Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

This method provides an efficient strategy for preparing functional scaffolds for bone tumor research. Decellularized the bone extracelullar matrix, showed variable serocompatibility for the survival and activities of osteosarcoma cells. The main advantage of this technique is that osteosarcoma cells culture in bone-derived matrix show highly heterogenous morphology similar to a clinical osteosarcoma histopathology.

The method provides ideal model for investigating the development, progression of drug sensitivities of bone tumors, such as osteosarcoma, Ewing sarcoma, and the other malignant tumors metastasis to bone. To begin, obtain four to six-week old BALB/c mice, after euthanizing a mouse using sterile surgical scissors, cut off fresh fibula, tibia, and femur from a hind limb. With the help of tweezers, peel off the epithelial tissue, and then remove as much of the soft tissue as possible.

Rinse the leg bones with sterile 10 mM PBS solution twice to remove blood in a six-centimeter dish. Immerse the bones in a dish with 75%ethanol for three minutes then rinse with PBS twice. Store the clean bones in a sterile 50 milliliter centrifuge tube with sterile PBS tube at 80 degrees Celsius.

Thaw the frozen bones at room temperature and then freeze again at 80 degrees Celsius for one hour. Subject the bones to more than two freeze-thaw cycles for cell lysis and tissue breakdown. Then, place the bones in a sterile 50 milliliter centrifuge tube filled with 0.5 normal HCl and incubate overnight at room temperature on an orbital shaker with gentle shaking to ensure complete and even coverage of the bones.

After decalcification, decant the hydrochloric acid solution completely and rinse the bones under running water for one hour. Then, use distilled water to wash the bones twice for 15 minutes per wash on an orbital shaker. Completely decant the solution.

To extract the lipids in a demineralized bones, place the bones in a 50 milliliter centrifuge tube with a 1:1 mixture of methanol and chloroform. Wrap the tube with tin foil to avoid light for prevention of chloroform decomposition. And put the tube on an orbital shaker for one hour.

Then, use tweezers to transfer the bones into another tube of methanol with tin foil for 30 minutes. Remove the methanol completely, and wash with distilled water twice for 15 minutes on an orbital shaker. Decant final wash water and proceed under sterile condition.

Rinse the bones in a six centimeter dish with sterile PBS for three minutes. Add 40 milliliters of sterile 0.05%TE solution into a 50 milliliter centrifuge tube and incubate bones for 23 hours in a carbon dioxide incubator at 37 degrees Celsius. Discard the TE solution, and rinse twice with sterile PBS supplemented with 90 g/mL ampicillin and 90 g/mL kanamycin for 15 minutes each on the orbital shaker.

After decanting the final wash completely, replenish again with 40 milliliters of sterile PBS with antibiotics. Wash thoroughly for 24 hours at room temperature with gentle shaking to achieve effective sterilization of porous spaces. Then, transfer the bones into a 50 milliliter centrifuge tube filled with sterile PBS with antibiotics.

The prepared Bone Extracellular Matrix can be stored at four degrees Celsius for two months. Immerse BEM in 75%ethanol in a dish and gently shake the dish by hand for 30 seconds. Then rinse with PBS for 30 seconds, twice.

Transfer the BEM onto a clean six-well cell culture plate. Add two milliliters of complete culture medium to each well. Incubate the BEM overnight in a carbon dioxide incubator at 37 degrees Celsius.

Obtain human OS cell lines in 100 microliters of pre-warmed PBS containing indicator phenol red. Use a pipette to suspend OS cells with the approximate concentration of 1 times 10 to the 5th. After the BEM is fully soaked in the medium, from proximal or distal epiphyses, pierce the needle to the medullary cavity of BEM and inject OS cells into BEM.

Incubate the OS-BEM model for a minimum of two hours in a humidified 5%carbon dioxide atmosphere at 37 degrees Celsius to ensure the injected cells firmly adhere to BEM. Then, take out the plate from the incubator. Add one milliliter culture medium onto the plate, and keep it in the incubator overnight to completely coat the surface of the BEM culture.

Gently transfer the OS-BEM model into a new well of a six-well plate with a sterile tweezer, and refeed one milliliter fresh culture medium. Culture the model for 14 days in the incubator. During the 14-day incubation, keep monitoring medium color.

If the medium turns into orange, or even yellow, immediately refresh the medium by discarding half of the old medium and adding in new medium to maintain a healthy environment for OS cells. Keep monitoring cell status under the inverted fluorescence microscope. When OS cells expand to plate, gently transfer the OS-BEM model to another new well with sterile tweezers.

After 14-day culture, gently rinse the OS-BEM model with PBS to remove the culture medium. Then transfer into a 15 milliliter centrifuge tube and add 10%buffered formalin to fix for histological identification. After demineralization and decellularization, BEM appears to be translucent with stronger resilience and tenacity compared to native mouse bone.

A little muscle residue in the space of medullary cavity can be clearly observed. Bright-field imaging of the native bone and the decellularized BEM, shows the thorough removal of cell nuclei. The natural porous structure in collagen network arrangement is well-maintained in decellularized BEM.

Additionally, immunohistochemical staining for Collagen I and Collagen IV shows the main components of extracellular matrix are preserved in mouse tibia after decellularization. During the 14-day culture, both periosteum and endosteum are infiltrated by the expansion of OS cells. OS cells on the decellularized BEM show highly heterogeneous morphology similar to the cytopathologic features of an OS section.

Immunohistochemical analysis after culturing in BEM model for 14 days shows great advantages in long term cultures. Also, OS cells and BEM culture, highly express bone matrix glycoprotein, which is specific for osteoid matrix. This in vitro's three-dimensional model has been used to demonstrate a phenotypic heterogeneity and a regulatory mechanism of osteosarcoma dedifferentiation with success.