weeks after operation(P value is 0.072,0.050,0.850 respectively).3 OD value of TNF-a in blood analysis:There are no significant difference between two groups (F=0.006, P=0.941), and among different time points (F=0.330, P=0.804). And interaction does not exist(F=0.491, P=0.691). Look at the time points, there is no significant difference between two groups at postoperative week 2,4,8,12 (t value is 0.772,0.233,1.718,0.048 respectively, P value is 0.462,0.822,0.124,0.963 correspondly). Look at the groups, statistic difference does exist among different time points both in two groups(F value is 0.793,0.230 respectively, P value is 0.515,0.847 correspondly).4 OD value of TNF-a in bone at the transplant site analysis:Significant differences exist both between two groups and among different time points(F value is 30.158,12.445 respectively, both P value are smller than 0.001). The interaction exist between groups and different time points (F=12.791, P<0.001). Look at the time points, OD value of study group are smaller than control group at postoperative week 2 and 4(t value is 5.871,3.107 respectively, P value is 0.000,0.015 correspondly), and there are no statistic difference between two groups both at 8 and 12 weeks after operation(t value is 0.181,0.170 respectively, P value is 0.861,0.869 correspondly). Look at the two groups, there is no significant difference among different time points in study group(F=0.072, P=0.974). But satistic difference exist in control group(F=21.797, P<0.001), post analysis were down by LSD method:OD value at postoperative 2 is smaller than the other time points(all P value are smaller than 0.001), and postoperative week 4 is bigger than 8(P=0.048), and there are no statistic difference between postoperative 4 and 8 and postoperative 8 and 12 (P value is 0.059,0.911 respectively).Third part Effects of cyclosporine-impregnaged versus freezed-dried bone allografts in repairing bone defects:a coparative study1 Gross observation:postoperative month 1:the bone defect of study group was conjuncted by bone, and CAB was surrounded by new bone, and doner bone and host bone mixed togethered tightly. New bone was growing from the two tips to the center of bone defect in control group, but quantity of new bone was less than study group and the mixing of doner bone and host bone was inferior to study group. Postoperative month 3:the bone defect of study group was conjuncted by compacted bone, and bone moulding have been finished partly. The bone conjuncted the defect of control group was pultaceouser than that of study group, and there was not obviously bone moulding. Postoperative month 6, for both two groups, the bone defect was conjuncted by compacted bone and obviously bone moulding could be observed.2 Obsercation and score of X-ray:postoperative month 1:new bone callus of study group was much more than that of control group. And fracture line of study group disappeared, but which of control group did not. Postoperative month 3:for study group, the bone defect healed almostly, and pulp chamber of repairing site recanalized partly and crotical bone of that moulded partly which was near normal bone in thickness. For control group:the bone callus at the defect site was more than postoperative month 3, and fracture line disappeared. Postoperative month 6:for both groups, the bone moulding were finished and the thickness of cortical bone was as same as normal bone and pulp chamber recanalized. X-ray score analysis:significant difference exist between two groups(F=49.741, P<0.001), and there is statistic difference among different time points(F=119.480, P<0.001), and the interaction exists(F= 10.344, P=0.001). Look at the time points, the score of study group are bigger than control group both at 1 and 3 month after operation(t value is 5.400, 4.628 respectively, P value is 0.001,0.002 correspondly), but there is no significant difference between two groups at postoperative month 6(t=0.891, P=0.399). Look at the different groups, there is significant difference among different time points in study group(F=51.256, P<0.001), post analysis were down by LSD method:the scores is increasing along with time(P value are 0.001,0.000,0.000 respectively). there is significant difference among different time points in control group(F=70.568, P<0.001), post analysis were down by LSD method:the scores is increasing along with time(P value are 0.001,0.000,0.000 respectively).3 Histological observation:postoperative month 1:lots of new bone formed in the inner part of CAB and the new collagen was compacted, but there was still doner bone can be seen. For the control group, lots of necrotic doner bone existed, which was full of plenty of inflammatory cell. Postoperative month 3, most of doner bone was absorbed and replaced by new bone. The quantity of mature bone collagen in study group was more than in the control group, which turned to be layered arrangement in the study group, which demonstrated better moulding of cortical bone. Postoperatice month 6, in both groups bone collagen were mature which turned to be tight layered arrangement.Conclusions1 Sustained-release effect of cyclosprin from CAB is good, and there are not brust release and postponement of drug release.2 When be transplanted, there is no significant difference between CAB and FDAB for general immunereaction, but located immuneraction of transplanted site of CAB is weaker than of FDAB.3 The CAB is superior to the FDAB in terms of the effect of repairing bone defects.
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