Method of inducing bone formation by ex-vivo osteoblast culturing for implantation
An ex-vivo culturing method of osteoblasts for implantation, comprising a culturing of adult live osteoblasts as an ex-vivo procedure. The ex-vivo culture, which leads to the formation of the active substance, further comprises the steps of isolation of osteo-progenitor cells, differentiation of osteo-progenitor cells in to osteoblasts, expansion culture, cell culture harvest and wash followed by filling and packaging. This method is instrumental in accelerating the process of bone formation.
1. A method of inducing bone formation by ex-vivo osteoblast culturing for implantation, comprising:
a) isolating osteo-progenitor cells from a harvest of a subject;
b) differentiating the osteo-progenitor cells in to osteoblasts;
c) expanding the osteoblasts for proliferation into an ex-vivo osteoblast growth culture;
d) harvesting and washing the ex-vivo osteoblast growth culture;
e) sub-culturing the ex-vivo osteoblast growth culture until the ex-vivo osteoblast growth culture reaches not less than 48 million cells to obtain an ex-vivo osteoblast culture;
f) selecting an ex-vivo osteoblast culture for implantation from the characterized ex-vivo osteoblast culture by flow cytometry (FACS) with CD44+, CD151+, and CD105− expression markers;
g) collecting the selected ex-vivo osteoblast culture in Dulbecco's Modified Eagle's Medium (DMEM) to obtain an ex-vivo osteoblast culture for implantation; and
h) injecting the collected ex-vivo osteoblast culture for implantation into a subject and examining bone formation and regeneration.
2. The method as claimed in claim 1 , wherein the harvest of a subject is from a posterior-superior iliac crest or sternum.
3. The method as claimed in claim 1 , wherein the isolating of osteo-progenitor cells includes separating of nucleated bone marrow cells.
4. The method as claimed in claim 3 , wherein the separating of nucleated bone marrow cells is followed by washing of the nucleated bone marrow cells with DMEM culture medium.
5. The method as claimed in claim 1 , wherein the differentiating the osteo-progenitor cells includes replenishing of differentiation medium in culture flasks.
6. The method as claimed in claim 1 , wherein the expanding the osteoblasts further includes transferring of culture flasks to a CO 2 incubator at a temperature range of 37-38 degree Celsius.
7. The method as claimed in claim 6 , wherein the CO 2 incubator is conditioned at a range of 5% CO 2 content.
8. The method as claimed in claim 6 , wherein the CO 2 incubator is humidified at 80%.
9. The method as claimed in claim 1 , wherein the harvesting step further includes centrifuging the ex vivo osteoblast growth culture or the ex-vivo osteoblast culture at a range of 1300-1500 rpm for five-six minutes.
10. The method as claimed in claim 9 , wherein the centrifuging is followed by washing of the ex-vivo osteoblast growth culture or the ex-vivo osteoblast culture with DMEM.
11. The method as claimed in claim 1 , wherein the ex-vivo osteoblast culture for implantation are filled in sterile vials, sealed effectively under aseptic conditions, transported at 2 to 8 degrees Celsius and used as active substance within 72 hours from date of manufacture.
12. The method as claimed in claim 1 further wherein the bone formation and regeneration at the site of induction of bone formation post implantation exhibits approximately ninety percent of bone regeneration on completion of ninety days.