In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

Abstract

Background: Bone grafts are required to repair large bone defects after tumour resection or

large trauma. The availability of patients?U? own bone tissue that can be used for these

procedures is limited. Thus far bone tissue engineering has not lead to an implant which

could be used as alternative in bone replacement surgery. This is mainly due to problems of

vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently

it was discovered that embryonic stem cells can form bone via the endochondral pathway,

thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the

potential of human adult mesenchymal stem cells to form bone via the endochondral

pathway. Methods: MSCs were cultured for 28 days in chondrogenic, osteogenic or control

medium prior to implantation. To further optimise this process we induced mineralisation in

the chondrogenic constructs before implantation by changing to osteogenic medium during

the last 7 days of culture. Results: After 8 weeks of subcutaneous implantation in mice, bone

and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic

medium. No bone was observed in any samples cultured in osteogenic medium. Switch to

osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of e_-

glycerophosphate to chondrogenic medium during the last 7 days in culture induced

mineralisation of the matrix and still enabled formation of bone and marrow in both human

and rat MSC cultures. To determine whether bone was formed by the host or by the

implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that

osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host

and donor origin. Conclusions: The preliminary data presented in this manuscript

demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both

human and rat cells. Furthermore, addition of e_-glycerophosphate to the chondrogenic

medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate

that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and

superior approach for bone tissue engineering.