Analyses Of Osteocyte Density, Microcrack, Microstructure And Biomechanical Properties In Ovariectomized Rat Vertebral Trabeculae

Part one:Associations between osteocyte density and biomechanics,microcrack and microstructure of vertebral trabeculae in OVX ratsObject:The study was aimed to examine the relationships between osteocyte density,biomechanics,bone mineral density,microcrack,and microstructure of vertebral trabeculae in OVX rats.Method:Forty 7-month-old female Sprague-Dawley rats were randomly divided into groups of ovariectomyzed(OVX),sham-operated (SHAM),17β-estradiol(10μg·kg^-1·d^-1,subcutaneously injected)treated (EST)or genisein(5mg·kg^-1·d^-1,subcutaneously injected)treated(GEN). At 15 weeks postoperatively the compressive test was performed on L5 vertebral body,additionally,micro-computed tomography(μ-CT) assessment was performed to estimate the 3D bone mineral density (BMD)and 3D microstructure parameters of L6 vertebral body.After fatigue damage testing,the L6 vertebral body was bulk-stained in 1% basic fuchsin and embedded in methylmethacrylate.Mounted bone slices were used to measure microcrack parameters and osteocyte density (Ot.N/T.Ar).Results:When data were pooled from all four groups,Ot.N/T.Ar positively correlated with maximum load(ML)and bone mineral content (BMC),but not with elastic modulus(EM)and BMD.There was a significant negative correlation between osteocyte density(Ot.N/T.Ar) and microcrack length(Cr.Le),but not between Ot.N/T.Ar and other microcrack indices as Cr.Dn or Cr.S.Dn.Ot.N/T.Ar positively correlated with trabecular connectivity density(Conn.D)and negatively correlated with trabecular separation(Tb.Sp).There were no statistically significant correlations between Ot.N/T.Ar,BV/TV,BS/BV,Tb.Th,Tb.N,SMI,and DA.Conclusion:Osteocyte could play a crucial role in maintaining bone mechanical quality and osteocyte density could be considered as an alternative index in assessing bone mechanical quality.There was significant negative correlation between osteocyte density and microcrack length,which indirectly reinforece the notion that osteocyte lacunae might act as a barrier to microcrack extension. Part two:Longitudinal changes of bone microstructure and biomechanical properties of vertebral trabeculae in OVX ratsObject:To characterize the longitudinal changes of cancellous bone microstructural deterioration of vertebral trabeculae and investigate the possible adaptative compensations in OVX rats.Method:50 Female Sprague-Dawley rats of seven-month-old were ovariectomized(OVX),SHAM-operated(SHAM)or left unoperated (baseline control).The vertebral bodies and artery blood samples were harvested at baseline,3 and 15 weeks postoperatively.The compressive test was performed on L5 vertebral body,and micro-computed tomography(μ-CT)assessment was performed to estimate 3D bone mineral density(BMD)and microstructure parameters of the L6 vertebral body.After fatigue damage testing,L6 vertebral body was bulk-stained in 1%basic fuchsin and embedded in methylmethacrylate.Mounted bone slices were used to measure osteocyte density(Ot.N/T.Ar).Blood samples were used to analysis serum tartrate-resistant acid phosphatase 5b(TRAP-5b).Results:At 3 weeks postoperatively,apparent BMD,trabecular volume fraction(TV/BV),trabecular thickness(Tb.Th),trabecular number(Tb.N),tartrate-resistant acid phosphatase 5b(TRAP-5b), maximum load(ML),elastic modulus(EM)and osteocyte density (Ot.N/T.Ar)were significantly decreased in OVX rats than that of SHAM and baseline control rats.Degree of anisotropy(DA)was significantly decreased in OVX rat than that of baseline control group.Bone area density(BS/BV),trabecular separation(Tb.Sp)and trabecular model index(SMI)were significantly increased in OVX rat than that of SHAM and baseline control rats.There were no significant differences between OVX and SHAM rats with regard to DA,trabecular connectivity density (Conn.D),ML,EM,Ot.N/T.Ar and TRAP-5b.There was no significant difference with regard to Conn.D between OVX and baseline control rats. At 15 weeks postoperatively,apparent BMD,TV/BV,Conn.D,Tb.N, TRAP-5b,ML and Ot.N/T.Ar were significantly decreased in OVX rat than that of SHAM and baseline control rats.SMI,Tb.Sp and DA were significantly increased in OVX rat than that of SHAM and baseline control rats.There were no significant differences with regard to BS/BV and Tb.Th.between OVX,SHAM and baseline control rats.BS/BV, Conn.D and Tb.N of OVX rat were significantly decreased at 15 weeks postoperatively than that at 3 weeks postoperatively.Ot.N/T.Ar,Tb.Sp and DA of OVX group were significantly increased at 15 weeks postoperatively than that at 3 weeks postoperatively.Conclusions:In early ovariectomized rats,bone mass of lumber vertebra lost rapidly while the microstructure of spongy bone deteriorated gradually.A decrease in serum TRAP-5b,and an increase in osteocyte density,trabecular degree of anisotropy and trabecular thickness might represent the adaptive changes to the attenuated bone quality accounting for maintenance of the strength and integrity of cancellous bone in OVX rats Part three:Effects of genistein on bone health in OVX rat vertebral trabeculaeObject:Effects of phytoestrogen on bone in both women and ovarian hormone-deficient animal models of osteoporosis remain uncertain.The study was aimed to investigate the effect of genistein (GEN)on trabecular bone quality in ovariectomized(OVX)rats.Method:90 female Sprague-Dawley were randomly divided into groups of ovariectomyzed(OVX),sham-operated(SHAM),17β-estradiol (10μg·kg^-1·d^-1)treated(EST)or genisein(5mg·kg^-1·d^-1)treated(GEN) and unoperated baseline controls.The vertebral bodies were harvested at baseline,3 and 15 weeks postoperatively.Compressive test was performed on L5 vertebral body,and micro-computed tomography(μ-CT) assessment was performed to estimate 3D bone mineral density(BMD) and microstructure parameters of L6 vertebral body.After fatigue damage testing,the L6 vertebral body was bulk-stained in 1%basic fuchsin and embedded in methylmethacrylate.Mounted bone slices were used to measure microcrack parameters,empty osteocyte lacuna density(e.Lc.Dn) and osteocyte density(Ot.N/T.Ar).The L4 vertebral body was embedded in methylmethacrylate for dynamic histomorphometry analysis without staining.Results:At 3 weeks postoperatively:Apparent BMD was significantly decreased in OVX,GEN and EST rats than that of SHAM rat.Structure model index(SMI)was significantly increased in GEN and EST rats than that of SHAM group.Trabecular volume fraction(TV/BV) was significantly decreased in OVX and GEN rats than that of SHAM rat. Bone area density(BS/BV)and trabecular separation(Tb.Sp)were significantly increased in OVX and GEN rats than that of SHAM rat. Trabecular thickness(Tb.Th)was significantly decreased in OVX rat than that of SHAM group.Trabecular number(Tb.N)was significantly decreased in OVX and GEN rats than that of SHAM rat.Trabecular number(Tb.N)was significantly decreased in GEN rat than that of EST rat.Degree of anisotropy(DA)was significantly increased in EST rat than that of OVX,GEN and SHAM rats.Trabecular connectivity density (Conn.D)was significantly decreased in GEN rat than that of SHAM and OVX rats.Maximum laod(ML)was significantly decreased in EST and GEN rats than that of SHAM rat.Maximum load was significantly decreased in GEN rat than that of OVX rat.There were no significant differences between OVX and SHAM rats with regard to ML.There were no significant differences between rats of four groups with regard to elastic modulus(EM)and osteocyte density(Or.N/T.Ar).At 15 weeks postoperatively:Apparent BMD,BV/TV and Tb.N were significantly decreased in OVX,GEN and EST rats than that of SHAM rat.BS/BV was significantly increased in EST rat than that of OVX and SHAM rats. SMI was significantly increased in EST,OVX and EST rats than that of SHAM rat.Tb.Sp was significantly increased in OVX and GEN rats than that of SHAM rat.Tb.Sp was significantly increased in OVX rat than that of EST rat.Tb.Th was significantly decreased in EST rat than that of SHAM and OVX rats.Conn.D was significantly decreased in OVX rat than that of SHAM and EST rats.Conn.D was significantly decreased in GEN rat than that of EST rat.DA was significantly increased in OVX and EST rats than that of SHAM rat.Maximum loading(ML)and Ot.N/T.Ar were significantly lower in OVX rat than that of other groups.E.Lc.Dn was significantly decreased in GEN and EST rats than that of OVX rat. Microcrack density(Cr.Dn),microcrack surface density(Cr.S.Dn)and microcrack length(Cr.Le)were significantly increased in OVX rat than that of other groups.Mineral apposition rate(MAR)was significantly decreased in OVX rat than that of SHAM,EST and GEN rats.Bone formation rate(BFR)was significantly decreased in OVX rat than that of other groups.There were no significantly differences with regard to mineralizing surface(MS/BS)between rats of four groups.Conclusions:The present study demonstrates that genistein preserved biomechanical quality of trabecular bone regardless of microstructure and bone mineral density in OVX rats.