gms | German Medical Science

Objectives: Bone healing disorders affect more than 15% of all fracture patients. While conventional treatments only focus on established non-unions, a direct application of cells with endogenous regenerative capacities is a promising alternative.

Sufficient angiogenesis and a balanced inflammation are essential in fracture healing. Peripheral blood (PB) derived CD31(+) cells play a role in angiogenic processes and immune modulation. Thus, a local administration of CD31(+) cells to a fracture gap may support endogenous regeneration and prevent healing disorders.

Methods: Flow-cytometric analyses of human PB cells were performed and CD31(+) cells isolated. Angiogenic capacities were evaluated in tube-formation-assays (co-culture with human endothelial cells) and a paracrine stimulus on osteogenesis of MSCs investigated. The impact of CD31(+) cells on cytokine expression levels of CD8(+)T-cells and monocytes was evaluated in respective co-cultures. Immunomodulatory capacity and angiogenic effects of CD31(+) cells were characterized at early healing time points on gene and protein level and via histology of fracture hematoma from a preclinical animal model. Healing progression after cell transplantation was monitored via microCT and histology over 42 days.

Results and Conclusion: The amount of viable CD31(+) cells (70-80% of leukocytes) in human PB is persistently high. Tube-formation-assays confirmed that CD31(+) cells show a high angiogenic potential (1.56foldchange, p<0.005) and analyses of MSC matrix calcification prove that CD31(+) cells stimulate osteogenic differentiation (1.65foldchange, p<0.05). In co-cultures with LPS-activated monocytes and stimulated CD8(+)T-cells, respectively, CD31(+) cells lead to anti-inflammatory signaling (monocyte culture: Il1a/IL1Ra ratio 0,06foldchange, CD8(+)T-cell culture: decreased IFNgamma, GM-CSF and IL-2 and increased IL-10 and IL-8 secretion, each compared to mono-cultures). Angiogenic factors such as vWF (1,33foldchange) and HIF1alpha (1,24foldchange) are upregulated on RNA level at early healing time points in the fracture hematoma of CD31(+) treated animals. A local application of CD31(+) cells enhances the formation of mineralized callus-tissue (2.14foldchange) compared to the control (p<0.05). Increased mineralization (1.96foldchange) and cartilage formation (2.6foldchange) in the osteotomy gap is visible in histology 42d after surgery, in accordance to a significantly reduced amount of fibrous tissue (0.73foldchange) in the CD31(+) treated animals (p<0.05).

This projects demonstrates the outstanding prospect of CD31(+) cell application in an early intervening treatment of healing disorders. The data highlights the regenerative potential of CD31(+) cells in angiogenic, immunomodulatory and osteogenic processes. The restorative effect of CD31(+) cells was validated with systematic radiological monitoring, histological evaluations and molecular screening in an animal model for biologically impaired bone healing.