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.
Averal, this is a Great post.
Let’s explore Osteocyte in the following context:
Gap junction intercellular communication in osteocytes plays an important role in bone remodeling in response to mechanical loading; however, the responsible molecular mechanisms remain largely unknown
http://mcb.asm.org/content/30/1/206.full
Prostaglandin Promotion of Osteocyte Gap Junction Function through Transcriptional Regulation of Connexin 43 by Glycogen Synthase Kinase 3/β-Catenin Signaling▿
PUT IT IN CONTEXT OF CANCER CELL MOVEMENT
The contraction of skeletal muscle is triggered by nerve impulses, which stimulate the release of Ca2+ from the sarcoplasmic reticuluma specialized network of internal membranes, similar to the endoplasmic reticulum, that stores high concentrations of Ca2+ ions. The release of Ca2+ from the sarcoplasmic reticulum increases the concentration of Ca2+ in the cytosol from approximately 10-7 to 10-5 M. The increased Ca2+ concentration signals muscle contraction via the action of two accessory proteins bound to the actin filaments: tropomyosin and troponin (Figure 11.25). Tropomyosin is a fibrous protein that binds lengthwise along the groove of actin filaments. In striated muscle, each tropomyosin molecule is bound to troponin, which is a complex of three polypeptides: troponin C (Ca2+-binding), troponin I (inhibitory), and troponin T (tropomyosin-binding). When the concentration of Ca2+ is low, the complex of the troponins with tropomyosin blocks the interaction of actin and myosin, so the muscle does not contract. At high concentrations, Ca2+ binding to troponin C shifts the position of the complex, relieving this inhibition and allowing contraction to proceed.
Figure 11.25
Association of tropomyosin and troponins with actin filaments. (A) Tropomyosin binds lengthwise along actin filaments and, in striated muscle, is associated with a complex of three troponins: troponin I (TnI), troponin C (TnC), and troponin T (TnT). In (more ) Contractile Assemblies of Actin and Myosin in Nonmuscle Cells
Contractile assemblies of actin and myosin, resembling small-scale versions of muscle fibers, are present also in nonmuscle cells. As in muscle, the actin filaments in these contractile assemblies are interdigitated with bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules, which produce contraction by sliding the actin filaments relative to one another (Figure 11.26). The actin filaments in contractile bundles in nonmuscle cells are also associated with tropomyosin, which facilitates their interaction with myosin II, probably by competing with filamin for binding sites on actin.
Figure 11.26
Contractile assemblies in nonmuscle cells. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions. Two examples of contractile assemblies in nonmuscle cells, stress fibers and adhesion belts, were discussed earlier with respect to attachment of the actin cytoskeleton to regions of cell-substrate and cell-cell contacts (see Figures 11.13 and 11.14). The contraction of stress fibers produces tension across the cell, allowing the cell to pull on a substrate (e.g., the extracellular matrix) to which it is anchored. The contraction of adhesion belts alters the shape of epithelial cell sheets: a process that is particularly important during embryonic development, when sheets of epithelial cells fold into structures such as tubes.
The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesisthe division of a cell into two following mitosis (Figure 11.27). Toward the end of mitosis in animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane. Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. The ring then disperses completely following cell division.
Figure 11.27
Cytokinesis. Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin II divides the cell in two.
http://www.ncbi.nlm.nih.gov/books/NBK9961/
This is good. I don’t recall seeing it in the original comment. I am very aware of the actin myosin troponin connection in heart and in skeletal muscle, and I did know about the nonmuscle work. I won’t deal with it now, and I have been working with Aviral now online for 2 hours.
I have had a considerable background from way back in atomic orbital theory, physical chemistry, organic chemistry, and the equilibrium necessary for cations and anions. Despite the calcium role in contraction, I would not discount hypomagnesemia in having a disease role because of the intracellular-extracellular connection. The description you pasted reminds me also of a lecture given a few years ago by the Nobel Laureate that year on the mechanism of cell division.