Name: evaluation of amino acid consumption in cultured bone cells and isolated bone shafts
Uploaded: 2022-04-13T13:00:00.000+00:00
Duration: 6 min 32 s
Description: Watch this Scientific Journal Video about Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts at JoVE.com

The Karner lab is supported by National Institute of Health R01 grants (AR076325 and AR071967) to C.M.K.

All mouse procedures described herein were approved by the Animal Studies Committees at the University of Texas Southwestern Medical Center at Dallas. The radiation protocol was approved by the Radiation Safety Advisory Committee at the University of Texas Southwestern Medical Center at Dallas.
1. Amino acid uptake in cells (Protocol I)
<ol>
	<li>Plate 5 x 104 ST2 cells in each well of a 12-well tissue culture plate. Plate cells in &#945;-MEM containing 10% FBS, 100 U/mL penicillin and 0.1 &#181;g/mL streptomycin (Pen/Strep). Plate extra wells of cells to quantify the cell number per condition for the normalizations in step 1.12. Incubate cells in a humidified cell culture incubator at 37 &#176;C with 5% CO2.</li>
	<li>Culture the cells for 2-3 days until confluent.</li>
	<li>On the day of the experiment, prepare the following solutions: 1x Phosphate Buffered Saline (PBS), pH 7.4 and Krebs Ringers HEPES (KRH) buffer, pH 8.0: (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, NaHCO3, 5 mM HEPES, 1 mM D-Glucose). Prewarm to 37 &#176;C.</li>
	<li>Aspirate the medium and wash the cells twice with 1 mL of 1x PBS, pH 7.4. 
	NOTE: This protocol is also appropriate for rapidly dividing non-confluent cells. In this case, it is important to normalize the radioactivity to either absolute cell number or DNA content. In addition, consider increasing the size of the culture plate or flask to increase the overall cell number and cpm values. This is important to determine empirically on an individual basis.</li>
	<li>Aspirate 1x PBS and wash the cells once with 1 mL KRH.</li>
	<li>Make 4 &#181;Ci/mL L-[3,4-3H]-Glutamine working media by diluting 4 &#181;L of [1 &#181;Ci &#181;L-1] L-[3,4-3H]-Glutamine stock per 1 mL of KRH. 
	NOTE: Before using radioactive materials, please contact the Office of Radiation Safety at your home institution to obtain approvals. All the procedures related to radiation must be performed behind the plexiglass shield.</li>
	<li>Incubate cells with 0.5 mL of KRH containing 4 &#181;Ci/mL L-[3,4-3H]-Glutamine working media for 5 min.</li>
	<li>Collect the radioactive medium and dispense into the liquid waste container. Wash the cells three times briefly with ice-cold KRH to terminate the reaction. Collect and discard all the washes in the radioactive liquid waste container.</li>
	<li>Add 1 mL of 1% SDS to each well and triturate 10x to lyse and homogenize the cells. Transfer the cell lysates to 1.5 mL tubes. Discard cell culture plates, serological pipettes, and pipette tips in the solid radioactive waste container.</li>
	<li>Centrifuge at &#62;10,000 x g for 10 min. Transfer the supernatants to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously. Discard tubes and pipette tips in solid radioactive waste container.</li>
	<li>Read radioactivity in counts per minute (cpm) using a Scintillation counter. Discard scintillation vials in scintillation vial waste container.</li>
	<li>Trypsinize, resuspend, and count the cells from the remaining non-radioactive plates of cells (see step 1.1) to estimate the number of cells in the lysed, radioactive cultures. Using a hemocytometer, count the number of cells per non-radioactive well for each experimental condition. Normalize the cpm from step 1.11 to the estimated cell number from the non-radioactive plates.</li>
	<li>After completion of the experiment, decontaminate the cell culture hood, bench, and all instruments with a radioactivity decontaminant spray. Finally, perform wipe tests to ensure the working area is radiation-free.</li>
</ol>
2. Amino acid uptake in freshly isolated bone tissues (Protocol II)
<ol>
	<li>Prewarm KRH to 37 &#176;C.</li>
	<li>Euthanize a 3-day-old mouse and remove the skin on the arms. Disarticulate the humeri from the shoulder using scissors and dissect out both humeri. Remove all extemporaneous tissues using a scalpel and forceps. Remove the epiphyses from the bone. 
	NOTE: In addition to humerii, this protocol can be adapted to neonatal femora, tibiae and calvariae as well as humerii, femora and tibiae isolated from 2- and 4-month-old mice (unpublished data).</li>
	<li>Flush out marrow from the bone and weigh the bone shafts to normalize in step 2.14.</li>
	<li>Boil one humerus in 1x PBS at 100 &#176;C for 10 min to decellularize the bone. The decellularized boiled bone is used as a negative control. 
	NOTE: As a quality control, paraffin embed the boiled and non-boiled bones for histological stains to visually confirm that boiling was efficacious in decellularizing the bone.</li>
	<li>Equilibrate both humeri in 1 mL of KRH for 30 min in the cell culture incubator at 37 &#176;C.</li>
	<li>Make a working solution of 4 &#181;Ci/mL L-[2,3,4-3H]-Arginine in KRH by diluting 4 &#181;L of 1&#181;Ci/&#181;L L-[2,3,4-3H]- Arginine stock per 1 mL KRH. 
	NOTE: This protocol is appropriate to evaluate the uptake of whichever radiolabeled nutrient is chosen. L-[2,3,4-3H]-glutamine, L-[14CU]-alanine, and L-[2,3-3H]-proline have been tested with similar results. Arginine data is shown to highlight the utility of this protocol. 
	CAUTION: All the procedures using radiation must be performed behind the plexiglass shield while using appropriate personal protective equipment (PPE).</li>
	<li>Incubate both the experimental and boiled control bone separately in KRH containing 4 &#181;Ci/mL L-[2,3,4-3H]-Arginine for up to 90 min at 37 &#176;C. 
	NOTE: It is important to empirically determine the incubation times for different conditions including the age of the mice, different skeletal elements and the types of radiolabeled amino acids by performing a time course. Saturation mostly occurs after approximately 3-4 h. Uptake should be evaluated at a timepoint in the linear rage of uptake.</li>
	<li>Remove the radioactive medium and discard in the liquid radioactive waste container. Wash humeri three times using ice cold 1 mL of KRH to terminate the reaction. Discard all the washes in the liquid radioactive waste container.</li>
	<li>Transfer each bone into a 1.5 mL tube. Add 500 &#181;L of RIPA buffer [150 mM NaCl; 5 mM EDTA; 50 mM Tris (pH 8.0); 0.5% NP40 (v/v); 0.5% DOX (w/v); 0.1% SDS (w/v)]. Discard the culture plates, serological pipettes, and pipette tips in a radioactive solid waste container.</li>
	<li>Homogenize the humeri by chopping 100 times with scissors in a RIPA buffer.</li>
	<li>Sonicate (Amplitude: 35%, Pulse 1 s) the resulting homogenized bone for 10 s. 
	NOTE: Sonication is also known to produce aerosols. Sonication steps can be performed in a biological safety cabinet. To reduce aerosol formation, it is important to not overfill the tubes. Sonication of small sample volumes can result in incomplete sonication or sample loss due to foaming. If foaming occurs, centrifuge the sample for 5 min to settle.</li>
	<li>Clarify the lysate by centrifugation for 10 min at &#62;10,000 x g at room temperature. Transfer 200 &#181;L of the supernatant to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously.&#160;Discard all solid radioactive waste (e.g., used tubes, pipette tips, plates, etc.) in the solid radioactive waste container.</li>
	<li>Read radioactivity (cpm) using the Scintillation counter. Discard used scintillation vials in the radioactive waste container designated for scintillation vials.</li>
	<li>Subtract the cpm of boiled bone (basal value) from the cpm of experimental bone. Normalize the radioactivity with bone weight from step 2.3.</li>
	<li>Spray the cell culture hood, instruments, and bench with radioactivity decontaminant. Perform wipe tests to confirm the working area is radiation-free.</li>
</ol>

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The authors have no disclosures.

The protocol described herein provides a fast and sensitive approach to evaluate amino acid uptake in response to various experimental permutations either in vitro or ex vivo. When compared to commercially available kits (e.g., Glutamine and Glutamate Determination Kit), this method is much more sensitive, quicker, and less labor intensive16,17,25. In our protocol, we evaluate uptake in the Krebs Ringers HEPES ...

Amino acids are organic compounds that contain an amino (-NH2) and carboxyl (-COOH) functional groups with a variable side chain that is specific to each amino acid. In general, amino acids are well known as the basic constituent of protein. More recently, novel uses, and functions of amino acids have been elucidated. For example, individual amino acids can be metabolized to generate intermediate metabolites that contribute to bioenergetics, function as enzymatic cofactors, regulate reactive oxygen species or are used to synthesize other amino acids1,2,3,4,5,6,7,8,9,10. Many studies demonstrate that amino acid metabolism is critical for cell pluripotency, proliferation, and differentiation in various contexts3,6,11,12,13,14,15,16,17.
Osteoblasts are secretory cells that produce and secrete the Collagen Type 1 rich extracellular bone matrix. To sustain high rates of protein synthesis during bone formation, osteoblasts demand a constant supply of amino acids. To meet this demand, osteoblasts must actively acquire amino acids. Consistent with this, recent studies reveal the importance of amino acid uptake and metabolism in osteoblast activity and bone formation15,16,17,18,19,20.
Osteoblasts acquire cellular amino acids from three major sources: extracellular milieu, intracellular protein degradation and de novo amino acid biosynthesis. This protocol will focus on the evaluation of amino acid uptake from extracellular environment. The most common methods to measure amino acid uptake rely on either radiolabeled (e.g., 3H or 14C) or heavy isotope labeled (e.g., 13C) amino acids. Heavy isotopomer assays can analyze amino acid uptake and metabolism more thoroughly and safely but are more time consuming taking multiple days to complete as it takes a day to prepare and derivatize samples and multiple days to analyze on the mass spectrometer depending on the number of samples21,22. By comparison, radiolabeled amino acid uptake assays are not informative about downstream metabolism but are cheap and relatively fast, being able to be completed within 2-3 h from the start of the experiment23,24. Here, we describe an easily modifiable basic protocol designed to evaluate radiolabeled amino acid uptake in cultured primary cells or cell lines in vitro or individual bone shafts ex vivo. The application of these two protocols can be extended to other radiolabeled amino acids and other bone associated cell types and tissues.

<table><tbody><tr><td>0.25% trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>1 mL syringe</td><td>BD precision</td><td>309628</td><td></td></tr><tr><td>30G Needle</td><td>BD precision</td><td>305106</td><td></td></tr><tr><td>Arginine Monohydrochloride L-[2,3,4-3H]-, 1mCi</td><td>PerkinElmer</td><td>NET1123001MC</td><td></td></tr><tr><td>Beckman LS6500 scintillation counter</td><td></td><td></td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>Choline chloride</td><td>Sigma</td><td>C7077</td><td></td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Dissection Tool</td><td></td><td></td><td>Forceps, scissors, scapels</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma</td><td>E9884</td><td></td></tr><tr><td>HEPES(1M)</td><td>Gibco</td><td>15630</td><td></td></tr><tr><td>L-[3,4-3H(N)]-Glutamine</td><td>PerkinElmer</td><td>NET551250UC</td><td></td></tr><tr><td>Liquid scintilation vials</td><td>Sigma</td><td>Z190535</td><td></td></tr><tr><td>Lithium chloride solution, 8M</td><td>Sigma</td><td>L7026</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>MEM&amp;alpha;</td><td>Gibco</td><td>12561</td><td></td></tr><tr><td>Microcentrifuge tube, 15mL</td><td>Biotix</td><td>89511-256</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>492016</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma</td><td>D6750</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>436143</td><td></td></tr><tr><td>Sonicator</td><td>Sonic&amp;amp;Materials</td><td>VCX130</td><td></td></tr><tr><td>Tris Base</td><td>Sigma</td><td>648311</td><td></td></tr><tr><td>Ultima Gold (Scintillation solution)</td><td>PerkinElmer</td><td>6013329</td><td></td></tr><tr><td>&amp;alpha;-(Methylamino)isobutyric acid</td><td>Sigma</td><td>M2383</td><td></td></tr></tbody></table>

evaluation of amino acid consumption in cultured bone cells and isolated bone shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

Amino acid transport is regulated by many membrane-bound amino acid transporters that have been categorized into distinct transport systems based on numerous characteristics, including substrate specificity, kinetics, as well as ion and pH dependence25. For example, glutamine uptake can be mediated by the Na+-dependent transport systems A, ASC, &#947;+L and N or the Na+-independent System L. The Na+-dependent systems are distinguished by the ability to substitute L...

Watch this Scientific Journal Video about Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts at JoVE.com

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

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1.7K Views.  University of Texas Southwestern Medical Center. This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

Video: Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Chemistry

Culturing and Measuring Fetal and Newborn Murine Long Bones

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Biology

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large ...

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

Source: Kampleitner, C. et al. <a href="https://www.jove.com/v/58077">Biological Compatibility Profile on Biomaterials for Bone Regeneration.</a> J. Vis. Exp. (2018)
In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.

Test mineralizacji in vitro: metoda kolorymetryczna do ilościowego oznaczania złogów wapnia w osteoblastach na substytucie przeszczepu kostnego przez barwienie i ekstrakcję czerwieni alizarynowej

Source: Kampleitner, C. et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018)
In this video, we describe a ...