Bone grafts are frequently used to augment bone healing. Autologous bone graft is the gold standard for osteogenesis but is limited by availability and donor site morbidity. The processing required to lower the immunogenicity of allograft also reduces the osteogeneic properties. Bone marrow contains mesenchymal stem cells (MSCs) which differentiate into osteoblasts, forming bone. Our study examined the use of bone marrow to enhance the osteogenic properties of allograft.

Bioactive proteins within allogenic bone graft stimulate marrow-derived MSCs to differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft.

After informed consent, bone marrow aspirates were taken from five patients during orthopaedic operations. Freeze-dried ethylene oxide treated allograft, from a number of donors, was obtained from the bone bank. MSCs isolated from each marrow aspirate were grown on eight samples of test allograft. Further allograft was heated to 70°C to denature the osteogenic proteins and MSCs from each aspirate were grown on 8 samples, as a negative control. Osteoblastic differentiation of MSCs cultured on the types of allograft was compared.

Scanning electron microscopy confirmed that MSCs covered the allograft after 14 days. Transmission electron microscopy showed that cells on the test allograft were characteristic of osteoblasts and produced collagen extracellular matrix. The levels of osteoblastic proteins, ALP, osteopontin and Type I pro-collagen, produced by cells on test allograft were significantly greater compared with heat-treated control (P< 0.005), after days 7 and 14.

Our study showed that marrow-isolated MSCs could be successfully cultured on allograft. As the levels of osteoblastic proteins increased significantly when MSCs were grown on allograft, osteogenic proteins within allograft caused MSCs to change into osteoblasts. This confirms that autologous marrow MSCs could be grown on allograft to increase its osteogenic prior to grafting, resulting in increased rate of bony healing.

Introduction: Bone grafts are frequently used in orthopaedic operations to augment bone healing. Autologous bone graft is the gold standard for osteogenesis, but the amount available from the patient’s iliac crest is often insufficient to fill the defect and donor site morbidity is a significant complication. Alternatively, allograft can be implanted into patients, however, processing is necessary to reduce the immunicity of the graft and the risk of transmission of infection, but this destroys osteoprogenitor cells and hence reduces the osteogenic properties of the graft. Mesenchymal stem cells (MSCs) are present in bone marrow and have the ability to differentiate into osteoblasts. Therefore our study examined the use of MCSs, from bone marrow, to enhance the osteogenic properties of allograft.

Hypothesis: MSCs cultured on freeze-dried ethylene oxide treated bone allograft differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft material.

Method: After informed consent, bone marrow aspirates were taken from 10 patients during elective orthopaedic operations. MSCs were characterized using Stro-1 antibody and grown on freeze-dried ethylene oxide treated bone allograft in vitro.

The hypothesis was tested on three groups of graft, with eight samples in each group. Firstly, freeze-dried ethylene oxide treated bone graft was tested (group 2). For a negative control, allograft was heated to 70°C to denature the osteogenic proteins (group 1). The final group tested the effect of additional osteogenic supplements (100nM dexamethasone, 0.05mM ascorbic acid and 10mM (-glycerol phosphate) on MSCs on allograft (group 3).

Osteoblastic differentiation of MSCs was observed under scanning (SEM) and transmission (TEM) electron microscopy, and by measuring protein levels: alkaline phosphatase (ALP), osteopontin and type I pro-collagen over 14 days.

Results: SEM confirmed that MSCs could be successfully cultured on bone allograft. Cells grown in groups 2 and 3 were characteristic of metabolically active osteoblasts and collagen extracellular matrix was observed under TEM. The amount of ALP protein produced by MSCs cultured in groups 2 and 3 increased significantly over 14 days (P< 0.05), but there was no increase in group 1. ALP, osteopontin and type I pro-collagen production was significantly greater for group 2 than for group 1 and for group 3 than for group 2 (P< 0.05).

Discussion and Conclusions: ALP, type I pro-collagen and osteopontin proteins are known to be produced by osteoblasts during increasing cell maturation and the levels of each of these proteins increased significantly when MSCs were cultured on allograft for 14 days compared with the negative control. The addition of osteogenic supplements significantly increased production of these proteins. Furthermore, MSCs cultured in groups 2 and 3 produced extracellular collagen matrix. These results are consistent with allograft causing MSCs to differentiate into osteoblasts and that this differentiation increases with additional osteogenic supplements.

This study confirms that MSCs, derived from autologous bone marrow, could be used to increase the osteogenic potential of allograft, thereby increasing bony healing in patients.