Bone Geconditioneerd Medium: Voorbereiding en Bioassay

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.