Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial f...Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.展开更多
目的通过体内外实验探究槲皮素对骨相关细胞衰老的影响,验证槲皮素通过抗骨相关细胞衰老作用对绝经后骨质疏松症的治疗作用。方法选取小鼠骨细胞样细胞MLO-Y4及成骨细胞样细胞MC3T3-E1,构建体外压力诱导的成熟前衰老(stress induced pre...目的通过体内外实验探究槲皮素对骨相关细胞衰老的影响,验证槲皮素通过抗骨相关细胞衰老作用对绝经后骨质疏松症的治疗作用。方法选取小鼠骨细胞样细胞MLO-Y4及成骨细胞样细胞MC3T3-E1,构建体外压力诱导的成熟前衰老(stress induced premature senescence, SIPS)模型,通过qRT-PCR法和细胞衰老相关β-半乳糖苷酶染色确定细胞衰老样变化,利用CCK-8法确定槲皮素体外工作浓度,验证槲皮素在骨相关细胞SIPS中的作用。建立去势小鼠骨质疏松模型,槲皮素灌胃给药,与经典雌激素疗法相比较,利用micro-CT扫描分析骨参数及骨微结构的变化,并通过qPCR检测小鼠皮质骨内衰老相关基因p21、p53,衰老相关分泌表型(senescence associated secretory phenotype, SASP)TNF-α、IL-6以及破骨相关基因TRAP、CTSK和成骨相关基因OCN、RUNX2的表达情况。结果体外实验证明槲皮素能有效抑制骨相关细胞SIPS样改变( P <0.05)。体内实验发现,给药12周后,相较对照组小鼠,槲皮素组小鼠股骨骨表面积与骨体积比值(BS/BV)和骨小梁分离度(Tb.Sp)显著降低( P <0.05),骨小梁数量(Tb.N)显著升高( P < 0.05),骨质疏松程度减轻,较雌激素疗法差异无统计学意义,并伴骨内SIPS相关基因p21、p53及SASP水平下调( P <0.05),成骨水平不受明显抑制。结论槲皮素可通过抑制细胞衰老挽救由雌激素缺乏导致的骨丢失;这可能成为绝经后骨质疏松症治疗的新手段。展开更多
基金the National Natural Science Foundation of China(Nos.U1601220,82072450,and 81672118)Chongqing Science and Technology Commission-Basic Science and Frontier Technology Key Project(No.cstc2015jcyjBX0119)Chongqing Medical University Intelligent Medicine Research Project(No.ZHYX202115).
文摘Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.
文摘目的通过体内外实验探究槲皮素对骨相关细胞衰老的影响,验证槲皮素通过抗骨相关细胞衰老作用对绝经后骨质疏松症的治疗作用。方法选取小鼠骨细胞样细胞MLO-Y4及成骨细胞样细胞MC3T3-E1,构建体外压力诱导的成熟前衰老(stress induced premature senescence, SIPS)模型,通过qRT-PCR法和细胞衰老相关β-半乳糖苷酶染色确定细胞衰老样变化,利用CCK-8法确定槲皮素体外工作浓度,验证槲皮素在骨相关细胞SIPS中的作用。建立去势小鼠骨质疏松模型,槲皮素灌胃给药,与经典雌激素疗法相比较,利用micro-CT扫描分析骨参数及骨微结构的变化,并通过qPCR检测小鼠皮质骨内衰老相关基因p21、p53,衰老相关分泌表型(senescence associated secretory phenotype, SASP)TNF-α、IL-6以及破骨相关基因TRAP、CTSK和成骨相关基因OCN、RUNX2的表达情况。结果体外实验证明槲皮素能有效抑制骨相关细胞SIPS样改变( P <0.05)。体内实验发现,给药12周后,相较对照组小鼠,槲皮素组小鼠股骨骨表面积与骨体积比值(BS/BV)和骨小梁分离度(Tb.Sp)显著降低( P <0.05),骨小梁数量(Tb.N)显著升高( P < 0.05),骨质疏松程度减轻,较雌激素疗法差异无统计学意义,并伴骨内SIPS相关基因p21、p53及SASP水平下调( P <0.05),成骨水平不受明显抑制。结论槲皮素可通过抑制细胞衰老挽救由雌激素缺乏导致的骨丢失;这可能成为绝经后骨质疏松症治疗的新手段。
