In our experiments on intercalary bone, we removed bone marrow content as much as possible in line with clinical practice.
After the tips of each bone were removed, bone marrow was collected by flushing out the content of femurs and tibias with phosphate buffered saline (PBS).
After the cement specimen was removed, bone marrow and hematoma material from the proximal portion of the tibia were thoroughly collected, placed into a sterile centrifuge tube, and weighed.
The treatment involved removing bone marrow from the infants, isolating the so-called stem cells, inserting normal genes to replace the defective ones, and then reinfusing the stem cells into the blood stream, where they were finally able to generate the cells for a functioning immune system.
Except for LCA, in which gene-carrying viruses are injected directly into eyes, these diseases are treated by removing bone marrow cells from patients, adding a gene to the cells, and reinfusing the cells back into the patient.
Each bone slice was placed in saline and flushed with a 23 gauge needle to remove bone marrow.
We did not use bone specimens where we could macroscopically see both solid bone and bone marrow, so we did not use any additional washing steps to remove bone marrow.
After washing in 0.1 M sodium cacodylate buffer, pH 7.4, the samples were first incubated in 5% sodium hypochlorite to remove bone marrow for 10 min at room temperature, and then treated with a 35% phosphoric acid gel (Temrex Gel Etch, Temrex Corporation, New York, USA) for 60 s to expose the collagen fibrils on the bone surface.
The femur was removed, the bone marrow expelled, cells dissociated into single cell suspensions and cultured for 10 days supplemented with 1000 units/ml of MCSF (Wyeth, Inc .. Cultured BMM proved to be 98% CD11b+ by flow cytometric analysis using a FACS Calibur flow cytometer (BD Biosciences).
C57BL/6 mice were killed by CO2 asphyxia and their limbs were removed for bone marrow isolation.
Femurs, tibiae, and humeri were removed, and bone marrow was harvested by flushing with HBSS (Hanks' balanced salt solution; without Ca2+/Mg2+), 30 mM Hepes, and 15 mM EDTA.
