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Modified enzymatic collagen digestion-mediated isolation of osteocytes

BIOCELL 2022年第4期46卷 1097-1104页

作者： WENJUAN XU GUANGMING DAI YIFEI LYU YINING ZHANG XIAOLIN TU Institute of Life and Science Research Chongqing Medical UniversityChongqing400016China Department of Bioengineering University of WashingtonSeattle98195USA

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone.Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times,and the digested cells and bone chips(BC)were cultured,digested,and passaged when cells were fully grown.The types of cells obtained were identified by morphology,viable cell counts,alkaline phosphatase staining,and biochemical activity analyses,and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction.Our results showed that among the cells obtained from the third digestion(fractions 7–9)of wild mice tibias and femurs and the remaining BCs,85%–90%of the cells were osteocytes.Moreover,their morphology was approximately one-tenth to one-fifth the size of osteoblasts,star-shaped or polygonal,with a dendritic structure,negative for alkaline phosphatase staining,and showed a high expression of dmp1 and sclerostin.Ninety percent of the cells in fractions 1–3 were osteoblasts,and were fusiform or polygonal shape.The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction,while the expression of osteocyte-specific dmp1 and sclerostin was not detected.In the second portion(fractions 4–6),a large number were osteoblasts,mixed with a small number of osteocytes,and had high alkaline phosphatase activity and osteocyte mRNA levels,a specific level of the osteocyte marker dmp1,and no sclerostin was detected.osteocytes in daβcatot mice were also successfully isolated by this method,and we found that Wnt signaling increased the proliferation of these osteocytes.The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone,which provides an avenue for the study of osteocyte function in vitro.

关键词： osteocytes Cell culture Collagen protease Sclerostin Alkaline phosphatase

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Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats

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Animal Models and Experimental Medicine 2019年第1期2卷 51-57页

作者： Priscilla Hakime Scalize Luiz Gustavo de Sousa Lígia Maria Napolitano Goncalves Dimitrius Leonardo Pitol Marcelo Palinkas Antonio Augusto Coppi Mariah Acioli Righeti Vitória Ricardo Karina Fittipaldi Bombonato-Prado Simone Cecílio Hallak Regalo Selma Siessere Department of Morphology Physiology and Basic PathologySchool of Dentistry of Ribeirao PretoUSP-University of Sao Paulo Faculty of Health and Medical Sciences School of Veterinary MedicineUniversity of Surrey

Background : Osteoporosis can make bone repair difficult. Low-level laser therapy( LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology. Methods : Fifty-four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups(n = ?6 per group), and 24 hours after the bone defects were created they received three, six or 12 sessions of LLLT at 0, 20 or 30 J/cm 2, using a 780-nm low-intensity GaAlAs laser. One-way ANOVA followed by Tukey ' s post hoc test was used for data processing. A difference of P osteocytes, especially with a laser energy density of 30 J/cm^2 given for six and 12 sessions.

关键词： bone low-level laser therapy osteocytes osteoporosis

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Elevated Intracellular Ca^(2+) Signals by Oxidative Stress Activate Connexin 43 Hemichannels in osteocytes

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Bone Research 2013年第4期1卷 355-361页

作者： Manuel A.Riquelme Jean X.Jiang Department of Biochemistry the University of Texas Health Science Center San Antonio

Elevated oxidative stress （OS） during aging leads to bone loss. OS increases intracellular Ca2＋（[Ca2＋]i）, resulting in cellular damage and death. We show earlier that Cx43 hemichannels open in response to OS, which serves as a protective mechanism for osteocytes. However, the underlying mechanism is unknown. Here, we found that treatment with H202 increased [Ca2＋]i in osteocytes with [Ca2＋]i being primarily derived from an extracellular Ca2~ source. Hemichannel opening induced by OS was inhibited by the depletion of [Ca2＋]i with BAPTA-AM, a Ca2＋ chelator, suggesting that [Ca2＋]i influenced the activity of Cx43 hemichannels. Conversely, blockade of hemichannels had no effect on [Ca2＋]i. A biotinylation assay showed that cell surface-expressed Cx43 was increased by OS, which could be inhibited by BAPTA-AM, suggesting that [Ca2＋]i is necessary for Cx43 migration to the cell surface in response to OS. Together, these data suggest that increased hemichannel activity induced by OS was likely to be caused by elevated [Ca2＋]i through increased Cx43 on the cell surface.

关键词： connexin hemichannels calcium osteocytes

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In silico analysis of RNA and small RNA sequencing data from human BM-MSCs and differentiated osteocytes, chondrocytes and tenocytes

Engineered Regeneration

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Engineered Regeneration 2021年第1期2卷 19-30页

作者： Srinivas V.Koduru Irina A.Elcheva Ashley N.Leberfinger Dino J.Ravnic Irvin S.Zubar Plastic Surgery Research Laboratory Penn State College of Medicine500 University DriveHersheyPA 17033-0850United States Department of Surgery Penn State Health Milton S.Hershey Medical CenterHersheyPAUnited States Department of Cellular and Molecular Physiology Penn State College of MedicineHersheyPAUnited States Division of Pediatric Hematology/Oncology Department of PediatricsPenn State College of MedicineHersheyPAUnited States

Background:Adult stem cells have a remarkable capacity of differentiating into various cell types necessary for tissue and organ regeneration.Multiple studies have focused on the differentiation potential of mesenchymal stem cells(MSCs),however little is known about the molecular characteristics of MSCs and their progenies obtained from donors of different ages.In this study,we analyzed publicly available sequencing data obtained from young(~22-year-old,n=8)and older(~65.5-year-old,n=8)donors of MSCs and their differentiated counterparts:osteocytes,chondrocytes and tenocytes.The raw mRNA and small RNA(non-coding RNA)sequencing data was downloaded from NIH BioProjects and systematically analyzed in order to identify uniquely expressed genes in MSC-derived osteocytes,chondrocytes and tenocytes of younger and older people.Results:We identified many commonly up-and downregulated genes are similar in both groups.However,the young group displayed a greater variety of differentially expressed genes in all analyzed MSC-derived cells.This discrepancy in gene expression profiles between younger and older groups may indicate a greater differentiation potential of MSCs isolated from younger donors.miRNA and mRNA integrated analysis showed key miRNAs that regulate mRNAs in both groups from all differentiated lineages.Conclusions:Our analysis provides additional information to previously reported data for identification of MSC markers of plasticity and engraftment.In addition,our data may shed light upon the molecular mechanisms of age-associated musculoskeletal diseases caused by a decreased capacity of MSCs to regenerate the locomotor system in elderly people.

关键词： BM-MSCs osteocytes Chondrocytes Tenocytes Small RNA miRNA-mRNA

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Irisin Attenuates Osteoarthritis by Inhibiting Apoptosis of osteocytes Through Activating Erk Signaling Pathway

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医用生物力学 2019年第A1期34卷 51-52页

作者： Zihao He Hanjun Li Feng Zhou Jingke Du Shuhong Zhang Tingting Tang Zhifeng Yu Department of Orthopedics Shanghai Key Laboratory of Orthopedic ImplantShanghai Ninth People’s HospitalShanghai Jiao Tong University School of Medicine

Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression in anterior cruciate ligament transection(ACLT)mice models and the mechanism of irisin therapy effect on OA by increase the resistance of apoptosis in MLO-Y4 cells induced by mechanical stretch in vitro.Methods For in vivo study,3-month-old male C57BL/6 J mice were randomized to three groups,sham-operated,anterior cruciate ligament transection(ACLT)-operated treated with vehicle,and ACLT-operated treated with irisin by intraperitoneal injection once a week.Cartilage erosion was observed by HE staining.Osteoarthritis Research Society International(OARSI)scores were evaluated according to the safranin O stai-ning.The microstructure of tibia cortical bone,trabecular bone,and subchondral bone was analyzed by micro-CT and the bone histomorphometry has been administrated including mineral apposition rate(MAR).Edu staining and cck-8 were used for the detection of the proliferation of MLO-Y4 cells.For mechanical stress,cells were seeded on the collagen-I coated chamber subjected with a peak biaxial stretch of 20%at 1 Hz for 16 hours to induce apoptosis.Flow cytometry was used for the detection of apoptosis and cell cycle.TUNNEL was used for staining the apoptotic cells and rt-PCR was applied for quantifying the expression of mRNA such as Bax,Bcl-2,SOST,c-myc,Opg.Western blot was utilized to confirm the mechanism of how irisin decrease the osteocyte apoptosis.Results In vivo,irisin can attenuate articular cartilage degeneration.Irisin maintains the proportion of hyaline cartilage and calcified cartilage and keep fewer cartilage erosions in ACLT-operated mice.For immunohistochemical(IHC)staining,irisin reduced the expression of caspase3,Bax and matrix metalloproteinase-13 in both cartilage and subchondral bone.Irisin-treated ACLT group shows higher Trabecular number(Tb.N)and bone volume fraction(BV/TV)compared

关键词： Irisin Attenuates Osteoarthritis Inhibiting Apoptosis osteocytes Activating Erk Signaling Pathway

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Osteocyte pericellular and perilacunar matrices as markers of bone-implant mechanical integrity

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BIOCELL 2022年第10期46卷 2209-2216页

作者： ReMY GAUTHIER HeLÈNE FOLLET ANA-MARIA TRUNFIO-SFARGHIU DELPHINE FARLAY NINA ATTIK SYLVAIN MEILLE JeRÔME CHEVALIER DAVID MITTON Universite de Lyon CNRSINSA de LyonUniversite Claude Bernard Lyon 1MATEISUMR5510VilleurbanneF-69621France Universite de Lyon Universite Claude Bernard Lyon 1INSERMLYOS UMR 1033LyonF-69008France Universite de Lyon INSA-LyonCNRS UMR5259LaMCoSVilleurbanneF-69621France Universite de Lyon Universite Claude Bernard Lyon 1UMR CNRS 5615Laboratoire des Multimateriaux et InterfacesVilleurbanneF-69622France Universite de Lyon Universite Claude Bernard Lyon 1Faculte d’OdontologieLyonF-69008France Universite de Lyon Universite Gustave EiffelUniv Claude Bernard Lyon 1LBMC UMR_T 9406LyonF-69622France

To develop durable bone healing strategies through improved control of bone repair,it is of critical importance to understand the mechanisms of bone mechanical integrity when in contact with biomaterials and implants.Bone mechanical integrity is defined here as the adaptation of structural properties of remodeled bone in regard to an applied mechanical loading.Accordingly,the authors present why future investigations in bone repair and regeneration should emphasize on the matrix surrounding the osteocytes.osteocytes are mechanosensitive cells considered as the orchestrators of bone remodeling,which is the biological process involved in bone homeostasis.These bone cells are trapped in an interconnected porous network,the lacunocanalicular network,which is embedded in a bone mineralized extracellular matrix.As a consequence of an applied mechanical loading,the bone deformation results in the deformation of this lacunocanalicular network inducing a shift in interstitial fluid pressure and velocity,thus resulting in osteocyte stimulation.The material environment surrounding each osteocyte,the so called perilacunar and pericellular matrices properties,define its mechanosensitivity.While this mechanical stimulation pathway is well known,the laws used to predict bone remodeling are based on strains developing at a tissue scale,suggesting that these strains are related to the shift in fluid pressure and velocity at the lacunocanalicular scale.While this relationship has been validated through observation in healthy bone,the fluid behavior at the bone-implant interface is more complex.The presence of the implant modifies fluid behavior,so that for the same strain at a tissue scale,the shift in fluid pressure and velocity will be different than in a healthy bone tissue.In that context,new markers for bone mechanical integrity,considering fluid behavior,have to be defined.The viewpoint exposed by the authors indicates that the properties of the pericellular and the perilacunar matrices have to be systematically investigated

关键词： Bone mechanical integrity osteocytes Pericellular Perilacunar Mechanosensitivity

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Combined OVX and Concurrent Mechanical Disuse Induced osteocytes Morphological Alteration, and Mitigation by Mechanobiology and Sclerostin Antibody

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医用生物力学 2019年第A1期34卷 29-29页

作者： Tony Zhang Yixian Qin Department of Biomedical Engineering Stony Brook University

Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed

关键词： OVX HLS Mitigation by Mechanobiology Sclerostin Antibody Sclerostin Combined OVX and Concurrent Mechanical Disuse Induced osteocytes Morphological Alteration

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Osteocyte viability and bone density in cadmium chloride-induced osteoporosis ameliorated with Pilostigma thonningii stem bark-extracted D-3-O-methy-chiroinositol

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Animal Models and Experimental Medicine 2019年第1期2卷 25-33页

作者： Edwin Aihanuwa Uwagie-Ero Clifford Nwabugwu Abiaezute Chinaka O.Nwaehujor Theophilus Okafor Nnaji Eugene Amienwanlen Odigie Oyetunde Kazeem Ekeolu Ohiemi Benjamin Ocheja Department of Surgery Faculty of Veterinary MedicineUniversity of Benin Department of Anatomy Faculty of Veterinary MedicineUniversity of Nigeria Department of Biochemistry College of Basic Medical SciencesUniversity of Calabar Department of Surgery Faculty of Veterinary MedicineUniversity of Nigeria Department of Anatomy Faculty of Veterinary MedicineUniversity of Benin Department of Physiology and Biochemistry Faculty of Veterinary MedicineUniversity of Benin

Background : This study examined the ameliorative effect of D-3-O-methylchiroinositol, isolated from the stem bark of Piliostigma thonningii, on cadmium chloride-induced osteoporosis in male Wistar rats. Methods : Thirty-six rats were assigned to three treatment groups(n = 12). Group A(2 mL distilled water), group B:(2.5 mg/kg b.w. CdCl_2) and group C:(2.5 mg/kg b.w. CdCl_2 and D-3-O-methyl-chiroinositol 2 mg/kg b.w.). Bone ash, calcium, phosphate, magnesium, and zinc content, as well as bone histological changes were determined at the end of months 1, 2, and 3. Results : There were significant differences( P ≤ 0.05) in the weight of the cervical, tibia, and femoral bones in all groups. The serum concentration of CdCl_2 was significantly different across the three groups with time. There was significant variation( P osteocytes, while the femur of Wistar rats treated with D-3-O-methyl-chiroinositol showed bone trabeculae and viable osteocytes. Conclusion : The study concluded that D-3-O-methyl-chiroinositol extract from Piliostigma thionningii stem bark ameliorated cadmium chloride-induced osteoporosis in male Wistar rats.

关键词： cadmium chloride D-3-O-methyl-chiroinositol osteocytes osteoporosis toxicity amelioration

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A Brief Review of Bone Adaptation to Unloading

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Genomics, Proteomics & Bioinformatics 2008年第1期6卷 4-7页

作者： Kazunori Hamamura Hiroki Yokota Department of Biomedical Engineering/Department of Anatomy and Cell Biology Indiana University-Purdue University IndianapolisIndianapolis IN 46202 USA

Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading （and not to loading） may exist.

关键词： weightlessness unloading osteocytes

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The multifunctional role of Notch signaling in multiple myeloma

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Journal of Cancer Metastasis and Treatment 2021年第1期7卷 300-314页

作者： Hayley MSabol Jesus Delgado-Calle Department of Physiology and Cell Biology University of Arkansas for Medical SciencesLittle RockAR 72205USA Winthrop P.Rockefeller Cancer Institute University of Arkansas for Medical SciencesLittle RockAR 72205USA

Multiple myeloma(MM)is a hematologic cancer characterized by uncontrolled growth of malignant plasma cells in the bone marrow and currently is incurable.The bone marrow microenvironment plays a critical role in MM.MM cells reside in specialized niches where they interact with multiple marrow cell types,transforming the bone/bone marrow compartment into an ideal microenvironment for the migration,proliferation,and survival of MM cells.In addition,MM cells interact with bone cells to stimulate bone destruction and promote the development of bone lesions that rarely heal.In this review,we discuss how Notch signals facilitate the communication between adjacent MM cells and between MM cells and bone/bone marrow cells and shape the microenvironment to favor MM progression and bone disease.We also address the potential and therapeutic approaches used to target Notch signaling in MM.

关键词： Notch tumor microenvironment multiple myeloma bone osteocytes osteoclasts osteoblasts γ-secretase inhibitors

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