CD133: enhancement of bone healing by local transplantation of peripheral blood cells in a biologically delayed rat osteotomy model.

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction

µCT analysis – 3D reconstructions of the bone healing progress.

<p>(<b>A</b>) 3D reconstructions from µCT scans from one representative animal after 2, 4, and 6 weeks of the three groups are shown. In the CD133(+) group (left) mineralized tissue was formed at the endosteal as well as at the periosteal regions. After 42 days the gap had almost been bridged; a thin layer of radiolucent tissue still separated the bone ends. In the ABC group (middle) only very little callus formation was observed, appearing between the 28th and 42nd day after the osteotomy. No bridging but a cap-formation was observed, sealing the medullary canal. In the CD34(+) group (right) bone formation was further progressed than in the ABC group. After 6 weeks however, bridging did not occur and the medullary canal was closed at the proximal end. (Resolution 28 µm; scale: 2 mm). (<b>B</b>) Bridging score for the ABC group is lower compared with the bridging in the CD34(+) or CD133(+) group. The bridging score for the CD34(+) group showed better results than the ABC group but lesser results compared with the CD133(+). 5 areas in the osteotomy gap were investigated, 12, 3, 6, and 9 o'clock position and the centre region. The sum of bridged areas for the healing course is indicated in the table.</p

<i>In vitro</i> analysis of angiogenic potential of progenitor cells.

<p>(A) Tube length evaluation for CD34 (+)/(−) and CD133(+)/(−) cells normalized to the tube length measured in the control group together with all PBMCs, note the significant difference in tube length between CD133(+) and CD133(−), n = 8, * p = 0.018; (B) Tube formation of CD133(+) and CD133(−) respectively, note the more pronounced tube formation in the CD133(+) fraction.</p

Histological analysis of bone formation and revascularization in the osteotomy gap.

<p>(<b>A</b>) Photomicrographs of histological sections 6 weeks post-surgery of the CD133(+) (left) and the ABC (right) group (Movat Pentachrome staining); In the CD133(+) group the osteotomy gap was predominantly filled with spongy mineralized tissue formations. A thin layer of hyaline cartilage (green) between the bone ends had not yet been mineralized. In the ABC group almost only fibrous tissue was found, completely lacking any cartilage or bone formation; (ABC: autologous blood clot group; CD133(+): CD133 cell treated group); cortical bone/yellow; bone marrow/dark red; cartilage/green; muscle/red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052650#pone.0052650-BPreininger1" target="_blank">[21]</a>. (<b>B</b>) Photomicrographs of immune histological sections 6 weeks post-surgery of the CD133(+) (left) and the ABC (right) group (factor VIII staining); In the CD133(+) group quite dense vessel formations (red) were observed, especially in the endostal region at the zone where the hyaline cartilage formations are mineralized. (ABC: autologous clot group; CD133: CD133+ cell treated group) (Scale bar: 1 mm).</p

µCT analysis – statistic evaluation of callus volume and bone mineral contend.

<p>An increase of callus volume (CV, [mm3]), as well as bone mineral contend (BMC, [mg]) on the basis of total mineral density of the callus was observed over time. Both parameters were significantly higher in the CD133(+) group when compared to the ABC group. Note that the differences occur not until after the 28th day and are most pronounced after the 42nd day. Comparing CV and BMC between the CD133(+) group and the CD34(+) group markedly higher values were found in the CD133(+) group. (CV: total volume, BMC: bone mineral content, wks: weeks); (CD133(+) group  =  grey; CD34(+) group  =  checkered; ABC (autologous blood clot) group  =  white).</p