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Posts Tagged ‘primary osteocyte’

Author: Aviral Vatsa PhD MBBS

This is the first post in a series of posts on mechanosensation and mechanotransduction and their role in physiology and disease.

Future posts in this category will focus on various aspects of role of mechanosensation and mechanotransduction in human physiology. These aspects will include among others: gene modulation, cellular mechanosensation, tissue regeneration, stem cell differentiation, cancer, disease models, nanomodulation, material science and therapeutics etc.

Based on Zhang et al [1]

Multicellular organisms such as humans require intricate orchestration of signals between cells to achieve global morphogenesis and organ function and thus maintain haemostasis. Three major ‘signalling modalities’ work in unison intracellularly and/or exrtacellularly to regulate harmonious functioning of the physiological milieu. These ‘modalities’ namely biochemical molecules, electrical currents or fields and mechanical forces (external or internal) cohesively direct the downstream regulation of physiological processes.

Traditionally most of the biological studies have focused on biochemical or electrical signalling events and relatively lesser resources have been dedicated towards exploring the role of mechanical forces in human health and disease. Despite early theories proposed by scientists such as Julius Wolff (Wolff’s law [2]) in the late nineteenth century “ that bone in a healthy person or animal will adapt to the loads under which it is placed”, relatively little has been studied about the role of external mechanical forces in maintaining haemostasis. However, recent important developments such as

  • identification of external force dependent regulation of signalling pathways [3]
  • determination of mechanosensing elements of cellular cytoskeleton [4]
  • manipulation of single molecules [5]

have reinstated the importance of external mechanical forces in physiology. As a result more recent investigations have demonstrated that external mechanical forces are major coordinators of development and haemostasis of organisms [6], [7] [8].

‘Mechanotransduction’ has been traditionally defined as the conversion of mechanical stimulus into chemical cues for the cells and thus altering downstream signalling e.g conformational changes in ion channels might lead to initiation of downstream signalling. However, with the accumulation of new knowledge pertaining to the effects of external mechanical loads on extracellular matrix or a cell or on subcellular structures, it is being widely accepted that mechanotransduction is more than merely a physical switch. Rather it entails the whole spectrum of cell-cell , cell-ECM, and intracellular interactions that can directly or indirectly modulate the functioning of cellular mechanisms involved in haemostasis. This modulation can function at various levels such as organism level, tissue level, cellular level and subcellular level.

Forces in cells and organisms

From biological point of view mechanical forces can be grouped into three categories

  • intracellular forces
  • intercellular forces
  • inter-tissue forces

In the eukaryotic cells these forces are generally generated by the the contractile cytoskeletal machinery of the cell that is comprised of

  • microfilaments : Diameter-6 nm; example- actin
  • intermediate filaments: Diameter-10 nm; example- vimentin, keratin
  • microtubules: Diameter-23 nm; example- alpha and beta tubulin

 

Actin labeling in single Osteocyte in situ in mouse bone. Source: Aviral Vatsa

Actin labeling in single Osteocyte in situ in mouse bone. Source: Aviral Vatsa

Actin (cytoskeleton) staining of single osteocyte in situ in mouse calvaria (source: Aviral Vatsa)

There are a range of forces generated in the biological milieu (adopted from Mammoto et al [8]): 

  • Hydrostatic pressure: mechanical force applied by fluids or gases (e.g. blood or air) that perfuse or infuse living organs (e.g. blood vessels or lung).
  • Shear stress: frictional force of fluid flow on the surface of cells. The shear stress generated by the heart pumping blood through the systemic circulation has a key role in the determination of the cell fate of cardiomyocytes, endothelial cells and hematopoietic cells.
  • Compressive force: pushing force that shortens the material in the direction of the applied force. Tensional force: pulling force that lengthens materials in the direction of the applied force.
  • Cell traction force: is exerted on the adhesion to the ECM and other cells as a result of the shortening of the contractile cytoskeletal actomyosin filaments, which transmit tensional forces across cell surface adhesion receptors (e.g. integrins, cadherins).
  • Cell prestress: stabilizing isometric tension in the cell that is generated by the establishment of a mechanical force balance within the cytoskeleton through a tensegrity mechanism. Pulling forces generated within contractile microfilaments are resisted by external tethers of the cell (e.g. to the ECM or neighboring cells) and by internal load-bearing structures that resist compression (e.g. microtubules, filipodia). Prestress controls signal transduction and regulates cell fate.

It is the interplay of these forces generated by the cellular cytoskeleton and the ECM that regulate physiological functions. Disruption in mechanotransduction has been implicated in a variety of diseases such as hypertension, muscular dystrophies, cardiomyopathies, loss of hearing, cancer progression and metastasis. Ongoing attempts at unravelling the finer details of mechanosensation hold promising potential for new therapeutic approaches.

 

References

[1] H. Zhang and M. Labouesse, “Signalling through mechanical inputs – a coordinated process,” Journal of Cell Science, vol. 125, no. 17, pp. 4172–4172, Oct. 2012.

[2] R. A. Brand, “Biographical Sketch: Julius Wolff, 1836–1902,” Clin Orthop Relat Res, vol. 468, no. 4, pp. 1047–1049, Apr. 2010.

[3] A. J. Hudspeth, “The cellular basis of hearing: the biophysics of hair cells,” Science, vol. 230, no. 4727, pp. 745–752, Nov. 1985.

[4] N. Wang, J. P. Butler, and D. E. Ingber, “Mechanotransduction across the cell surface and through the cytoskeleton,” Science, vol. 260, no. 5111, pp. 1124–1127, May 1993.

[5] J. T. Finer, R. M. Simmons, and J. A. Spudich, “Single myosin molecule mechanics: piconewton forces and nanometre steps,” , Published online: 10 March 1994; | doi:10.1038/368113a0, vol. 368, no. 6467, pp. 113–119, Mar. 1994.

[6] P. A. Janmey and R. T. Miller, “Mechanisms of mechanical signaling in development and disease,” J Cell Sci, vol. 124, no. 1, pp. 9–18, Jan. 2011.

[7] R. Keller, L. A. Davidson, and D. R. Shook, “How we are shaped: The biomechanics of gastrulation,” Differentiation, vol. 71, no. 3, pp. 171–205, Apr. 2003.

[8] T. Mammoto and D. E. Ingber, “Mechanical control of tissue and organ development,” Development, vol. 137, no. 9, pp. 1407–1420, May 2010.

 

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Curator, Reporter: Aviral Vatsa, PhD, MBBS

Isolation of primary osteocytes from skeletally mature mice bones: A report on “Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice” (BioTechniques 52:361-373 ( June 2012) doi 10.2144/0000113876)

A new study by Stern et al reports a technique where in the authors have isolated primary osteocytes from mature and aged mice.

Osteocytes are deeply embedded in the mineralised matrix of bone. They form the majority cell types of bone and play vital function in maintenance of bone homeostasis. However their study has been limited by their location in the bone and that they are terminally differentiated cells.

Osteocytes are the most abundant of the three bone cell types; however, the least is known about them. While their location deep within the bone matrix makes them ideally situated to sense bone strain, it also makes their observation and study in vivo difficult. Additionally, primary osteocytes, particularly those within the long bones of skeletally mature animals, have proven difficult to obtain and study ex vivo. Furthermore, once primary osteocytes are obtained, their study is often limited by their inability to proliferate as they are considered terminally differentiated cells. ”

As a result majority of the studies on osteocytes in vitro have used either cell lines and/or primary cells from new-born animals such as chicken, rat and mouse.

The MLO-Y4 cell line is well-characterized and represents the phenotype of early osteocytes ”

“Although the MLO-Y4 cell line is a very powerful tool for the study of osteocytes in vitro, there are known differences between primary osteocytes and the immortalized MLO-Y4 cell line. For example, MLO-Y4 cells express low to undetectable levels of Dentin matrix protein 1 (Dmp1) and Sclerostin (Sost), while osteocytes are known to express these genes in vivo .”

Primary osteocytes have most commonly been isolated from 16- or 18-day-old chick calvaria or from newborn through 4-day-old rat calvaria, 12-day-old mouse calvaria, and 3- to 4-week-old mouse calvaria and long bones.”

Studies utilizing these primary osteocytes can provide insight to the behavior of osteocytes during development but do not aid in the study of osteocytes from skeletally mature animals or enable the comparison between osteocytes isolated from skeletally mature but relatively young mice (4- to 6-month) and aged mice (>22-month-old).”

To circumvent the above mentioned limitations the authors utilised multi-step digestion technique. They subjected mouse long bone pieces (from 4-month old mouse and 22-month old mouse) to collagenase and EDTA alternatively for 25 minutes and collected the aspirate after each step for plating and culture of cells. (as described in the table, which has been taken from the study).

Table 1. Osteocyte isolation from murine long bone (courtesy: Stern et al)

They collected cells from nine such alternate steps in total and also the left over bone. These cells were then cultured for 7 days. Following parameters were tested to characterise the osteocytes.

  • E11/GP38 staining – early osteocyte specific protein
  • Alkaline Phosphatase (ALP) staining – indicator of osteoblastic state
  • COL 1 – major component of bone and produced by osteoblasts
  • Gene expression of E11, SOST, MEPE, Dmp1 – markers of osteogencity in different stages of osteogenesis
    • Osteoid osteocytes are known to express E11, Phex, and Mepe, while mineralizing osteocytes express Dmp1, and mature osteocytes encased in a mineralized matrix express Sost and Fgf23”

The authors were able to demonstrate that the isolated cells indeed expressed osteogenic markers. It was observed that cells isolated from later digestion steps (6-9) were more osteocyte like. This was also the case with the cells isolated from the left over bone pieces.

In this study, we were able to success fully isolate primary cells displaying several characteristics of osteocytes from the long bones of skeletally mature 4-month-old and 22-month-old mice through a process of sequential digestions and the use of a tissue homogenizer. From both the 4-month-old and 22-month-old mice, approximately 250,000 cells per osteocyte-enriched digestion (digestions 7–9) were obtained. These cells expressed E11/GP38 protein, and they lacked ALP and COL1A1 expression found in osteoblasts. Furthermore, several genes known to be expressed in osteocytes were also expressed in the cells obtained using our methodology. These include E11/gp38, Sost, Cox2, Mepe, Phex, and Dmp1.”

Limitations: 

As the authors pointed out, their study characterised the cells ensemble from separate digestion steps. This could lead to having a mixed population from each step.

The authors did not mention about the proliferation (or the absence of it) of the isolated cells. Since osteocytes are terminally differentiated cells, theoretically they should not proliferate. In addition when such primary cells are co-cultured with dividing cells, such as osteoblasts and fibroblasts in this case, the dividing population tends to over grow in culture leaving behind very few primary osteocytes. A detailed characterisation of these cells at different stages of digestion along with progressive time points will be very helpful.

Possibilities:

As authors claim, in future, this technique can help scientists to answer tricky questions about osteocytes such as comparing osteocytes from animals grouped on the basis of age, disease, bone characteristics, and therapies.

Reference: Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. BioTechniques 52:361-373 ( June 2012) doi 10.2144/0000113876 .

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