Name: Bone Conditioned Medium: Preparation and Bioassay (Video) | JoVE Journal
Uploaded: 2015-07-08T14:00:00.000+00:00
Duration: 7 min 18 s
Description: Learn to prepare bone-conditioned medium from pig mandible chips and test its biological activity on cultured cells. This method enables gene expression analysis via quantitative real-time PCR for bone biology research.

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5 minutes

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/pubmed?term=((Long-term+Stability+of+Early+Implant+Placement+with+Contour+Augmentation%5BTitle%5D)+AND+%22Journal+of+dental+research%22%5BJournal%5D)">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/pubmed?term=((augmentation+procedures+in+implant+dentistry%5BTitle%5D)+AND+%22The+International+journal+of+oral+%26+maxillofacial+implants.+24+Suppl%22%5BJournal%5D)">augmentation procedures in implant dentistry.</a> The International journal of oral & 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/pubmed?term=((substitutes%3A+an+update.+Injury%5BTitle%5D)+AND+%2236%2C+Suppl+3.+S20-S27%22%5BJournal%5D)">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/pubmed?term=((healing+and+graft+resorption+of+autograft%2C+anorganic+bovine+bone+and+beta-tricalcium+phosphate.+A+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs%5BTitle%5D)+AND+%22Clinical+oral+implants+research%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+a+novel+biphasic+calcium+phosphate+in+standardized+bone+defects%3A+a+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs%5BTitle%5D)+AND+%22Clinical+oral+implants+research%22%5BJournal%5D)">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/pubmed?term=((graft+and+bone+graft+substitutes+in+spine+surgery%3A+current+concepts+and+controversies%5BTitle%5D)+AND+%22The+Journal+of+the+American+Academy+of+Orthopaedic+Surgeons%22%5BJournal%5D)">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/pubmed?term=((The+biology+of+bone+grafting%5BTitle%5D)+AND+%22The+Journal+of+the+American+Academy+of+Orthopaedic+Surgeons%22%5BJournal%5D)">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/pubmed?term=((The+osteogenic+effect+of+bone+transplants+in+rabbits.+The+Journal+of+bone+and+joint+surgery%5BTitle%5D)+AND+%22British%22%5BJournal%5D)">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/pubmed?term=((formation+by+autoinduction%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">formation by autoinduction.</a> Science. 150, 893-899 (1965).</li>
<li>Caballé-Serrano, J. D., Buser, D., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+analysis+of+porcine+bone+conditioned+medium%5BTitle%5D)+AND+%22The+International+journal+of+oral+%26+maxillofacial+implants%22%5BJournal%5D)">Proteomic analysis of porcine bone conditioned medium.</a> The International journal of oral & maxillofacial implants. , (2014).</li>
<li>Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone-Conditioned+Medium+Inhibits+Osteogenic+and+Adipogenic+Differentiation+of+Mesenchymal+Cells+In+Vitro%5BTitle%5D)+AND+%22Clinical+implant+dentistry+and+related+research%22%5BJournal%5D)">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/pubmed?term=((Human+bone+chips+release+of+sclerostin+and+FGF-23+into+the+culture+medium%3A+an+in+vitro+pilot+study%5BTitle%5D)+AND+%22Clinical+oral+implants+research%22%5BJournal%5D)">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>Caballé-Serrano, J., Bosshardt, D. D., Gargallo-Albiol, J., Buser, D., Bone Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((conditioned+medium+enhances+osteoclastogenesis+in+murine+bone+marrow+cultures.+Clin+Oral+Impl+Res%3B+in%5BTitle%5D)+AND+%22Int+J+Oral+Maxillofac+Surg%22%5BJournal%5D)">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/pubmed?term=((Bone-conditioned+medium+changes+gene+expression+in+bone-derived+fibroblasts%29%5BTitle%5D)+AND+%22IJOMI+accepted%22%5BJournal%5D)">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/pubmed?term=((Myelopoiesis+is+regulated+by+osteocytes+through+Gsalpha-dependent+signaling%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Demineralized+bone+matrix+mediates+differentiation+of+bone+marrow+stromal+cells+in+vitro%3A+effect+of+age+of+cell+donor%5BTitle%5D)+AND+%22Journal+of%22%5BJournal%5D)">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/pubmed?term=((Proteomic+techniques+for+characterisation+of+mesenchymal+stem+cell+secretome%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">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/pubmed?term=((Osteoblastic+cell+secretome%3A+A+novel+role+for+progranulin+during+risedronate+treatment%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment.</a> , Bone. (2013).</li>
<li>Schuldt Filho,, G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditioned+medium+of+demineralized+bone+matrix+activates+TGF-%CE%B2+signaling+pathways+in+mesenchymal+cells+in+vitro%5BTitle%5D)+AND+%22J+Cranio+Maxill+Surg%22%5BJournal%5D)">Conditioned medium of demineralized bone matrix activates TGF-β 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/pubmed?term=((Early+implant+placement+following+single-tooth+extraction+in+the+esthetic+zone%3A+biologic+rationale+and+surgical+procedures%29%5BTitle%5D)+AND+%22The+International+journal+of+periodontics+%26+restorative+dentistry%22%5BJournal%5D)">Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures).</a> The International journal of periodontics & restorative dentistry. 28, 441-451 (2008).</li>
<li>Stoecklin-Wasmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absorbable+collagen+membranes+for+periodontal+regeneration%3A+a+systematic+review%5BTitle%5D)+AND+%22Journal+of+dental+research%22%5BJournal%5D)">Absorbable collagen membranes for periodontal regeneration: a systematic review.</a> Journal of dental research. 92, progress. 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><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 &amp;amp; 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>16030074</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen Corporation</td><td>21885-025</td><td></td></tr><tr><td>&lt;br/&gt; High Pure RNA Isolation Kit</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>Transcriptor First Strand cDNA Synthesis Kit</td><td>Roche</td><td>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>4673484001</td><td></td></tr><tr><td>PBS</td><td>Roche</td><td>11666789001</td><td></td></tr></tbody></table>

bone conditioned medium: preparation and bioassay

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...

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 ...

The overall goal of this video is to describe how to prepare bone conditioned medium, and test its activity in vitro. This is accomplished by first harvesting bone chips from a pig mandible using a bone scraper. The second step is to heat treat demineralize or do nothing to the bone chips prior to incubating them in culture medium for 24 hours.Next, the bone conditioned medium is collected containing all the factors released from the bone chips. The final step is to stimulate cells with the bone conditioned medium or to pre incubate a biomaterial with the bone conditioned medium and seed cells on it following the incubation period. Ultimately, quantitative real-time PCR is used to show changes in gene expression of the cells following treatment.Utilization of autologous bone grafts alone or in combination with other biomaterials stimulates bone regeneration in peri-implant bone defects, and thus ensures good long-term results. This video presents an in vitro bioassay, which is useful in determining the genetic response of massal cells to the biomolecules within bone condition. Medium Bone condition medium can help answer key questions in the trauma maxillofacial and dental field, such as why autologous bone is the goal of standard graft in bone regeneration.Preparing condition medium from processed bone, like heat treated bone auto graft or ized bone matrix can be difficult to standardize. Therefore, it's important to be precise. To begin obtain fresh pig mandibles from a local butcher and place them onto a firm surface.Next, use a periosteum to release a full thickness flap of periosteum while paying special attention not to leave any of the soft tissue attached to the bone. Once the periosteum is removed, firmly grip the bone scrapper and use long movements to harvest bone chips from the buccal side of the mandible. Discard any of the bone chips that are smaller than one millimeter in length.Prevent the bone chips from drying out by quickly covering them with culture medium in a 10 centimeter Petri dish. After enough bone chips have been collected, prepare a thermal control batch by pasteurizing five grams of the bone chips. Pasteurize the chips by heating them for 30 minutes at 80 degrees Celsius or autoclave them for 20 minutes at 121 degrees Celsius.Next, prepare a demineralized control by adding five grams of bone chips into a one molar solution of hydrochloric acid and placing them on a shaker for four to six hours at four degrees Celsius. Then wash the bone chips repeatedly with culture medium until a neutral pH reached. Place five grams of fresh bone chips and five grams of each of the control preparations into separate dishes, and add 10 milliliters of fresh culture medium to each dish.Then incubate the samples in a humidified atmosphere at 37 degrees Celsius for 24 hours in order to create the conditioned medium the next day. Remove the media from each dish and place it in a 15 milliliter conical tube. Centrifuge the bone conditioned media at 200 times gravity for 10 minutes to remove any debris.Then remove the supernatant and pass it through a 0.2 micron sterile filter store the Ali quads frozen at minus 80 degrees Celsius until they are needed. Start by seeding human mesenchymal cells such as bone cells or gingival and periodontal ligament fibroblasts into a 12 whale plate at a concentration of 30, 000 cells per square centimeter. Cover the cells with growth medium and let the cells attach to the plate overnight the next morning.Discard the culture medium and wash the cells with prewarm PBS at 37 degrees Celsius. Then stimulate the cells by adding prewarm serum free culture medium with and without 20%bone conditioned medium. If also testing the absorption ability of a biomaterial, add the bone conditioned medium, including any controls to tubes containing the biomaterial of interest, and incubate the tubes at 37 degrees Celsius for one hour.Then rinse the biomaterial vigorously with prewarm PBS and seed human mesenchymal cells at a concentration of 30, 000 cells per square centimeter onto the material. Incubate the cells alone or those seeded on the biomaterials in a humidified atmosphere at 37 degrees cells CS for 24 hours. Then discard the culture medium.Rinse the cells with prewarm PBS and extract the cells RNA using a standard RNA isolation protocol. Once the RNA has been isolated, prepare CD NA samples of each treatment group by starting with equal concentrations of the RNA extracted and running a reverse transcriptase reaction on each sample. Then perform quantitative real-time PCR on the samples to look for levels of selected genes using the primers and conditions listed in the accompanying text protocol.Finally, calculate the quality of the bone conditioned medium based on the relative gene expression levels by normalizing to cycle threshold levels of the genes to the housekeeping gene Gabb dh using the delta delta CT method. Here are some typical results showing the changes in gene expression of oral fibroblasts exposed to bone conditioned medium for 24 hours. Two genes adrenalin and pent trax and three were both significantly downregulated to 40%of original levels, whereas interleukins 11 and 33, along with N-A-D-P-H oxidase four and prote glycan four were all upregulated as much as 200 fold.Interestingly, collagen barrier membranes used to shield the bone chips from the surrounding soft tissue absorb much of the bone conditioned medium that is responsible for the changes in gene expression. The collagen membranes, however, failed to absorb the factors that control interleukin 33 expression. Therefore, interleukin 33 expression is not regulated in this setting.The protocol described here can be adapted to study the response of different types of cells involving bone regeneration. Moreover, the protocol can be used to prepare condition medium from process bone and other bone fillers. The clinical relevance Of this bioassay remains unclear.The cellular response to BCM in vivo is likely more complex and includes a large spectrum of genes and different target cells extending those presented here. Nevertheless, our bioassay provides first insights into the presumably complex composition of bone RAF molecules and the in vitro cellular response.

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Learn to prepare bone-conditioned medium from pig mandible chips and test its biological activity on cultured cells. This method enables gene expression analysis via quantitative real-time PCR for bone biology research.

Bone Conditioned Medium: Preparation and Bioassay

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