| 1 Objective1.1 The clinical research:To evaluate the mid-term clinical efficacy of the Hybrid surgery,which composed of the artificial cervical disc replacement(ACDR)and the anterior cervical discectomy and fusion(ACDF),for the treatment of cervical spondylosis.Postoperative heterotopic ossification(HO)was recorded,the cause and its potential impact on clinical results was explored.1.2 The clinical basic research:To explore the potential mechanism of the osteogenic degeneration of the cervical spine and the role of TGF-β/Smads signaling pathway in the osteogenic degeneration.1.3 Basic experimental research:To explore the effectiveness and the potential mechanism of Panax notoginsenosides(PNS)in targeting prevention of the heterotopic ossification based on the TGF-β/Smads signaling pathway.2 Methods2.1 The clinical research:A retrospective cohort study was conducted.The clinical and radiological data of the patients with cervical spondylosis who has underwent Hybrid surgery or ACDF surgery in recent years were recorded and analyzed.The mid-term clinical and radiological results of each group were compared.Postoperative heterotopic ossification and other complications were also recorded and analyzed.2.2 The clinical basic research:Using case-control method,based on the preoperative radiological examination,patients with severe cervical disc degenerative disease were divided into osteogenic group and control group according to whether they had osteogenic degeneration.Samples of the degenerated disc related tissue were collected during their surgery treatment.Three different histological staining methods were used to compare the histological differences between the two groups.The immunohistochemical staining with semi-quantitative detection was used to detect and compare the expressions of TGF-β1,p-Smad2 and p-Smad3 between the two groups.2.3 Basic experimental research:A prospective randomized controlled trial was conducted.A classical heterotopic ossification model of Achilles tendon transection in rats was established and was intervened by different concentrations of PNS.The rats in each group were tested at 2w,5w and 10w after operation,respectively.The X-ray examination was performed to observe the occurrence of heterotopic ossification in each group.The morphological examination was performed to observe the healing and growth of the Achilles tendon in each group,and the volume of the Achilles tendon in each group was recorded and compared.The histological staining was performed to compare the histological differences of each group.Finally,the immunohistochemical staining was used to compare the expression differences of TGF-β/Smads related signal factors in the Achilles tendon tissues of each group.3 Results3.1 The clinical research:(1)General information:A total of 83 patients were included in this study.Fifty-nine patients were enrolled and followed up for an average of 6 years(79.69±16.89 months),the follow-up rate was about 71%.There were 27 males and 32 females,aged 34-78 years,with an average of 56.67 years.(2)Clinical and radiological outcomes:At the last follow-up,the VAS score,JOA score and NDI score in both groups were significantly improved compared with those before operation(P<0.05),but there was no significant difference between the Hybrid group and the ACDF group both pre-operation,2-week post-operation and at the last follow-up(P>0.05).The range of motion(ROM)of the Byran disc index level in Hybrid group(7.19 ± 3.83)was higher than that before operation(6.48±3.28),but there was no statistic difference(P>0.05).And the cervical lordosis of the two groups was significantly higher than that before operation(P<0.05).The ROM of C2-7 in both groups was significantly decreased(P<0.05),and the ACDF group(25.06±12.99)was significantly smaller than that of the Hybrid group(34.15 ± 14.43,P<0.05).In ACDF group,the ROM of superior adjacent segment(13.57±5.37)was significantly increased(P<0.05);it was also significantly higher than that of the Hybrid group(10.41± 5.01)at the last follow-up(P<0.05).(3)Heterotopic ossification:At the last follow-up,there were 5 cases of severe heterotopic ossification at the Byran disc index level in the Hybrid group,including 4 cases of grade III and 1 case of grade Ⅳ heterotopic ossification.The overall incidence was about 15.6%.3.2 The clinical basic research:(1)General information:Twenty-two patients,include 14 males and 8 females,with an average age of 55.12 years were included in this study.The operative levels consisted of 3 cases of single-level,15 cases of double-level and 4 cases of three-level,with a total of 45 operative levels.And they were divided into two groups,25 in the osteogenic group and 20 in the control group(non-osteogenic group).(2)Histological results:In the osteogenic group,the count of cells was more than the control group both in the outer annulus fibrosus(OAF),the inner annulus fibrosus(IAF)and the nucleus pulposus(NP)of the intervertebral disc(P<0.05).In IAF and NP,the average optical density(AOD)of safranin O staining in the osteogenic group was significantly lower than that of the control group(P<0.05).The immunohistochemical staining showed that the TGF-β1 and its downstream p-Smad2 and p-Smad3 were expressed in all parts of the intervertebral disc and the osteophytes in the osteogenic group.In OAF and NP of the osteogenic group,the AOD of TGF-β1 was higher than that of the control group,the difference was statistically significant(P<0.05).The p-Smad2 in the IAF of the osteogenic group was significantly higher than that in the control group(P<0.05).For p-Smad3,the AOD in the OAF of osteogenic group was significantly higher than that of the control group(P<0.05).3.3 Basic experimental research:(1)Postoperative X-ray outcomes:The incidence of HO,5-week after operation,in the HP group,MP group,LP group,X group and M group were 67%,67%,50%,50%and 67%respectively.At 10w after operation,the incidence in the HP group was 83%and 100%in all of the other surgical groups.The integral optical density(IOD)of HO in group M was higher than that of other groups at 5w after operation(P<0.05),and the IOD in group MP was significantly higher than that of the other groups at 10w after operation(P<0.05).(2)Morphological outcomes:All the transected tendons had healed at 2w after the operation From 2w to 10w,the hardness of the tendon increased gradually in the operation group,and the volume of all groups increased in varying degrees.At 2w after operation,the volume in the experimental group was significantly higher than that of the group M,J and Z(P<0.05).The LP group was significantly higher than that of the group HP and X(P<0.05).At 5w after operation,the experimental groups and group X were higher than group M,J and Z,the difference was statistically significant(P<0.05).Group LP and group X were significantly larger than group MP,the difference was statistically significant(P<0.05).At 10w after operation,the volume of tendon in the operation groups was larger than that of group Z(P<0.05),and the volume in the LP group and M group was larger than that of group HP and MP(P<0.05)(3)Routine histological staining outcomes:The count of cells in the Achilles tendon tissue was significantly increased in all the operation groups,and the fibers were relatively more slender and arranged disorderly.At 5w after operation,the ectopic bone tissue and the surrounding area of endochondral ossification could be seen in all operation groups.At 10w after operation,the ectopic bone tissue of each operation group was further matured.(4)Immunohistochemical outcomes:TGF-β1 and its downstream p-Smad2 and p-Smad3 were expressed in each group,and the IOD was significantly different between each groups both at 2w,5w and 10w after operation(P<0.001).At 2w after operation,the IOD of TGF-β1 in the HP group and LP group was significantly higher than that of group J(P<0.05).For p-Smad2,the IOD in the HP group was significantly larger than that of group Z(P<0.05).Group LP and group M were significantly larger than that of group Z and J(P<0.05).The IOD of p-Smad3 in all the operation groups were higher than that of group J and Z(P<0.05).Group MP was higher than that of group LP,X and M(P<0.05).The IOD in the LP group was higher than that of group X(P<0.05).At 5w after operation,the expression of TGF-β1 in the MP group was statistically higher than that of group J,Z and X(P<0.05);Group LP and group M were statistically higher than that of group J(P<0.05).For p-Smad2,the IOD in group HP,MP,X and M were statistically higher than that in group J and Z(P<0.05);The IOD in group HP and M were statistically higher than that in group MP and LP(P<0.05);and group X was also statistically higher than that of group LP(P<0.05).For p-Smad3,the IOD in group MP,LP and M were statistically higher than that of group J and Z(P<0.05).At 10w after operation,for TGF-β1,group M was higher than group J and Z,with statistical significance(P<0.05);The IOD in group HP and LP were higher than that of group J,with statistical significance(P<0.05)For p-Smad2,the IOD in group MP and LP were significantly higher than that of group J(P<0.05);and group X was significantly higher than that of group J and group Z(P<0.05).For p-Smad3,the IOD in group HP was higher than that of group Z(P<0.05).4 Conclusions(1)For the treatment of double-level severe cervical spondylosis,the Hybrid surgery can achieve satisfied mid-term clinical outcomes,and it yielded superior mid-term radiological outcomes compared with the ACDF.(2)The incidence of postoperative severe heterotopic ossification is low.It can significantly limit the ROM of the artificial disc index level,and there is a potential risk of re-compression of the spinal cord and nerve root.However,it has no obvious impact on the mid-term clinical efficacy,and its further impact needs a long-term follow-up study.(3)The TGF-β/Smads signaling pathway is involved both in the cervical disc degeneration and the ectopic bone formation,but there may be a difference between the p-Smad2 and p-Smad3 for the effect on osteogenic degeneration of the cervical spine.(4)PNS may have bidirectional effects on TGF-β/Smads signaling pathway,and there may be a window threshold for its inhibition on the signaling pathway.(5)PNS has the potential to reduce the incidence of heterotopic ossification and reduce the quality of the ectopic bone under certain conditions,but it also has the risk of aggravating the heterotopic ossification. |
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