Introduction

Problematic bone defects are encountered regularly in orthopaedic practice particularly in fracture non-union, revision hip and knee arthroplasty, following bone tumour excision and in spinal fusion surgery. At present the optimal source of graft to ‘fill’ these defects is autologous bone but this has significant drawbacks including harvest site morbidity and limited quantities.

Bone marrow has been proposed as the main source of osteogenic stem cells for the tissue-engineered cell therapy approach to bone defect management. Such cells constitute a minute proportion of the total marrow cell population and their isolation and expansion is a time consuming and expensive strategy.

In this study we investigated human bone marrow stem cells as a potential treatment of bone defect by looking at variability in patient osteogenic cell populations as a function of patient differences. We produced a model to predict which patients would be more suited to cell based therapies and propose possible methods for improving the quality of grafts.

Introduction: Osteoblasts precursors reside in the marrow and small numbers circulate in the blood. Our previous work demonstrated an increase in circulating cells following fracture in humans. Skeletal injury is recognised to stimulate a distant osteogenic response.

We hypothesised that in response to fracture, some integral osteoblasts are recruited via the circulation from remote bone marrow sites.

Method: We established a fracture union model in 3-month-old, male, New Zealand White rabbits and reimplanted labelled autologous osteoblast precursors. At date of submission we have 20 rabbits allocated into 4 groups. Three groups had labelled cells re-implanted, whilst the fourth control group did not receive cells. In groups I, II and III the cells were re-implanted into the fracture gap, into the circulation and into a remote bone marrow cavity respectively. There were six animals in groups I and IV, and four in both II and III.

All animals had bone marrow harvested from their right tibia by saline flush. The mononuclear cells were isolated and culture-expanded in osteogenic medium for 3 weeks. Fluorescent reporter molecules were incorporated into the cell membranes, 24 hours prior to re-implantation of the cells into the fracture model. A 3 mm ulnar defect was preformed in all the animals. In groups I–III this was established 48 hours prior to cell re-implantation.

The animals were sacrificed at least 3 weeks after fracture surgery. Representative samples of the fracture callous, lung, liver, spleen and kidney were harvested from all animals and cryo-sectioned. Using confocal microscopy, the labelled cells were expressed as the average in 5 high power fields for each solid tissue. In addition, cyto-spins were made from blood and marrow and the cell number expressed as a percentage of the total cells.

Results: In group I, labelled cells were identified in the fracture callous, establishing their viability in vivo. Following intravenous re-implantation a smaller number of labelled cells were identified in the callous. When the cells were re-implanted into a remote marrow site, the number of cells in the callous was greater than after venous reimplantation, but less numerous than those in group I.

In all sections, these labelled cells appeared on trabecular surfaces in an osteoblastic fashion, but occasionally they were surrounded by osteoid, corresponding to osteocytes.

A small number of labelled cells were found in the blood, bone marrow, lung, liver and spleen of all animals in groups I–III. No labelled cells were identified in the kidney tissue.

Discussion and Conclusions: We have demonstrated that cells from remote sites are integral in fracture healing. Their presence in callous following venous administration supports recruitment via the circulation. This preliminary data is a proof of concept. This is an exciting new phenomenon, which could provide alternatives for harvesting skeletal progenitor cells and for their delivery in the treatment of bony pathology.