These methods can help answer the key questions in the field of biomaterials. Particularly for bone regeneration. They're used to evaluate the effectiveness of biomaterials and potential immune responses.
The main advantage of this multidisciplinary platform is that it provides a range of parameters for predicting biocompatibility of biomaterials used for bone repair. The implications of these in vitro and in vivo bone techniques extend towards other bone disorders. Due to the need for preclinical screening of novel orthopedic biomaterials.
The dermatological methodology can provide insights into biomaterials for bone healing, it can also be applied to biomaterial assessments for any other clinical indication. Aspirate culture medium from primary osteoblasts 14 days after the addition of osteogenic mineralization medium. Rinse twice with 0.5 milliliters of 1X PBS.
Then fix the cells by adding 0.5 milliliters of 10%buffered formalin solution and incubating at room temperature for 10 minutes. After 10 minutes, aspirate the 10%buffered formalin with a single-use pipet and wash twice with 0.5 milliliters of ultrapure water. Then add 250 microliters of 40 millimolar Alizarin Red S staining solution to the fixed osteoblasts.
And incubate the plate at room temperature for 10 minutes on a shaker at 100 shakes per minute. Following the incubation with staining solution, aspirate the staining solution with a single-use pipet and rinse with one milliliter of ultrapure water. Repeat this step five to 10 times until the rinsing solution comes off clear to remove nonspecific staining.
After aspirating the final wash, add one milliliter of cold PBS. And incubate the plate at room temperature for 10 minutes on a shaker as before. Next, transfer the stained beta tricalcium phosphate discs to a new well and scan the plates with a flatbed scanner to record mineralization.
After image capture, add 250 microliters of 10%cetylpyridinium chloride solution and shake for 15 minutes to extract the Alizarin Red S dye. Next, transfer the solution from the wells to individual 1.5 milliliters tubes and centrifuge at 17, 000 x g for five minutes at room temperature. Dilute the extract with 10%cetylpyridinium chloride solution at a dilution ration of 1:10 to 1:20 in a 1.5 milliliter tube.
Then transfer 300 microliters of each diluted sample into the wells of the 96-well plate. Include two blank wells containing only 10%cetylpyridinium chloride. Next, prepare seven Alizarin Red S reference standards ranging from 4 400 micromolar by diluting 40 milliomolar Alizarin Red S staining solution with 10%cetylpyridinium chloride solution to generate a standard curve.
Load 300 microliters of each standard into the plate. Lastly, read the absorbents of the samples, blanks, and reference standards at 520 nanometers. Isolate bone marrow osteoplast precursors from the femurs and tibiae of a euthanized mouse by using sterile scissors to remove the legs from the body at the hip joint.
Cut the limbs at the knee and ankle joints and remove the soft tissue with sterile scalpel and forceps. Cut off the epiphyses. Insert a 27 gauge needle attached to a syringe filled with one milliliter of bone growth medium into the lumen and flush out the marrow into a six centimeter sterile Petri dish.
After repeating this for all femurs and tibiae, transfer the cell suspension to a 50 milliliter conical tube. Wash the six centimeter Petri dish with five milliliters of bone growth medium and transfer it to the same 50 milliliter conical tube. Centrifuge at 350 x g at four degrees Celsius for five minutes.
Following centrifugation re-suspend the cells in one milliliter per well of osteoclast differentiation medium. One BALB/c mouse provides sufficient numbers of osteoclast precursors for one 24-well culture plate. Add primary osteoblasts suspended in bone growth medium onto the biomaterial and the controls at 8.8*10^4 cells per square centimeter.
Beta tricalcium phosphate discs, controlled bovine bone, and tissue culture plastic are used here. Incubate at 37 degrees celsius in five percent carbon dioxide for 24 hours. After 24 hours, add freshly isolated bone marrow osteoclast precursors and osteoclast differentiation medium to the cultured primary osteoblasts and return to the incubator for five days of culture at 37 degrees celsius at five percent carbon dioxide.
Replace the medium with freshly-prepared osteoclast differentiation medium every other day. Then stain osteoclasts for tartrate resistant acid phosphatase to evaluate differentiation. Shave the scalp of an anesthetized eight-week-old BALB/c mouse and clean the surface with 7.5%povidone iodine solution and 70%ethanol.
Cover the eyes with ophthalmic ointment to prevent drying during the procedure. Ensure an appropriate level of anesthesia by lack of a reaction to a toe and tail pinch. Then after making a midline sagittal incision remove the paracranium connective tissues above the right parietal bone by scraping it with a scalpel.
Then use a sterile dental trephine with a diameter of four millimeters at 2000 rotations per minute to create a critical-sized defect in the right parietal bone. Constantly irrigate the region with sterile saline solution while notching the right parietal bone and cutting through the ectocortext and some endocortex. To prevent damage to the Dera Mater, use a small periosteal elevator to break through the remaining endocortex.
Then use forceps to carefully lift the calvarial bone and remove it. The resulting defect should be circular and approximately 3.5 millimeters in diameter. Next, fill the calvarial defect with beta tricalcium phosphate collagen foam, presoaked in sterile PBS.
To keep the biomaterial in place, apply 0.5 microliters of tissue adhesive at the end of the defect at two opposite points. Then close the skin with a nonabsorbable suture. Place an anesthetized mouse on its back, shave the abdominal fur and clean the shaved surface with 7.5%povidone iodine solution and 70%ethanol.
After making an eight millimeter-long midline incision through the skin along the linea alba of the abdomen, implant PBS soaked biomaterial under the skin. Then suture the skin with a nonabsorbable suture. Eight weeks post-implantation, excise the implantation site with surrounding tissue from the euthanized mouse.
This image demonstrates that growth on beta tricalcium phosphate discs shown in open bars does not affect osteoblast viability compared to growth on tissue culture plastic at seven and 14 days of culture. Cultured osteoblasts were stained with Alizarin Red S after 14 days. Mineralization was higher for mineralization medium controls compared to osteoblasts cultured in medium alone.
And on beta tricalcium phosphate discs in suspension culture wells. When a surgically-induced critical-sized calvarial defect was left empty, a thin layer covering the entire defect was observed, but no significant bone formation was present at 12 weeks post-operation. In contrast, when the defect contained beta tricalcium phosphate collagen foam, foam remnants surrounded by dense fibrous tissue including some blood vessels and inflammatory cells bridged the defect area without evidence of bone formation.
Following subcutaneous beta tricalcium phosphate collagen foam implantation, an inflammatory response with foreign body giant cells was observed on hematoxylin and Eosin-stained sections an evidence of fibrosis on Masson's trichrome stained sections at eight weeks. In contrast, the implantation site of the sham controls had minimal inflammation and no fibrosis. While attempting this procedure it's important to do precise and reproducible notching of the calvarium without hurting the animal.
Following this procedure's other methods like osteoclast differentiations assays and high throughput into peritoneal immune models can be preformed in order to answer additional questions like osteoclast's response to biomaterials and type of immune response.
