Leonid Kandel

Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells.

The culture expansion of human mesenchymal stem cells (hMSCs) may alter their characteristics and is a costly and time‐consuming stage. This study demonstrates for the first time that immunoisolated noncultured CD105‐positive (CD105+) hMSCs are multipotent in vitro and exhibit the capacity to form bone in vivo. hMSCs are recognized as promising tools for bone regeneration. However, the culture stage is a limiting step in the clinical setting. To establish a simple, efficient, and fast method for applying these cells for bone formation, a distinct population of CD105+ hMSCs was isolated from bone marrow (BM) by using positive selection based on the expression of CD105 (endoglin). The immunoisolated CD105+ cell fraction represented 2.3% ± 0.45% of the mononuclear cells (MNCs). Flow cytometry analysis of freshly immunoisolated CD105+ cells revealed a purity of 79.7% ± 3.2%. In vitro, the CD105+ cell fraction displayed significantly more colony‐forming units‐fibroblasts (CFU‐Fs; 6.3 ± 1.4) than unseparated MNCs (1.1 ± 0.3; p < .05). Culture‐expanded CD105+ cells expressed CD105, CD44, CD29, CD90, and CD106 but not CD14, CD34, CD45, or CD31 surface antigens, and these cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. In addition, freshly immunoisolated CD105+ cells responded in vivo to recombinant bone morphogenetic protein‐2 by differentiating into chondrocytes and osteoblasts. Genetic engineering of freshly immunoisolated CD105+ cells was accomplished using either adenoviral or lentiviral vectors. Based on these findings, it is proposed that noncultured BM‐derived CD105+ hMSCs are osteogenic cells that can be genetically engineered to induce tissue generation in vivo.

75-kd sirtuin 1 blocks tumor necrosis factor α–mediated apoptosis in human osteoarthritic chondrocytes

OBJECTIVE Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. METHODS The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. RESULTS Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. CONCLUSION These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

Set7/9 Impacts COL2A1 Expression Through Binding and Repression of SirT1 Histone Deacetylation

Type II collagen is a key cartilaginous extracellular protein required for normal endochondral development and cartilage homeostasis. COL2A1 gene expression is positively regulated by the NAD‐dependent protein deacetylase Sirtuin 1 (SirT1), through its ability to bind chromatin regions of the COL2A1 promoter and enhancer. Although SirT1/Sox9 binding on the enhancer site of COL2A1 was previously demonstrated, little is known about its functional role on the gene promoter site. Here, we examined the mechanism by which promoter‐associated SirT1 governs COL2A1 expression. Human chondrocytes were encapsulated in three‐dimensional (3D) alginate beads where they exhibited upregulated COL2A1 mRNA expression and increased levels of SirT1 occupancy on the promoter and enhancer regions, when compared to monolayer controls. Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA‐binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site. ChIP reChIP assays revealed that SirT1 and Set7/9 form a protein complex on the COL2A1 promoter region of 3D‐cultured chondrocytes, which also demonstrated elevated trimethylated lysine 4 on histone 3 (3MeH3K4), a hallmark of Set7/9 methyltransferase activity. Advanced passaging of chondrocytes yielded a decrease in 3MeH3K4 and Set7/9 levels on the COL2A1 promoter and reduced COL2A1 expression, suggesting that the SirT1/Set7/9 complex is preferentially formed on the COL2A1 promoter and required for gene activation. Interestingly, despite SirT1 occupancy, its deacetylation targets (ie, H3K9/14 and H4K16) were found acetylated on the COL2A1 promoter of 3D‐cultured chondrocytes. A possible explanation for this phenotype is the enrichment of the histone acetyltransferases P300 and GCN5 on the COL2A1 promoter of3 D‐cultured chondrocytes. Our study indicates that Set7/9 prevents the histone deacetylase activity of SirT1, potentiating euchromatin formation on the promoter site of COL2A1 and resulting in morphology‐dependent COL2A1 gene transactivation.

Detecting cathepsin activity in human osteoarthritis via activity-based probes

IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Effect of Severe Aortic Stenosis on the Outcome in Elderly Patients Undergoing Repair of Hip Fracture

Background: The perioperative assessment and management of elderly patients with hip fracture and significant aortic stenosis (AS) is an increasingly common clinical problem with little data available to guide perioperative management. Objectives: It was the aim of this study to examine the incidence of perioperative events in an elderly population of patients with severe AS undergoing repair of hip fracture as compared with controls without severe AS. Methods: Patients over the age of 70 with an echocardiographic diagnosis of severe AS defined as an aortic valve area ≤1.0 cm2 who underwent surgery for hip fracture repair were retrospectively identified. An age-matched group of patients without a history of AS who underwent surgical repair of hip fracture was the control group. The primary outcome of the incidence of postoperative cardiac events defined as death, acute coronary syndrome or pulmonary edema within 30 days was compared. Results: Thirty-two patients with AS (median age 84.5 years, range 72–94; 27 females and 5 males) and 88 controls (median age 86 years, range 80–95; 67 females and 21 males) were entered into the study. There were no significant differences between the AS group and controls for 30-day mortality (6.2 vs. 6.8%) or for the total cardiac event rate (18.7 vs. 11.8%). Conclusions: Our results demonstrate that elderly patients with severe AS can safely undergo repair of hip fractures with a mortality and morbidity comparable with a control population. These patients should not be denied surgery on the basis of their aortic valve disease.

75kDa SirT1 Blocks TNFα-Mediated Apoptosis in Human Osteoarthritic Chondrocytes

OBJECTIVE Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. METHODS The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. RESULTS Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. CONCLUSION These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes.

Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age‐related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three‐dimensional alginate microbeads (3D). SOX9 was hypo‐acetylated in 3D cultures and displayed enhanced binding to a −10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co‐immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin β by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.

Navigation-Assisted Minimally Invasive Two-Incision Total Hip Arthoplasty

Flouroscopy-based navigation can increase accuracy in two-incision minimally invasive THA, a novel technique develop for promoting fast recovery.

Bipolar hip hemiarthroplasty in a patient with an above knee amputation: a case report

The treatment of an above knee amputee who has sustained a fracture of femoral neck is a challenge for both the orthopaedic surgeon and the rehabilitation team. We present a case of such a patient and discuss different difficulties in his treatment.

Extensive Regulatory Changes in Genes Affecting Vocal and Facial Anatomy Separate Modern from Archaic Humans

Changes in gene regulation are broadly accepted as key drivers of phenotypic differences between closely related species. However, identifying regulatory changes that shaped human-specific traits is a very challenging task. Here, we use >60 DNA methylation maps of ancient and present-day human groups, as well as six chimpanzee maps, to detect regulatory changes that emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes affecting vocalization and facial features went through particularly extensive methylation changes. Specifically, we identify silencing patterns in a network of genes (SOX9, ACAN, COL2A1 and NFIX), and propose that they might have played a role in the reshaping of the human face, and in forming the 1:1 vocal tract configuration that is considered optimal for speech. Our results provide insights into the molecular mechanisms that may have shaped the modern human face and voice, and suggest that they arose after the split from Neanderthals and Denisovans.Summary Regulatory changes are broadly accepted as key drivers of phenotypic divergence. However, identifying regulatory changes that underlie human-specific traits has proven very challenging. Here, we use 63 DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes affecting the face and vocal tract went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-affecting genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract.Identifying changes in gene regulation that shaped human-specific traits is critical to understanding human adaptation. Here, we use dozens of ancient and present-day DNA methylation maps to detect regulatory changes that emerged in modern humans. We show that genes affecting vocalization and facial features went through particularly extensive changes in methylation. Especially, we identify expansive changes in a network of genes regulating skeletal development (SOX9, ACAN and COL2A1), and in NFIX, which controls facial projection and voice box (larynx) development. We propose that these changes played a key role in shaping the human face, and in forming the human 1:1 vocal tract configuration that is considered optimal for speech. Our results provide insights into the molecular mechanisms that shaped the modern human face and voice, and suggest that they arose after the split from Neanderthals and Denisovans.