Process for ex vivo formation of mammalian bone and uses thereof
View Patent ↗The present invention concerns methods for the ex vivo formation of mammalian bone and subsequent uses of the bone. A critical and distinguishing feature of the present invention are defined tissue culture conditions and factors resulting in the formation of bone cell spheroids. The invention also provides for methods of implanting into subjects the ex vivo formed bone. Also described are methods for genetically altering the bone cell spheroids to affect bone formation, identification of candidate modulators of bone formation, and identification of genes involved in bone formation.
1. A method for identifying a modulator of bone formation comprising the steps of:
a) obtaining an osteogenic cell or bone precursor cell;
b) culturing said cell under serum-free conditions in the presence of a candidate compound, in the absence of osteogenic growth factors;
c) measuring bone cell spheroid formation; and
d) comparing the formation of bone cell spheroid with that observed in the absence of the candidate compound;
wherein an increase or decrease in bone cell spheroid formation indicates the candidate compound is a modulator of bone cell spheroid formation.
2. The method of claim 1 , further comprising the steps of:
e) obtaining an osteogenic cell or bone precursor cell;
f) culturing said cell under serum-free conditions in the presence of growth factors of the TGF-β gene superfamily;
g) maintaining the cell culture at cell densities that allows the formation of a bone cell spheroid;
h) measuring bone cell spheroid formation; and
i) comparing the bone spheroid formation measured in step c) to the bone spheroid formation measured in step h).
3. The method of claim 2 , wherein the culture conditions of step f) are free of the candidate compound.
4. The method of claim 2 , wherein the culture conditions of step f) further comprise the candidate compound.
5. A method for producing a modulator of bone formation, comprising the steps of:
a) obtaining an osteogenic cell or bone precursor cell;
b) culturing said cell under serum-free conditions in the presence of a candidate compound, in the absence of osteogenic growth factors;
c) measuring bone cell spheroid formation; and
d) comparing the formation of bone cell spheroid with that observed in the absence of the candidate compound, wherein an increase or decrease in bone cell spheroid formation indicates the candidate compound is a modulator of bone cell spheroid formation;
e) identifying the candidate compound as a modulator of bone cell formation; and
f) producing the modulator so identified.