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We thank Ming Ruan, Glenda L. bone tissue formation when compared with vehicle-treated mice. research confirmed that senescent cell-conditioned moderate impaired osteoblast mineralization and improved osteoclast progenitor success, leading to elevated osteoclastogenesis. Collectively, these data set up a causal function for senescent cells in bone tissue loss with maturing and demonstrate that concentrating on these cells provides both anti-resorptive and anabolic results on bone tissue. As getting rid of senescent cells and/or inhibiting their pro-inflammatory secretome boosts cardiovascular function4 also, enhances insulin awareness3, and decreases frailty7, concentrating on this fundamental system to avoid age-related bone tissue reduction suggests a book treatment strategy not merely for osteoporosis also for multiple age-related co-morbidities. appearance in mouse osteocytes boosts markedly after ~18 a few months old in both sexes (Supplementary Fig. 1a,b), coinciding using the timing of accelerated age-related bone tissue reduction in both feminine and male mice (Supplementary Fig. 1cCj)22,23. Getting rid of a relatively little percentage (~30%) of senescent cells utilizing a suicide transgene, that allows inducible eradication of transgenic mice2C4 had been randomized to either automobile or AP20187 treatment double every week for 4 a few months, beginning at 20 a few months old (Fig. 1a). As expected, AP20187 treatment led to markedly lower mRNA appearance (by ?59%) in bone tissue in accordance with vehicle-treated mice (Fig. 1b) aswell as lower mRNA (by ?48%) encoded with the transgene2C4 (Fig. 1c), in keeping with clearance of senescent cells. This is verified by fewer senescent osteocytes in AP20187- versus vehicle-treated mice (by ?46%), as assessed by a recognised senescence biomarker (senescence-associated distension of satellites [SADS]9,16 (Fig. 1dCf); discover Supplementary Fig. 3 and tale for an additional, detailed validation from the SADS assay using major osteocyte CLTB civilizations)9,16. Remember that we utilized three procedures of senescent cell burden in bone tissue (mRNA, mRNA encoded with the transgene, and SADS-positive osteocytes), all with concordantly lower beliefs in AP20187- versus vehicle-treated mice. The systemic clearance of senescent cells by AP20187 treatment was additional confirmed by lower (Fig. 1g) and (Fig. 1h) mRNA amounts in adipose tissues. Open in another home window Fig. 1 Clearance of senescent cells prevents age-related bone tissue reduction. (a) Experimental style for testing the result of senescent cell clearance utilizing a transgenic strategy on age-related bone tissue reduction: 20-month-old feminine mice had been randomized to either automobile (= 13) or AP20187 (= 16) remedies (intraperitoneally [i.p] double regular) for 4 a few months. rt-qPCR evaluation of (b) and (c) (encoded with the transgene) mRNA appearance amounts in osteocyte-enriched cells produced from bones from the mice. Representative pictures ( 30 pictures per pet, 13 automobile- and 16 AP20187-treated) of (d) a senescent (SEN) osteocyte (magnification 100) versus (e) a non-senescent (non-SEN) osteocyte (magnification 100) based on the senescence-associated distension of satellites (SADS, discover arrows [in d]) assay in cortical bone tissue diaphysis (size pubs, 2 m). (f) Quantification from the percentage of senescent osteocytes in mice treated with either automobile or AP20187 based on the SADS assay. rt-qPCR evaluation of (g) and (h) mRNA manifestation amounts in perigonadal adipose cells. (i) Consultant micro-computed tomography (CT) pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture in the lumbar backbone of automobile- versus AP20187-treated mice. Quantification of CT-derived (j) bone tissue volume small fraction (BV/Television; %), (k) trabecular quantity (Tb.N; 1/mm), (l) trabecular width (Tb.Th; mm), (m) trabecular parting (Tb.Sp; mm), and (n) framework model index (SMI, a way of measuring plate/pole morphology, with lower amounts being better) in the lumbar spine. (o) Consultant CT pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture in the femur. Quantification of CT-derived (p) cortical width (Ct.Th, mm) and (q) micro-finite-element evaluation (FEA)-derived failure fill (N, Newton [a way of measuring bone tissue power]). Histomorphometric quantification in the femoral endocortical surface area of (r) osteoclast amounts per bone tissue perimeter (N.Oc/B.Pm;/mm), (s) osteoblast amounts per bone tissue perimeter (N.Ob/B.Pm;/mm), (t) endocortical nutrient apposition price (MAR; mcm/d), and (u) endocortical bone tissue formation price per bone tissue surface area (BFR/BS; mcm3/mcm2/d) (= 8/group). (v) Mineralization of osteoblastic MC3T3 cells subjected to control (CON) or senescent (SEN) conditioned moderate (CM) (= 3/group), with quantification of eluted alizarin reddish colored dye in (w). Data stand for Mean SEM (mistake pubs). * 0.05; ** 0.01; *** 0.001 (individual samples mice with AP20187 didn’t alter bone tissue guidelines (Supplementary Fig. 6aCo), indicating that strategy is particular for avoidance of age-related bone tissue loss. Trabecular bone tissue histomorphometry in the older mice demonstrated considerably lower bone tissue resorption (osteoclast amounts per bone tissue perimeter; Supplementary Fig. 7a) in AP20187- versus vehicle-treated mice, with out a coupled decrease in bone tissue development indices (osteoblast amounts, mineral apposition price, and bone tissue formation price [Supplementary Fig. 7bCompact disc]). We excluded feasible direct ramifications of AP20187 on osteoclasts by demonstrating that.1 Clearance of senescent cells prevents age-related bone tissue reduction. to vehicle-treated mice. The helpful effects of focusing on senescent cells had been because of lower bone tissue resorption with either taken care of (trabecular bone tissue) or more (cortical bone tissue) bone tissue formation when compared with vehicle-treated mice. research proven that senescent cell-conditioned moderate impaired osteoblast mineralization and improved osteoclast progenitor success, leading to improved osteoclastogenesis. Collectively, these data set up a causal part for senescent cells in bone tissue loss with ageing and demonstrate that focusing on these cells offers both anti-resorptive and anabolic results on bone tissue. As removing senescent cells and/or inhibiting their pro-inflammatory secretome also boosts cardiovascular function4, enhances insulin level of sensitivity3, and decreases frailty7, focusing on this fundamental system to avoid age-related bone tissue reduction suggests a book treatment strategy not merely for osteoporosis also for multiple age-related co-morbidities. manifestation in mouse osteocytes raises markedly after ~18 weeks old in both sexes (Supplementary Fig. 1a,b), coinciding using the timing of accelerated age-related bone tissue reduction in both feminine and male mice (Supplementary Fig. 1cCj)22,23. Removing a relatively little percentage (~30%) of senescent cells utilizing a suicide transgene, that allows inducible eradication of transgenic mice2C4 had been randomized to either automobile or AP20187 treatment double every week for 4 weeks, beginning at 20 weeks old (Fig. 1a). As expected, AP20187 treatment led to markedly lower mRNA manifestation (by ?59%) in bone tissue in accordance with vehicle-treated mice (Fig. 1b) aswell as lower mRNA (by ?48%) encoded from the transgene2C4 (Fig. 1c), in keeping with clearance of senescent cells. This is verified by fewer senescent osteocytes in AP20187- versus vehicle-treated mice (by ?46%), as assessed by a recognised senescence biomarker (senescence-associated distension of satellites [SADS]9,16 (Fig. 1dCf); discover Supplementary Fig. 3 and tale for an additional, detailed validation from the SADS assay using major osteocyte ethnicities)9,16. Remember that we utilized three methods of senescent cell burden in bone tissue (mRNA, mRNA encoded with the transgene, and SADS-positive osteocytes), all with concordantly lower beliefs in AP20187- versus vehicle-treated mice. The systemic clearance of senescent cells by AP20187 treatment was additional showed by lower (Fig. 1g) and (Fig. 1h) mRNA amounts in adipose tissues. Open in another screen Fig. 1 Clearance of senescent cells prevents age-related bone tissue reduction. (a) Experimental style for testing the result of senescent cell clearance utilizing a transgenic strategy on age-related bone tissue reduction: 20-month-old feminine mice had been randomized to either automobile (= 13) or AP20187 (= 16) remedies (intraperitoneally [i.p] double regular) for 4 a few months. rt-qPCR evaluation of (b) and (c) (encoded with the transgene) mRNA appearance amounts in osteocyte-enriched cells produced from bones from the mice. Representative pictures ( 30 pictures per pet, 13 automobile- and 16 AP20187-treated) of (d) a senescent (SEN) osteocyte (magnification 100) versus (e) a non-senescent (non-SEN) osteocyte (magnification 100) based on the senescence-associated distension of satellites (SADS, find arrows [in d]) assay in cortical bone tissue diaphysis (range pubs, 2 m). (f) Quantification from the percentage of senescent osteocytes in mice treated with either automobile or AP20187 based on the SADS assay. rt-qPCR evaluation of (g) and (h) mRNA appearance amounts in perigonadal adipose tissues. (i) Consultant micro-computed tomography (CT) pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture on the lumbar backbone of automobile- versus AP20187-treated KC01 mice. Quantification of CT-derived (j) bone tissue volume small percentage (BV/Television; %), (k) trabecular amount (Tb.N; 1/mm), (l) trabecular width KC01 (Tb.Th; mm), (m) trabecular parting (Tb.Sp; mm), and (n) framework model index (SMI, a way of measuring plate/fishing rod morphology, with lower quantities being better) on the lumbar spine. (o) Consultant CT pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture on the femur. Quantification of CT-derived (p) cortical width (Ct.Th, mm) and (q) micro-finite-element evaluation (FEA)-derived failure insert (N, Newton [a way of measuring bone tissue power]). Histomorphometric quantification on the femoral endocortical surface area of (r) osteoclast quantities per bone tissue perimeter (N.Oc/B.Pm;/mm), (s) osteoblast quantities per bone tissue perimeter (N.Ob/B.Pm;/mm), (t) endocortical nutrient apposition price (MAR; mcm/d), and (u) endocortical bone tissue formation price per bone tissue surface area (BFR/BS; mcm3/mcm2/d) (= 8/group). (v) Mineralization of osteoblastic MC3T3 cells.Ice-cold PBS was utilized to avoid the enzymatic reaction after that. helpful effects of concentrating on senescent cells had been because of lower bone tissue resorption with either preserved (trabecular bone tissue) or more (cortical bone tissue) bone tissue formation when compared with vehicle-treated mice. research showed that senescent cell-conditioned moderate impaired osteoblast mineralization and improved osteoclast progenitor success, leading to elevated osteoclastogenesis. Collectively, these data set up a causal function for senescent cells in bone tissue loss with maturing and demonstrate that concentrating on these cells provides both anti-resorptive and anabolic results on bone tissue. As getting rid of senescent cells and/or inhibiting their pro-inflammatory secretome also increases cardiovascular function4, enhances insulin awareness3, and decreases frailty7, concentrating on this fundamental system to avoid age-related bone tissue reduction suggests a book treatment strategy not merely for osteoporosis also for multiple age-related co-morbidities. appearance in mouse osteocytes increases markedly after ~18 months of age in both sexes (Supplementary Fig. 1a,b), coinciding with the timing of accelerated age-related bone loss in both female and male mice (Supplementary Fig. 1cCj)22,23. Eliminating a relatively small proportion (~30%) of senescent cells using a suicide transgene, that permits inducible elimination of transgenic mice2C4 were randomized to either vehicle or AP20187 treatment twice weekly for 4 months, starting at 20 months of age (Fig. 1a). As anticipated, AP20187 treatment resulted in markedly lower mRNA expression (by ?59%) in bone relative to vehicle-treated mice (Fig. 1b) as well as lower mRNA (by ?48%) encoded by the transgene2C4 (Fig. 1c), consistent with clearance of senescent cells. This was confirmed by fewer senescent osteocytes in AP20187- versus vehicle-treated mice (by ?46%), as assessed by an established senescence biomarker (senescence-associated distension of satellites [SADS]9,16 (Fig. 1dCf); see Supplementary Fig. 3 and legend for a further, detailed validation of the SADS assay using primary osteocyte cultures)9,16. Note that we used three steps of senescent cell burden in bone (mRNA, mRNA encoded by the transgene, and SADS-positive osteocytes), all with concordantly lower values in AP20187- versus vehicle-treated mice. The systemic clearance of senescent cells by AP20187 treatment was further exhibited by lower (Fig. 1g) and (Fig. 1h) mRNA levels in adipose tissue. Open in a separate windows Fig. 1 Clearance of senescent cells prevents age-related bone loss. (a) Experimental design for testing the effect of senescent cell clearance using a transgenic approach on age-related bone loss: 20-month-old female mice were randomized to either vehicle (= 13) or AP20187 (= 16) treatments (intraperitoneally [i.p] twice weekly) for 4 months. rt-qPCR analysis of (b) and (c) (encoded by the transgene) mRNA expression levels in osteocyte-enriched cells derived from bones of the mice. Representative images ( 30 images per animal, 13 vehicle- and 16 AP20187-treated) of (d) a senescent (SEN) osteocyte (magnification 100) versus (e) a non-senescent (non-SEN) osteocyte (magnification 100) according to the senescence-associated distension of satellites (SADS, see arrows [in d]) assay in cortical bone diaphysis (scale bars, 2 m). (f) Quantification of the percentage of senescent osteocytes in mice treated with either vehicle or AP20187 according to the SADS assay. rt-qPCR analysis of (g) and (h) mRNA expression levels in perigonadal adipose tissue. (i) Representative micro-computed tomography (CT) images (= 13 vehicle- and 16 AP20187-treated mice) of bone microarchitecture at the lumbar spine of vehicle- versus AP20187-treated mice. Quantification of CT-derived (j) bone volume fraction (BV/TV; %), (k) trabecular number (Tb.N; 1/mm), (l) trabecular thickness (Tb.Th; mm), (m) trabecular separation (Tb.Sp; mm), and (n) structure model index (SMI, a measure of plate/rod morphology, with lower numbers being better) at the lumbar spine. (o) Representative CT images (= 13 vehicle- and 16 AP20187-treated mice) of bone microarchitecture at the femur. Quantification of CT-derived (p) cortical thickness (Ct.Th, mm) and (q) micro-finite-element analysis (FEA)-derived failure load (N, Newton [a measure of bone strength]). Histomorphometric quantification at the femoral endocortical surface of (r) osteoclast numbers per bone perimeter (N.Oc/B.Pm;/mm), (s) osteoblast numbers per bone perimeter (N.Ob/B.Pm;/mm), (t) endocortical mineral apposition rate (MAR; mcm/d), and (u) endocortical bone formation rate per bone surface (BFR/BS; mcm3/mcm2/d) (= 8/group). (v) Mineralization of osteoblastic MC3T3 cells exposed to control (CON) or senescent (SEN) conditioned medium (CM) (= 3/group), with quantification of eluted alizarin red dye in (w). Data represent Mean SEM (error bars). * 0.05; ** 0.01; *** 0.001 (independent samples mice with AP20187 did not alter bone parameters (Supplementary Fig. 6aCo), indicating that this strategy is specific for prevention of age-related bone loss. Trabecular bone histomorphometry in the aged mice demonstrated significantly lower bone resorption (osteoclast numbers per bone perimeter; Supplementary Fig. 7a) in AP20187- versus vehicle-treated mice, without a coupled reduction in bone formation indices (osteoblast numbers, mineral apposition rate, and bone formation rate [Supplementary Fig. 7bCd]). We excluded possible direct effects.Cultures were incubated for 10 days and colonies were identified as monocyte/macrophage (M) or granulocyte-monocyte/macrophage (GM). bone formation as compared to vehicle-treated mice. studies demonstrated that senescent cell-conditioned medium impaired osteoblast mineralization and enhanced osteoclast progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. As eliminating senescent cells and/or inhibiting their pro-inflammatory secretome also improves cardiovascular function4, enhances insulin sensitivity3, and reduces frailty7, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis but also for multiple age-related co-morbidities. expression in mouse osteocytes increases markedly after ~18 months of age in both sexes (Supplementary Fig. 1a,b), coinciding with the timing of accelerated age-related bone loss in both female and male mice (Supplementary Fig. 1cCj)22,23. Eliminating a relatively small proportion (~30%) of senescent cells using a suicide transgene, that permits inducible elimination of transgenic mice2C4 were randomized to either vehicle or AP20187 treatment twice weekly for 4 months, starting at 20 months of age (Fig. 1a). As anticipated, AP20187 treatment resulted in markedly lower mRNA expression (by ?59%) in bone relative to vehicle-treated mice (Fig. 1b) as well as lower mRNA (by ?48%) encoded by the transgene2C4 (Fig. 1c), consistent with clearance of senescent cells. This was confirmed by fewer senescent osteocytes in AP20187- versus vehicle-treated mice (by ?46%), as assessed by an established senescence biomarker (senescence-associated distension of satellites [SADS]9,16 (Fig. 1dCf); see Supplementary Fig. 3 and legend for a further, detailed validation of the SADS assay using primary osteocyte cultures)9,16. Note that we used three measures of senescent cell burden in bone (mRNA, mRNA encoded by the transgene, and SADS-positive osteocytes), all with concordantly lower values in AP20187- versus vehicle-treated mice. The systemic clearance of senescent cells by AP20187 treatment was further demonstrated by lower (Fig. 1g) and (Fig. 1h) mRNA levels in adipose tissue. Open in a separate window Fig. 1 Clearance of senescent cells prevents age-related bone loss. (a) Experimental design for testing the effect of senescent cell clearance using a transgenic approach on age-related bone loss: 20-month-old female mice were randomized to either vehicle (= 13) or AP20187 (= 16) treatments (intraperitoneally [i.p] twice weekly) for 4 months. rt-qPCR analysis of (b) and (c) (encoded by the transgene) mRNA expression levels in osteocyte-enriched cells derived from bones of the mice. Representative images ( 30 images per animal, 13 vehicle- and 16 AP20187-treated) of (d) a senescent (SEN) osteocyte (magnification 100) versus (e) a non-senescent (non-SEN) osteocyte (magnification 100) according to the senescence-associated distension of satellites (SADS, see arrows [in d]) assay in cortical bone diaphysis (scale bars, 2 m). (f) Quantification of the percentage of senescent osteocytes in mice treated with either vehicle or AP20187 according to the SADS assay. rt-qPCR analysis of (g) and (h) mRNA expression levels in perigonadal adipose tissue. (i) Representative micro-computed tomography (CT) images (= 13 vehicle- and 16 AP20187-treated mice) of bone microarchitecture at the lumbar spine of vehicle- versus AP20187-treated mice. Quantification of CT-derived (j) bone volume fraction (BV/TV; %), (k) trabecular number (Tb.N; 1/mm), (l) trabecular thickness (Tb.Th; mm), (m) trabecular separation (Tb.Sp; mm), and (n) structure model index (SMI, a measure of plate/rod morphology, with lower numbers being better) at the lumbar spine. (o) Representative CT images (= 13 vehicle- and 16 AP20187-treated mice) of bone microarchitecture in the femur. Quantification of CT-derived (p) cortical thickness (Ct.Th, mm) and (q) micro-finite-element analysis (FEA)-derived failure weight (N, Newton [a measure of bone strength]). Histomorphometric quantification in the femoral endocortical surface of (r) osteoclast figures per bone perimeter (N.Oc/B.Pm;/mm), (s) osteoblast figures.(a) Representative images (from three individual biological replicates) of tartrate-resistant acid phosphatase (Capture) stained osteoclast ethnicities (magnification 4; level bars, 1000 m) and (b) osteoclast figures per well following four days of osteoclast differentiation of bone marrow cells pre-treated with vehicle (bad control), M-CSF (25ng/mL), control (CON) conditioned medium (CM), or senescent (SEN) CM (= 3/group). microarchitecture compared to vehicle-treated mice. The beneficial effects of focusing on senescent cells were due to lower bone resorption with either managed (trabecular bone) or higher (cortical bone) bone formation as compared to vehicle-treated mice. studies shown that senescent cell-conditioned medium impaired osteoblast mineralization and enhanced osteoclast progenitor survival, leading to improved osteoclastogenesis. Collectively, these data establish a causal part for senescent cells in bone loss with ageing and demonstrate that focusing on these cells offers both anti-resorptive and anabolic effects on bone. As removing senescent cells and/or inhibiting their pro-inflammatory secretome also enhances cardiovascular function4, enhances insulin level of sensitivity3, and reduces frailty7, focusing on this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis but also for multiple age-related co-morbidities. manifestation in mouse osteocytes raises markedly after ~18 weeks of age in both sexes (Supplementary Fig. 1a,b), coinciding with the timing of accelerated age-related bone loss in both female and male mice (Supplementary Fig. 1cCj)22,23. Removing a relatively small proportion (~30%) of senescent cells using a suicide transgene, that permits inducible removal of transgenic mice2C4 were randomized to either vehicle or AP20187 treatment twice weekly for 4 weeks, starting at 20 weeks of age (Fig. 1a). As anticipated, AP20187 treatment resulted in markedly lower mRNA manifestation (by ?59%) in bone relative to vehicle-treated mice (Fig. 1b) as well as lower mRNA (by ?48%) encoded from the transgene2C4 (Fig. 1c), consistent with clearance of senescent cells. This was confirmed by fewer senescent osteocytes in AP20187- versus vehicle-treated mice (by ?46%), as assessed by an established senescence biomarker (senescence-associated distension of satellites [SADS]9,16 (Fig. 1dCf); observe Supplementary Fig. 3 and story for a further, detailed validation of the SADS assay using main osteocyte ethnicities)9,16. Note that we used three actions of senescent cell burden in bone (mRNA, mRNA encoded from the transgene, and SADS-positive osteocytes), all with concordantly lower ideals in AP20187- versus vehicle-treated mice. The KC01 systemic clearance of senescent cells by AP20187 treatment was further shown by lower (Fig. 1g) and (Fig. 1h) mRNA levels in adipose cells. Open in a separate windowpane Fig. 1 Clearance of senescent cells prevents age-related bone tissue reduction. (a) Experimental style for testing the result of senescent cell clearance utilizing a transgenic strategy on age-related bone tissue reduction: 20-month-old feminine mice had been randomized to either automobile (= 13) or AP20187 (= 16) remedies (intraperitoneally [i.p] double regular) for 4 a few months. rt-qPCR evaluation of (b) and (c) (encoded with the transgene) mRNA appearance amounts in osteocyte-enriched cells produced from bones from the mice. Representative pictures ( 30 pictures per pet, 13 automobile- and 16 AP20187-treated) of (d) a senescent (SEN) osteocyte (magnification 100) versus (e) a non-senescent (non-SEN) osteocyte (magnification 100) based on the senescence-associated distension of satellites (SADS, find arrows [in d]) assay in cortical bone tissue diaphysis (range pubs, 2 m). (f) Quantification from the percentage of senescent osteocytes in mice treated with either automobile or AP20187 based on the SADS assay. rt-qPCR evaluation of (g) and (h) mRNA appearance amounts in perigonadal adipose tissues. (i) Consultant micro-computed tomography (CT) pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture on the lumbar backbone of automobile- versus AP20187-treated mice. Quantification of CT-derived (j) bone tissue volume small percentage (BV/Television; %), (k) trabecular amount (Tb.N; 1/mm), (l) trabecular width (Tb.Th; mm), (m) trabecular parting (Tb.Sp; mm), and (n) framework model index (SMI, a way of measuring plate/fishing rod morphology, with lower quantities being better) on the lumbar spine. (o) Consultant CT pictures (= 13 automobile- and 16 AP20187-treated mice) of bone tissue microarchitecture on the femur. Quantification of CT-derived (p) cortical width (Ct.Th, mm) and (q) micro-finite-element evaluation (FEA)-derived failure insert (N, Newton [a way of measuring bone tissue power]). Histomorphometric quantification on the femoral endocortical surface area of (r) osteoclast quantities per bone tissue perimeter (N.Oc/B.Pm;/mm), (s) osteoblast quantities per bone tissue perimeter (N.Ob/B.Pm;/mm), (t) endocortical nutrient apposition price (MAR; mcm/d), and (u) endocortical bone tissue formation price per bone tissue surface area (BFR/BS; mcm3/mcm2/d) (= 8/group). (v) Mineralization of osteoblastic MC3T3 cells subjected to control (CON) or senescent (SEN) conditioned moderate (CM) (= 3/group), with quantification of.