Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Transglutaminase-2 mediates the biomechanical properties of the colorectal cancer tissue microenvironment that contribute to disease progression

Colorectal cancer is the third most common cancer worldwide, and the fourth leading cause of malignancy-related mortality. This highlights the need to understand the processes driving this disease in order to develop new treatments and improve patient outcomes. A potential therapeutic target is the increased stiffness of the tumour microenvironment, which is linked to aggressive cancer cell behaviour by enhancing biomechanical signalling. In this study, we used an siRNA-based approach to investigate the contribution of the protein cross-linking enzyme transglutaminase-2 (TG2) to matrix remodelling and biomechanical properties of the tumour microenvironment. TG2 inhibited cancer cell growth in organotypic 3D fibroblast/SW480 co-culture models, and biomechanical analysis demonstrated that colorectal cancer cells induced fibroblast-mediated stiffness which was inhibited by silencing TG2. These biomechanical changes were associated with observed alterations to collagen fibre structure, notably fibre thickness. Our in vitro findings of collagen composition changes were also seen with imaging biopsied tissues from patients with colorectal cancer, with TG2 correlating positively with thicker collagen fibres, and associating with poor outcome as determined by disease recurrence post-surgery and overall survival. In conclusion, this study demonstrates a role for TG2 in the stromal response to invading tumour, leading to tissue stiffening and poor outcome in patients.</jats:p

Electrospun fiber alignment guides osteogenesis and matrix organization differentially in two different osteogenic cell types

Biomimetic replication of the structural anisotropy of musculoskeletal tissues is important to restore proper tissue mechanics and function. Physical cues from the local micro-environment, such as matrix fiber orientation, may influence the differentiation and extracellular matrix (ECM) organization of osteogenic progenitor cells. This study investigates how scaffold fiber orientation affects the behavior of mature and progenitor osteogenic cells, the influence on secreted mineralized-collagenous matrix organization, and the resulting construct mechanical properties. Gelatin-coated electrospun poly(caprolactone) fibrous scaffolds were fabricated with either a low or a high degree of anisotropy and cultured with mature osteoblasts (MLO-A5s) or osteogenic mesenchymal progenitor cells (hES-MPs). For MLO-A5 cells, alkaline phosphatase (ALP) activity was highest, and more calcium-containing matrix was deposited onto aligned scaffolds. In contrast, hES-MPs, osteogenic mesenchymal progenitor cells, exhibited higher ALP activity, collagen, and calcium deposition on randomly orientated fibers compared with aligned counterparts. Deposited matrix was isotropic on random fibrous scaffolds, whereas a greater degree of anisotropy was observed in aligned fibrous constructs, as confirmed by second harmonic generation (SHG) and scanning electron microscope (SEM) imaging. This resulted in anisotropic mechanical properties on aligned constructs. This study indicates that mineralized-matrix deposition by osteoblasts can be controlled by scaffold alignment but that the early stages of osteogenesis may not benefit from culture on orientated scaffolds

Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.

We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253

Mesenchymal stem cell responses to mechanical stimuli

Mesenchymal stem cells (MSCs) have the potential to replace or restore the function of damaged tissues and offer much promise in the successful application of tissue engineering and regenerative medicine strategies. Optimising culture conditions for the predifferentiation of MSCs is a key goal for the research community, and this has included a number of different approaches, one of which is the use of mechanical stimuli. Mesenchymal tissues are subjected to mechanical stimuli in vivo and terminally differentiated cells from the mesenchymal lineage respond to mechanical stimulation in vivo and in vitro. MSCs have also been shown to be highly mechanosensitive and this may present an ideal method for controlling MSC differentiation. Here we present an overview of the response of MSCs to various mechanical stimuli, focusing on their differentiation towards the mesenchymal tissue lineages including bone, cartilage, tendon/ligament, muscle and adipose tissue. More research is needed to elucidate the complex interactions between biochemically and mechanically stimulated differentiation pathway

Mechanical and physical guidance of osteogenic differentiation and matrix production

Summary Tissue engineering and regenerative medicine strategies until now have mostly relied on static culture using chemical stimulation to induce cell differentiation. However, these strategies neglect the dynamic environment in which cells reside in the body where they are surrounded by a chemically and physically well-defined threedimensional (3D) topography. Not only does this environment control cellular differentiation, but its structure also determines the mechanical function of that tissue. Alongside physical cues, external mechanical forces play an essential role in the homeostasis of many tissues, particularly bone. In order to develop tissue engineered constructs that are suitable for implantation, it may be important to incorporate these essential cues into pre-culture methods and in order to do this, a better understanding of the cellular responses is required. The main aim of this research was to understand how physical and mechanical cues affect cell behaviour, differentiation and matrix production, with particular emphasis on osteogenesis and collagen organisation. In order to achieve this, electrospun scaffolds were fabricated with controllable fibre orientation for studies involving fibroblast matrix organisation, and the affect on the differentiation of osteoprogenitor cells. Short bouts of tensile loading were conducted using a previously established bioreactor model for conditioning collagen-producing cells. A simple rocking platform method for subjecting cells to fluid-flow was also investigated for its potential to enhance osteogenesis and collagen organisation. This system was further used to study the role of the primary cilium for the mechanotransduction of bone cells. The overall goal was to understand how to manipulate cell differentiation and matrix production in order to develop a more suitable construct with correct tissue structure in a rapid manner. Monitoring of the major structural matrix protein collagen was achieved using the minimally-invasive technique of second harmonic generation, which was optimised. Electrospun scaffolds with a random architecture caused cells to deposit matrix in a similar random manner, however highly aligned scaffolds caused deposited collagen to orientate in the fibre direction giving superior tensile properties. Further to this, random fibres appeared to be more favourable for the differentiation of osteoprogenitor cells than highly aligned substrates. 9 Short bouts of tensile stimulation of collagen producing cells on 3D substrates caused an increase in collagen deposition. Another stimulation method, a simple rocking platform, created oscillatory fluid shear stress (FSS) suitable for stimulation of osteogenic cells and enhanced collagen organisation. Further to this, human dermal fibroblasts could be induced to form a mineralised matrix when cultured in osteogenic media, which was further enhanced with FSS. It was also demonstrated that this simple rocking system could be used to test a wide variety of loading parameters. Finally, rocking was used to examine the role of the primary cilium in the load-induced mineral deposition response of bone cells. When mature bone cells were subjected to FSS, primary cilia shortened in length and removal of primary cilia resulted in loss of the load-induced matrix response suggesting that primary cilia are mechanosensors in bone cells

A simple rocker-induced mechanical stimulus upregulates mineralization by human osteoprogenitor cells in fibrous scaffolds.

Biodegradable electrospun polycaprolactone scaffolds can be used to support bone-forming cells and could fill a thin bony defect, such as in cleft palate. Oscillatory fluid flow has been shown to stimulate bone production in human progenitor cells in monolayer culture. The aim of this study was to examine whether bone matrix production by primary human mesenchymal stem cells from bone marrow or jaw periosteal tissue could be stimulated using oscillatory fluid flow supplied by a standard see-saw rocker. This was investigated for cells in two-dimensional culture and within electrospun polycaprolactone scaffolds. From day 4 of culture onwards, samples were rocked at 45 cycles/min for 1 h/day, 5 days/week (rocking group). Cell viability, calcium deposition, collagen production, alkaline phosphatase activity and vascular endothelial growth factor secretion were evaluated to assess the ability of the cells to undergo bone differentiation and induce vascularisation. Both cell types produced more mineralized tissue when subjected to rocking and supplemented with dexamethasone. Mesenchymal progenitors and primary human mesenchymal stem cells from bone marrow in three-dimensional scaffolds upregulated mineral deposition after rocking culture as assessed by micro-computed tomography and alizarin red staining. Interestingly, vascular endothelial growth factor secretion, which has previously been shown to be mechanically sensitive, was not altered by rocking in this system and was inhibited by dexamethasone. Rocker culture may be a cost effective, simple pretreatment for bone tissue engineering for small defects such as cleft palate

HDF morphology on PCL fibres.

<p>Nucleus (DAPI in blue) and cytoskeleton (phalloidin-TRITC in red) of HDFs seeded on random and aligned scaffolds. HDF morphology was visualised at day 7 (top) and 21 (bottom) imaged at different depths (5–40 µm). Cells on random fibres were generally attached in multiple directions while cells on aligned fibres were spindle shaped and aligned in the fibre direction. White arrows indicate direction of scaffold fibre alignment. Scale bars are 50 µm.</p

Fibroblasts and secreted collagen grow into constructs and align with scaffold fibre orientation.

<p>Fibroblast-secreted collagen and scaffold fibres were visualised using SHG emissions collected at depths of 10–40 µm (A). Yellow arrow heads indentify PCL fibres of the scaffold. Fibroblast-seeded constructs were fixed and visualised via SHG emissions (collagen and scaffold fibres in yellow) and DAPI staining (cell nucleus in blue) at depths of 10–40 µm (B). Fixed constructs were also visualised for fibroblast cytoskeleton (phalloidin-TRITC in red) proximity to secreted collagen (SHG in green) (C). SHG was obtained using 800 nm illumination and all images were collected after 21 days of culture. White arrows indicate scaffold fibre orientation.</p

HDF viability and migration on PCL fibres.

<p>HDFs seeded into the centre of random and aligned fibres and assayed with MTT at days 3, 7 and 12 to observe cell migration and viability. Cells on random fibres migrated equally in all directions whereas cells on aligned fibres migrated along the direction of the fibre. Data is mean ± SD (n = 6), <i>*p<0.05</i>.</p