Osterix-driven LINC complex disruption in vivo diminishes osteogenesis at 8 weeks but not at 15 weeks.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a crucial connective component between the nuclear envelope and the cytoskeleton involving various cellular processes including nuclear positioning, nuclear architecture, and mechanotransduction. How LINC complexes regulate bone formation in vivo, however, is not well understood. To start bridging this gap, here we created a LINC disruption murine model using transgenic mice expressing Cre recombinase enzyme under the control of the Osterix (Osx-Cre) which is primarily active in pre-osteoblasts and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Tg(CAG-LacZ/EGFP-KASH2) mice contain a lox-STOP-lox flanked LacZ gene which is deleted upon cre recombination allowing for the overexpression of an EGFP-KASH2 fusion protein. This overexpressed protein disrupts endogenous Nesprin-Sun binding leading to disruption of LINC complexes. Thus, crossing these two lines results in an  Osx- driven  LINC  disruption (ODLD) specific to pre-osteoblasts. In this study, we investigated how this LINC disruption affects exercise-induced bone accrual. ODLD cells had decreased osteogenic and adipogenic potential in vitro compared to non-disrupted controls and sedentary ODLD mice showed decreased bone quality at 8 weeks. Upon access to a voluntary running wheel, ODLD animals showed increased running time and distance; however, our 6-week exercise intervention did not significantly affect bone microarchitecture and bone mechanical properties.

Nuclear actin structure regulates chromatin accessibility.

Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

Porcine computational modeling to investigate developmental dysplasia of the hip.

While it is well-established that early detection and initiation of treatment of developmental dysplasia of the hip (DDH) is crucial to successful clinical outcomes, research on the mechanics of the hip joint during healthy and pathological hip development in infants is limited. Quantification of mechanical behavior in both the healthy and dysplastic developing joints may provide insight into the causes of DDH and facilitate innovation in treatment options. In this study, subject-specific three-dimensional finite element models of two pigs were developed: one healthy pig and one pig with induced dysplasia in the right hindlimb. The objectives of this study were: (1) to characterize mechanical behavior in the acetabular articular cartilage during a normal walking cycle by analyzing six metrics: contact pressure, contact area, strain energy density, von Mises stress, principal stress, and principal strain; and (2) to quantify the effect on joint mechanics of three anatomic abnormalities previously identified as related to DDH: variation in acetabular coverage, morphological changes in the femoral head, and changes in the articular cartilage. All metrics, except the contact area, were elevated in the dysplastic joint. Morphological changes in the femoral head were determined to be the most significant factors in elevating contact pressure in the articular cartilage, while the effects of acetabular coverage and changes in the articular cartilage were less significant. The quantification of the pathomechanics of DDH in this study can help identify key mechanical factors that restore normal hip development and can lead to mechanics-driven treatment options.

Diet-Stimulated Marrow Adiposity Fails to Worsen Early, Age-Related Bone Loss.

INTRODUCTION: Longitudinal effect of diet-induced obesity on bone is uncertain. Prior work showed both no effect and a decrement in bone density or quality when obesity begins prior to skeletal maturity. We aimed to quantify long-term effects of obesity on bone and bone marrow adipose tissue (BMAT) in adulthood. METHODS: Skeletally mature, female C57BL/6 mice (n = 70) aged 12 weeks were randomly allocated to low-fat diet (LFD; 10% kcal fat; n = 30) or high-fat diet (HFD; 60% kcal fat; n = 30), with analyses at 12, 15, 18, and 24 weeks (n = 10/group). Tibial microarchitecture was analyzed by µCT, and volumetric BMAT was quantified via 9.4T MRI/advanced image analysis. Histomorphometry of adipocytes and osteoclasts, and qPCR were performed. RESULTS: Body weight and visceral white adipose tissue accumulated in response to HFD started in adulthood. Trabecular bone parameters declined with advancing experimental age. BV/TV declined 22% in LFD (p = 0.0001) and 17% in HFD (p = 0.0022) by 24 weeks. HFD failed to appreciably alter BV/TV and had negligible impact on other microarchitecture parameters. Both dietary intervention and age accounted for variance in BMAT, with regional differences: distal femoral BMAT was more responsive to diet, while proximal femoral BMAT was more attenuated by age. BMAT increased 60% in the distal metaphysis in HFD at 18 and 24 weeks (p = 0.0011). BMAT in the proximal femoral diaphysis, unchanged by diet, decreased 45% due to age (p = 0.0002). Marrow adipocyte size via histomorphometry supported MRI quantification. Osteoclast number did not differ between groups. Tibial qPCR showed attenuation of some adipose, metabolism, and bone genes. A regulator of fatty acid ß-oxidation, cytochrome C (CYCS), was 500% more abundant in HFD bone (p < 0.0001; diet effect). CYCS also increased due to age, but to a lesser extent. HFD mildly increased OCN, TRAP, and SOST. CONCLUSIONS: Long-term high fat feeding after skeletal maturity, despite upregulation of visceral adiposity, body weight, and BMAT, failed to attenuate bone microarchitecture. In adulthood, we found aging to be a more potent regulator of microarchitecture than diet-induced obesity.