Authors would like to thank Catherine Solioz for her skillful assistance.

1. BCM Preparation
<ol>
	<li>Obtain pig mandibles from the local butcher as fresh as possible. Place the mandibles onto a firm surface and release a full thickness flap paying special attention not to leave any soft tissue or periosteum attached to the bone. Work in a clean environment without the need to work under the flow hood.</li>
	<li>Once a full thickness flap is released, use a bone scraper to harvest the bone chips from the buccal side. Please note that the bone scraper has to be sharp. Handle firmly the bone scraper and with long movements collect the bone. Discard bone chips smaller that 1mm.
	<ol>
		<li>To maintain native bone chips, place directly the bone chips in plastic dishes of 10cm diameter with Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) supplemented with 1% antibiotics and antimycotics not letting them to dry out.</li>
		<li>To evaluate the impact of thermal processing, subject bone chips to pasteurization for 30 min at 80 &#176;C or autoclave for 20 min at 121 &#176;C.</li>
		<li>To evaluate the impact of demineralization, shake bone chips in 1 M HCL for 4-6 hr at 4 &#176;C and wash repeatedly with culture medium until the pH is neutral.</li>
	</ol>
	</li>
	<li>Place a total of 5 g of bone chips per 10 ml fresh DMEM supplemented with 1% antibiotics and antimycotics into a new plastic dish.</li>
	<li>Place the plastic dishes in a humidified atmosphere at 37 &#176;C for 24 hr. Then, harvest BCM. Centrifuge the BCM at 200 x g for 10 min to remove debris, filter it sterile (0.2 nm), and keep aliquots frozen at -80 &#176;C.</li>
	<li>Thaw the BCM stock immediately before use and avoid repeated cycles of freezing and thawing.</li>
	<li>For indicated experiments, soak collagen membranes with BCM or serum-free medium for 1 hr at RT. Wash vigorously the membranes with PBS and place them into 96 well plates. Wet membranes are seeded with cells.</li>
	<li>BCM preparation process is summarized in Figure 1.</li>
</ol>
2. Bioassays Based on Mesenchymal Cells
<ol>
	<li>Seed human mesenchymal cells (for example bone cells, gingival and periodontal ligament fibroblasts) into a 12-well plate with a concentration of 30,000 cells/cm2. To seed the cells use growth medium consisting of DMEM, 10% fetal calf serum and antibiotics. Let the cells attach to the plate O/N.</li>
	<li>Discard the culture medium and wash the cells with pre-warmed PBS at 37 &#176;C. Stimulate the cells by adding pre-warmed serum-free culture medium with and without 20% BCM. Place the cells in a humidified atmosphere at 37 &#176;C for 24 hr.</li>
	<li>Discard the culture medium, rinse the cells with pre-warmed PBS and extract the RNA according to your preferred protocol.</li>
	<li>Adjust the concentration of RNA in order to have the same amount of RNA in each sample. Prepare cDNAs and perform a qRT-PCR to analyse the selected genes using the primers shown in Table 1. 
	NOTE: These are the dilutions of every component: 2x SYBR Green, 20x primer forward, 20x primer reverse, 5x sterile DD water, 5x cDNA. The qRT-PCR is performed in 40 cycles of 95 &#176;C 15 sec and 60 &#176;C 1 min.</li>
	<li>Calculate the relative expression levels by normalizing to the housekeeping gene GAPDH using the &#916;(&#916;Ct) method where &#916;CT is CT target - CT GAPDH and &#916;(&#916;CT) is &#916;CT stimulated - &#916;CT control.</li>
	<li>After this quality control, add BCM to culture medium to stimulate all types of cells including mesenchymal cells, hematopoietic cells or endothelial cells.</li>
</ol>

<ol>
	<li>Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Stability+of+Early+Implant+Placement+with+Contour+Augmentation.">Long-term Stability of Early Implant Placement with Contour Augmentation.</a> Journal of dental research. , (2013).</li><li>Chiapasco, M., Casentini, P., Bone Zaniboni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=augmentation+procedures+in+implant+dentistry.">augmentation procedures in implant dentistry.</a> The International journal of oral &amp; maxillofacial implants. 24 Suppl. , 237-259 (2009).</li><li>Giannoudis, P. V., Dinopoulos, H., Bone Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=substitutes:+an+update.+Injury.">substitutes: an update. Injury.</a> 36, Suppl 3. S20-S27. , (2005).</li><li>Jensen, S. S., Broggini, N., Hjorting-Hansen, E., Schenk, R., Bone Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=healing+and+graft+resorption+of+autograft,+anorganic+bovine+bone+and+beta-tricalcium+phosphate.+A+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 17, 237-243 (2006).</li><li>Jensen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+biphasic+calcium+phosphate+in+standardized+bone+defects:+a+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 18, 752-760 (2007).</li><li>Grabowski, G., Bone Cornett, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=graft+and+bone+graft+substitutes+in+spine+surgery:+current+concepts+and+controversies.">graft and bone graft substitutes in spine surgery: current concepts and controversies.</a> The Journal of the American Academy of Orthopaedic Surgeons. 21, 51-60 (2013).</li><li>Khan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+bone+grafting.">The biology of bone grafting.</a> The Journal of the American Academy of Orthopaedic Surgeons. 13, 77-86 (2005).</li><li>Bohr, H., Ravn, H. O., Werner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteogenic+effect+of+bone+transplants+in+rabbits.+The+Journal+of+bone+and+joint+surgery.">The osteogenic effect of bone transplants in rabbits. The Journal of bone and joint surgery.</a> British. 50, 866-873 (1968).</li><li>Bone Urist, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=formation+by+autoinduction.">formation by autoinduction.</a> Science. 150, 893-899 (1965).</li><li>Caball&#233;-Serrano, J. D., Buser, D., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+porcine+bone+conditioned+medium.">Proteomic analysis of porcine bone conditioned medium.</a> The International journal of oral &amp; maxillofacial implants. , (2014).</li><li>Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-Conditioned+Medium+Inhibits+Osteogenic+and+Adipogenic+Differentiation+of+Mesenchymal+Cells+In+Vitro.">Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro.</a> Clinical implant dentistry and related research. , (2014).</li><li>Brolese, E., Buser, D., Kuchler, U., Schaller, B., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+chips+release+of+sclerostin+and+FGF-23+into+the+culture+medium:+an+in+vitro+pilot+study.">Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study.</a> Clinical oral implants research. , (2014).</li><li>Caball&#233;-Serrano, J., Bosshardt, D. D., Gargallo-Albiol, J., Buser, D., Bone Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=conditioned+medium+enhances+osteoclastogenesis+in+murine+bone+marrow+cultures.+Clin+Oral+Impl+Res;+in.">conditioned medium enhances osteoclastogenesis in murine bone marrow cultures. Clin Oral Impl Res; in.</a> Int J Oral Maxillofac Surg. , (2015).</li><li>Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-conditioned+medium+changes+gene+expression+in+bone-derived+fibroblasts).">Bone-conditioned medium changes gene expression in bone-derived fibroblasts).</a> IJOMI accepted. , (2014).</li><li>Fulzele, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelopoiesis+is+regulated+by+osteocytes+through+Gsalpha-dependent+signaling.">Myelopoiesis is regulated by osteocytes through Gsalpha-dependent signaling.</a> Blood. 121, 930-939 (2013).</li><li>Becerra, J., Andrades, J. A., Ertl, D. C., Sorgente, N., Nimni, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demineralized+bone+matrix+mediates+differentiation+of+bone+marrow+stromal+cells+in+vitro:+effect+of+age+of+cell+donor.">Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.</a> Journal of. 11, 1703-1714 (1996).</li><li>Kupcova Skalnikova, ., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+techniques+for+characterisation+of+mesenchymal+stem+cell+secretome.">Proteomic techniques for characterisation of mesenchymal stem cell secretome.</a> Biochimie. , (2013).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cell+secretome:+A+novel+role+for+progranulin+during+risedronate+treatment.">Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment.</a> , (2013).</li><li>Schuldt Filho, ., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+of+demineralized+bone+matrix+activates+TGF-&#946;+signaling+pathways+in+mesenchymal+cells+in+vitro.">Conditioned medium of demineralized bone matrix activates TGF-&#946; signaling pathways in mesenchymal cells in vitro.</a> J Cranio Maxill Surg. , (2015).</li><li>Buser, D., Chen, S. T., Weber, H. P., Belser, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+implant+placement+following+single-tooth+extraction+in+the+esthetic+zone:+biologic+rationale+and+surgical+procedures).">Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures).</a> The International journal of periodontics &amp; restorative dentistry. 28, 441-451 (2008).</li><li>Stoecklin-Wasmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbable+collagen+membranes+for+periodontal+regeneration:+a+systematic+review.">Absorbable collagen membranes for periodontal regeneration: a systematic review.</a> Journal of dental research. 92, 773-781 (2013).</li></ol>

The authors have nothing to disclose. Jordi Caball&#233;-Serrano received a scholarship from the Foundation of Dental Research and Education, Basel, Switzerland.

Bone-conditioned medium reflects the released activity of bone grafts during the early stages of bone regeneration. The protocol described here can be adapted to study the response of different types of cells involved in bone regeneration. Furthermore, the protocol can be used to prepare conditioned medium from processed bone or bone fillers. The methods are easy to perform and rely on a simple concept: the factors released from various native and processed bone. Understanding how BCM affects mesenchymal cells can help t...

Autologous bone is widely used to bridge defects that occurred as a consequence of malformation, resective surgery, reconstructive trauma surgery, and prior to implant placement 1,2. Understanding the biological principles of how bone grafts support the process of graft consolidation is not only key to understand why autografts are considered to be the gold standard in reconstructive surgery, it is also bionic to the improved design of bone substitutes 3. Still, graft consolidation is faster with autologous bone compared to bone substitutes 4,5. Thus, it is imperative to reveal the molecular and cellular mechanisms that make autologous bone so effective to support bone regeneration.
There are three textbook characteristics of autografts that are considered to support the consolidation process 6,7. First, autologous bone is osteoconductive, providing guidance for the newly formed bone to grow into the defect. Secondly, autologous bone is osteogenic, meaning that it contains mesenchymal cells that can differentiate into osteoblasts 8. Third, autologous bone is osteoinductive as growth factors like bone morphogenetic proteins entombed in the matrix can initiate the process of endochondral or even intramembranous bone formation 9. There is another aspect: freshly prepared bone chips hold a paracrine function based on the in vitro observations with &#8220;bone-conditioned medium&#8221; 10-15. Also the impact of myelopoiesis should be mentioned 16. A similar term &#8220;demineralized bone matrix-conditioned medium&#8221; was already coined in 1996 and supports the overall concept of a paracrine function of bone, even when processed by demineralization 17. For our purposes, BCM can be prepared from fresh pig mandibles 10,11. Proteomic analyses of BCM revealed the complex composition, including growth factors and constituents of the extracellular matrix 10, also extending existing knowledge on the proteasome of whole bone 18,19. Thus, BCM should reflect the released activity of various modifications of bone grafts in vitro.
What happens when mesenchymal cells, for example those isolated from bone chips or from oral soft tissue, are exposed to BCM? In vitro, BCM reduces osteogenic and adipogenic differentiation, and provokes a strong increase of IL11 expression 11. Genome wide microarray revealed more genes to be differentially expressed in mesenchymal cells in response to BCM. Among these genes are adrenomedullin (ADM), IL11, IL33, NADPH oxidase 4 (NOX4), proteoglycan 4 (PRG4, or lubricin) and pentraxin 3 (PTX3) 15. BCM obtained from autoclaved bone chips failed to change the expression of the respective genes 14. BCM from bone chips that underwent pasteurization and freezing was able to change gene expression 14. Also conditioned medium of demineralized bone matrix (DBM-CM) changes the expression of TGF-&#946;-regulated genes 20. Interestingly, collagen barrier membranes used to shield the bone chips from the surrounding soft tissue 21,22, adsorbed those parts of BCM that are responsible for the changes in gene expression 23. BCM research can be extended to other cell types involved in bone regeneration such as bone-resorbing osteoclasts and endothelial cells, to name a few. Overall, the accumulating in vitro data provide the scientific basis for the design of a preclinical study.
The present protocol is two-fold: First, it shows how to prepare BCM. Secondly, it shows how to test its biological activity based on mesenchymal cells in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pig Mandibles</td>
			<td>Local bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Scraper</td>
			<td>Hu-Friedy</td>
			<td>PPBUSE2/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics &#38; Antimicotics</td>
			<td>All life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Membranes (Bio-Gide)</td>
			<td>Geistlich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Invitrogen Corporation</td>
			<td align="right">16030074</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Corporation</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			High Pure RNA Isolation Kit</td>
			<td>Roche</td>
			<td align="right">11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche</td>
			<td align="right">4379012001</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Microsynth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (for Q-RT-PCR)</td>
			<td>Roche</td>
			<td align="right">4673484001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td align="right">11666789001</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bone Conditioned Medium is prepared from fresh porcine bone chips. General overview of the process to prepare BCM and to use biomaterials in combination with BCM is shown in Figure 1 and Figure 2 respectively. During the BCM preparation, it is important to obtain large bone chips with long movements as short movements or very small bone chips can affect the quality of the final BCM. Quality of BCM can be controlled by analyzing the gene expression of BCM target genes: ADM, PTX3, IL11, IL...

Watch this Scientific Journal Video about Bone Conditioned Medium: Preparation and Bioassay at JoVE.com

Bone Conditioned Medium: Preparation and Bioassay

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 ...

bone-conditioned-medium-preparation-and-bioassay

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11.0K Views.  School of Dental Medicine, University of Bern. We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Video: Bone Conditioned Medium: Preparation and Bioassay

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

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, ...