| BackgroundSenile osteoporosis is a systemic bone disease related to aging.Its pathological features are bone mass reduction,bone microstructure damage,bone fragility increase and easy to fracture.Although the etiology of senile osteoporosis is diverse,cell senescence is one of the important factors.Studies have shown that senescent bone tissue cells,such as bone marrow-derived mesenchymal stem cells,osteoblasts,osteocytes,bone marrow monocytes and osteoclasts in bone microenvironment,will gradually accumulate in local tissues with aging.However,the aging changes of major cell types in bone tissue microenvironment have not been fully revealed,and the effects of aging on the biological functions of major cell types in bone tissue are not fully understood.Also,it is still unclear whether aging changes exist in the whole cell population or in specific cell subtypes.Low magnitude vibration（acceleration<1g）as a new option for the treatment of osteoporosis,provides a non-invasive and tolerable treatment,which can inhibit bone resorption,increase bone strength and eliminate the adverse reactions associated with drug treatment.In clinical application,low magnitude vibration is highly recognized and accepted by doctors and patients.However,whether low magnitude vibration is a potential anti-aging therapy,whether it can regulate the aging changes of bone tissue,and the specific mechanism of regulating the aging changes of bone tissue have not been reported.Therefore,from the aged rat model level,the cell population level and the single cell level,the purpose of this study is to systematically study:1)The aging changes of bone tissue cells in the bone microenvironment of aged rats,the effect of bone cells aging on the biological function of bone tissue cells,bone mass and bone structure;2)The effect and mechanism of mechanical stimulation produced by low magnitude vibration on the aging of bone tissue cells,inhibition of osteoporosis and promotion of bone formation in aged rats.This study is expected to clarify the aging characteristics and functional changes of bone tissue cells,reveal the pathogenesis of senile osteoporosis regulated by bone tissue cell aging,and find the intervention measures for intervening bone tissue cell aging,delaying senile osteoporosis and providing an effective,non-drug,non-invasive,acceptable and long-term tolerable method for the prevention and treatment of senile osteoporosis.Methods and results1.Aging changes of bone tissue in aged rats1.1 Aging and functional changes of bone tissue cells at cell population level1.1.1 Methods Three months old normal SD rats（n=20,body weight 260 g±15 g,half male and half female）were selected as young rats group;22 months old SD rats（n=20,body weight 500 g±25 g,half male and half female）were used as aged rats group.BMSCs,bone marrow monocytes,osteoblasts and osteocytes were isolated and cultured in each group.（1）Detection of the aging parameters of bone tissue cells:CCK8 method was used to detect cell proliferation;immunofluorescence assay was used to detect aging related heterochromatin aggregation（H3k9me3 labeling）and aging related DNA damage（53BP1 labeling）;The senescence marker proteins(p16INK4aand p21)were detected by Western blot,and the number of aging cells was detected byβ-galactosidase staining.（2）Detection of the functional parameters of bone tissue cells:The osteogenic differentiation ability of BMSCs was detected by alizarin red staining,the adipogenic differentiation ability of BMSCs was detected by oil red O staining;the osteogenic activity of osteoblasts was detected by alizarin red staining,alkaline phosphatase staining,bone morphogenetic protein detection and ALP activity detection;the osteoclastic differentiation ability of bone marrow monocytes was detected by tartrate resistant acid phosphatase（TRAP）staining and TRAP activity assay.1.1.2 Results Compared with young rats,the proliferation of BMSCs,osteoblasts,osteocytes and bone marrow monocytes in aged rats was significantly decreased.DNA damage marker 53BP1 and senescence related heterochromatin marker h3k9me3 were highly expressed in all kinds of cells in aged rats,and the senescence markers p16INK4aand p21（P<0.01）were highly expressed in all kinds of cells;The number of aging cells with positiveβ-galactosidase staining in all kinds of cells in aged rats increased significantly（P<0.05）.Meantime,compared with young rats,the number of calcium nodules,the activity of alkaline phosphatase and the expression of bone morphogenetic protein were significantly decreased in aged rats（P<0.01）;the negative regulator of bone formation,osteosclerotin,was highly expressed in osteocytes of aged rats（P<0.05）;the number of calcium nodules in BMSCs of aged rats was significantly reduced after osteogenic differentiation;the number of osteoclasts induced by bone marrow monocytes decreased significantly（P<0.05）,but the TRAP activity of osteoclasts induced by bone marrow monocytes did not change significantly（P>0.05）.1.2 Aging changes of bone tissue cells at single cell level1.2.1 Methods Single cell RNA sequencing was performed on femur bone marrow cells of young rats（n=1,female）and aged rats（n=1,female）.The sequencing data were collected for bioinformatics analysis.Bone marrow cells were clustered by spatial clustering algorithm.The cell groups were defined according to the marker genes of each cell group.The aging changes of bone marrow monocytes and BMSCs were detected according to the expression of aging marker gene TP53.The types and numbers of aging bone marrow monocytes and aging BMSCs were identified by flow cytometry.The osteoclastic differentiation ability of bone marrow monocytes was detected according to the expression of osteoclast differentiation genes MITF,SPI1,Fos and csf1r;the osteogenic differentiation ability of BMSCs in each group was detected according to the expression of osteogenic differentiation genes Alpl,BGLAP and COL1A1;and the adipogenic differentiation ability of BMSCs in each group was detected according to the expression of adipogenic differentiation gene LPL.1.2.2 Results Single cell RNA sequencing results showed that bone marrow cells of young and aged rats could be divided into 23 cell groups.According to the marker genes of each cell type,bone marrow monocytes（group 10,11,12,13）and BMSCs（group 22）were defined.In the four cell subsets of bone marrow monocytes,cell group10 expressed TP53 gene highly,while cell groups 11,12 and 13 expressed TP53 gene lowly.It is suggested that cell group 10 is an aging cell subset in bone marrow monocytes of aged rats and young rats,while cell groups 11,12 and 13 are bone marrow monocyte subsets with no obvious aging change.In order to effectively define and classify senescent and non-senescent bone marrow monocyte subsets,it is necessary to find cell surface markers that can distinguish senescent and non-senescent bone marrow monocyte subsets.We found that the ITGB2 gene was negatively expressed in cell group 10,and positively expressed in other monocyte subsets（cell group 11,12,13）,suggesting that ITGB2 is a cell surface marker for distincting senescent cell subset（cell group 10）and non-senescent cell subsets in bone marrow monocytes.ITGB2-bone marrow monocytes are a subset of senescent cells,and ITGB2+bone marrow monocytes are a subset of non-senescent cells.Compared with ITGB2-bone marrow monocytes,the osteoclast differentiation genes Mitf,Spi1,Fos and Csf1r are highly expressed in ITGB2+bone marrow monocytes.Compared with young rats,the expression of TP53 gene in ITGB2-bone marrow monocytes in aged rats was up-regulated,and the expression of osteoclast differentiation gene was down-regulated;the expression of TP53 gene and osteoclast differentiation gene in ITGB2+bone marrow monocytes in aged rats had no significant change.In addition,the proportion of ITGB2-and ITGB2+bone marrow monocytes in bone marrow cells decreased significantly with aging,while the proportion of ITGB2+bone marrow monocytes in bone marrow monocytes increased.Compared with young rats,the expression of TP53 gene of BMSCs in aged rats was higher（P<0.01）,and the expression of Alpl,BGLAP and COL1A1 was lower（P<0.01）,while LPL was higher（P<0.01）.1.3 Changes of bone mass and structure of femur1.3.1 Methods The changes of bone mass and structure of femur were detected by micro CT and bone histomorphology staining;ALP activity and TRAP activity were used to detect the changes of osteogenic and osteoclastic capacity in rats.1.3.2 Results Compared with young rats,bone mineral density（BMD）,bone trabecular volume fraction（BV/TV）and trabecular number per unit volume（TB.N）in aged rats were decreased（P<0.01）,and trabecular separation（TB.SP）increased（P<0.01）;trabecular structure in cancellous bone was sparse;ALP activity in serum of aged rats was significantly decreased（P<0.05）,while TRAP activity had no significant difference（P>0.05）.2.Low magnitude vibration regulates the senescence of bone marrow monocytes from aged rats2.1 Comparison of ITGB2 and its downstream genes between ITGB2+bone marrow monocytes and ITGB2-bone marrow monocytes2.1.1 Methods Based on the bioinformatics analysis of single cell sequencing data in part 1.2,Feature Plot function was used to visualize the expression of ITGB2,ACTG1,actb,ICAM1,ptk2b,LMNA,mapk3,lmnb1 in bone marrow monocytes.2.1.2 Results Single cell RNA sequencing results showed that ITGB2,ACTG1,actb,ICAM1,ptk2b,LMNA,mapk3 and lmnb1 were highly expressed in ITGB2+bone marrow monocytes,but lowly expressed in ITGB2-bone marrow monocytes.It is suggested that ITGB2 may be related to inhibition of the senescence of ITGB2+bone marrow monocytes,and ITGB2 may be one of the important molecules regulating the senescence of bone marrow monocytes.2.2 The effects of knockdown or overexpression of ITGB2 on the senescence and osteoclastic differentiation of bone marrow monocytes from aged rats2.2.1 Methods The ITGB2 si RNA or ITGB2 vector were transfected into the bone marrow monocytes from aged rats,to knockdown or overexpress ITGB2.Western blot was used to detect the expression of ITGB2 and its downstream protein.The number of senescent cells was detected byβ-galactosidase staining.The osteoclastic differentiation activity of bone marrow monocytes was detected by NFATc1immunoblotting and TRAP staining.2.2.2 Results Compared with the scramble RNA（Scr）group,the expression of ITGB2 protein in ITGB2 knockdown group was down regulated（P<0.01）,the number of aging cells increased（P<0.05）,the expression of osteoclast related transcription factor NFATc1 was down regulated（P<0.05）,and the number of osteoclasts formed after osteoclast differentiation induction decreased（P<0.05）.Compared with the blank vector roup,the expression of ITGB2 protein in ITGB2overexpression group were up regulated（P<0.01）;the number of aging cells was decreased（P<0.05）,the expression of NFATc1 was increased（P<0.05）,the number of osteoclasts formed after osteoclast differentiation induction was increased（P<0.05）.2.3 The effect of low magnitude vibration on senescence and osteoclastic differentiation of bone marrow monocytes from aged rats2.3.1 Methods Vibration stimulation was applied to bone marrow monocytes from aged rats,bone marrow monocytes from aged rats with ITGB2 knockdown or ITGB2overexpression:0.3g×90hz,30 min/day×Five days.Western blot was used to detect the expression of ITGB2 and its downstream proteins.The number of senescent cells was detected byβ-galactosidase staining,and osteoclastic differentiation activity was detected by NFATc1 immunoblotting and TRAP staining.2.3.2 Results Compared with non vibration group,the expression of ITGB2 protein in bone marrow monocytes treated with vibration was down regulated（P<0.01）,the number of aging cells was increased（P<0.01）,the expression of osteoclast related transcription factor NFATc1 was down regulated（P<0.01）,and the number of osteoclasts formed after osteoclast differentiation induction decreased（P<0.01）.ITGB2 knockdown further increased the low magnitude vibration-induced senescence and osteoclastic differentiation inhibition of bone marrow monocytes from aged rats.On the contrary,over expression of ITGB2 attenuated the low magnitude vibration-induced senecence and osteoclastic differentiation inhibition of bone marrow monocytes from aged rats.3.Low magnitude vibration regulates the senescence of BMSCs from aged rats3.1 Comparison of ITGB2 and its downstream genes between BMSCs from young rats and BMSCs from aged rats3.1.1 Methods Based on the bioinformatics analysis of single cell sequencing data in part 1.2,Feature Plot function was used to visualize the expression of ITGB2,ACTG1,actb,ICAM1,ptk2b,LMNA,mapk3,lmnb1 in BMSCs.3.1.2 Results Single cell RNA sequencing results showed that ITGB2,ACTG1,actb,ICAM1,ptk2b,LMNA,mapk3 and lmnb1 were lowly expressed in aged BMSCs,but highly expressed in young BMSCs.It is suggested that ITGB2 may be one of the important molecules regulating the senescence of BMSCs.3.2 The effects of knockdown or overexpression of ITGB2 on the senescence and osteogenic differentiation of BMSCs from aged rats3.2.1 Methods The ITGB2 si RNA or ITGB2 vector were transfected into the BMSCs from aged rats,to knockdown or overexpression ITGB2.Western blot was used to detect the expression of ITGB2 and its downstream protein.The number of senescent cells was detected byβ-galactosidase staining,the osteogenic differentiation activity of BMSCs was detected by Runx2 immunoblotting and Alizarin red staining.3.2.2 Results Compared with the scramble RNA（Scr）group,the expression of ITGB2 protein in ITGB2 knockdown group was down regulated（P<0.01）,the number of aging cells was increased（P<0.05）,the expression of osteoblast related transcription factor Runx2 was down regulated（P<0.05）,and the number of calcium nodules induced by osteogenic differentiation decreased（P<0.05）.Compared with the blank vector control group,the expression of ITGB2 protein in ITGB2 overexression group were up regulated（P<0.01）;the number of aging cells was decreased（P<0.05）,the expression of Runx2 was increased（P<0.05）,and the number of calcium nodules induced by osteogenic differentiation was increased（P<0.05）.3.3 The effect of low magnitude vibration on senescence and osteogenic differentiation of BMSCs from aged rats3.3.1 Methods Vibration stimulation was applied to BMSCs from aged rats,BMSCs from aged rats with ITGB2 knockdown or ITGB2 overexpression:0.3g×90Hz,30min/day×Five days.Western blot was used to detect the expression of ITGB2 and its downstream proteins.The number of senescent cells was detected byβ-galactosidase staining,and the osteogenic differentiation activity was detected by Runx2immunoblotting and alizarin red staining.3.3.2 Results Compared with non vibration group,the expression of ITGB2 protein in vibration group was up regulated（P<0.01）,the number of aging cells was decreased（P<0.01）,the expression of osteogenic transcription factor Runx2 was up regulated（P<0.01）,and the number of calcium nodules induced by osteogenic differentiation was increased（P<0.01）.Over expression of ITGB2 further increased the low magnitude vibration-induced osteogenic differentiation and senescence inhibition of BMSCs.On the contrary,ITGB2 knockdown attenuated the low magnitude vibration-induced osteogenic differentiation and senescence inhibition of BMSCs.4.In vivo verification of the effect of low magnitude vibration on regulating senescence of bone cells in aged rats4.1 In vivo verification of the effect of low magnitude vibration on senescence and osteoclastic differentiation of bone marrow monocytes in aged rats4.1.1 Methods Aged rats（n=20,female）were randomly divided into two groups:1)vibration group（n=10）:during the experiment,rats were placed on the vibration platform and loaded with low magnitude whole-body vibration（0.3g×90Hz,30min/day,5 days/week,lasting for 12 weeks）;2)static group（n=10）:rats were placed on the vibration platform without vibration（30min/day,5 days/week,lasting for 12weeks）.Aged rats were used to isolate and culture primary femur bone marrow monocytes.Western blot was used to detect the expression of ITGB2 related proteins;the number of aging cells was detected byβ-galactosidase staining;the osteoclastic differentiation activity of bone marrow monocytes was detected by NFATc1immunoblotting and TRAP staining.4.1.2 Results Compared with non vibration group,low magnitude vibration could inhibit the expression of ITGB2 protein in bone marrow monocytes in aged rats（P<0.05）,increase the number of aging cells（P<0.05）,inhibit the expression of osteoclast related transcription factor NFATc1（P<0.01）,and decrease the number of osteoclasts induced by osteoclast differentiation（P<0.05）.4.2 In vivo verification of the effect of low magnitude vibration on senescence and osteogenic differentiation of BMSCs in aged rats4.2.1 Methods Aged rats were used to isolate and culture primary femur BMSCs.Western blot was used to detect the expression of ITGB2 and its downstream protein;the number of aging cells was detected byβ-galactosidase staining;the osteogenic differentiation activity of BMSCs was detected by Runx2 Western blot;the number of calcium nodules was detected by alizarin red staining.4.2.2 Results Compared with non vibration group,low magnitude vibration could promote ITGB2 protein expression in BMSCs in aged rats（P<0.01）,decrease the aging cells（P<0.05）,promote the expression of osteogenic related transcription factor Runx2（P<0.01）,and increase the number of calcium nodules induced by osteogenic differentiation（P<0.05）.4.3 In vivo verification of the effect of low magnitude vibration on bone mass and bone structure in aged rats4.3.1 Methods The rats were killed and the bilateral femurs were separated.The bone mass and morphological changes of femurs were detected by micro CT and bone histomorphology staining;the changes of osteogenic and osteoclastic capacity of rats in each group in vivo were detected by serum ALP activity assay and TRAP activity assay.4.3.2 Results Compared with the non vibration group,the bone mineral density,trabecular volume fraction and trabecular number per unit volume in the vibration group were significantly increased（P<0.05）,while the trabecular separation was decreased（P<0.05）;trabecular structure in cancellous bone was restored;the activity of serum ALP was increased（P<0.05）,and the activity of TRAP was decreased（P<0.05）.Conclusion1.ITGB2 may be one of the important molecules regulating the senescence of bone marrow monocytes.According to the expression of ITGB2 in bone marrow monocytes,the aging cell subsets（ITGB2-）and non aging cell subsets（ITGB2+）can be accurately distinguished.Inhibition of ITGB2 can promote the senescence of bone marrow monocytes of aged rats and inhibit the differentiation of bone marrow monocytes of aged rats into osteoclasts;Activation of ITGB2 can inhibit the senescence of bone marrow monocytes of aged rats and promote the differentiation of bone marrow monocytes of aged rats into osteoclasts.2.ITGB2 may also be one of the important molecules regulating BMSCs senescence.Inhibition of ITGB2 can promote senescence of BMSCs of aged rats and inhibit BMSCs to differentiatie into osteoblasts;Activation of ITGB2 can inhibit senescence of BMSCs of aged rats and promote BMSCs to differentiate into osteoblasts.3.Compared with the young rats,senescence of ITGB2-bone marrow monocytes of aged rats was obvious,while the osteoclastic differentiation ability and the proportion of ITGB2-bone marrow monocytes decreased;senescence and osteoclastic differentiation ability of ITGB2+bone marrow monocytes of aged rats had no obvious change,while the proportion of ITGB2+bone marrow monocytes increased.Because ITGB2+bone marrow monocytes with strong osteoclast differentiation ability had no obvious aging changes and increased their proportion in bone marrow monocytes,the bone resorption in aged rats was higher than the bone formation,leading to osteoporosis.4.Compared with the young rats,the proliferative activity of BMSCs,osteoblasts and osteocytes decreased and the senescence markers were up regulated,showing obvious senescent changes.Meantime,the osteogenic activities decreased,the bone mineral density decreased and bone structure changed,showing obvious osteoporosis.5.Low magnitude vibration could down regulate the expression of ITGB2,promote the senescence and inhibit the osteoclastic differentiation of bone marrow monocytes in aged rats.Low magnitude vibration could increase the expression of ITGB2,inhibit the senescence and promote the osteogenic differentiation of BMSCs in aged rats.Low magnitude vibration could enhance the bone mass and structure of aged rat. |
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