No abstract

Cells respond to mechanical cues from their environment through a process of mechanosensing and mechanotransduction. Cell stretching devices are important tools to study the molecular pathways responsible for cellular responses to mechanobiological processes. We describe the development and testing of a uniaxial cell stretcher that has applications for microscopic as well as biochemical analyses. By combining simple fabrication techniques with adjustable control parameters, the stretcher is designed to fit a variety of experimental needs. The stretcher can be used for static and cyclic stretching. As a proof of principle, we visualize stretch induced deformation of cell nuclei via incremental static stretch, and changes in IEX1 expression via cyclic stretching. This stretcher is easily modified to meet experimental needs, inexpensive to build, and should be readily accessible for most laboratories with access to 3D printing.

Autologous chondrocyte implantation (ACI) is a cell therapy to repair cartilage defects. In ACI a biopsy is taken from a non-load bearing area of the knee and expanded in-vitro. The expansion process provides the benefit of generating a large number of cells required for implantation; however, during the expansion these cells de-differentiate and lose their chondrocyte phenotype. In this review we focus on examining the de-differentiation phenotype from a mechanobiology and biophysical perspective, highlighting some of the nuclear mechanics and chromatin changes in chondrocytes seen during the expansion process and how this relates to the gene expression profile. We propose that manipulating chondrocyte nuclear architecture and chromatin organization will highlight mechanisms that will help to preserve the chondrocyte phenotype.

The layer of stem cells surrounding developing limbs is essential for bone formation and regeneration. Our work addresses the critical question of how these stem cells and bone template communicate to ensure that limbs form correctly. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor whose mutations are linked to bone and joint pathologies. Here, we show the abundant expression of LRP1 in skeletal progenitor cells, especially in the perichondrium - the dense layer of fibrous connective tissue enveloping the cartilage of the developing limb bud. Our mouse models reveal that LRP1 deficiency in these stem cells (Lrp1flox/flox/Prrx1Cre) but not in chondrocytes (Lrp1flox/flox/AcanCreERT2) causes disrupted articulation and cavitation starting at as early as embryonic stage 16.5. LRP1 deficiency is also associated with aberrant accumulation of LRP1 ligands including tissue-inhibitor of metalloproteinase 3 and CCN2. These early abnormalities result in severe defects in multiple joints, plus markedly deformed and low-density long bones leading to dwarfism and impaired mobility. Ourin vitroexploration shows unique regulation of non-canonical WNT components by LRP1 that may explain the malformation of long bones. Mechanistically, we found that LRP1 facilitates cell-association, endocytic recycling but not degradation, and graded distribution of Wnt5a in the developing limbs. We propose that LRP1-mediated endocytic regulation of availability and distribution of extracellular signalling molecules play a critical role in limb development. This provides a novel mechanism for crosstalk among skeletal elements.