Fig 1: Relative Vegf gene expression in cultured hPDL cells was significantly upregulated following treatment with 0.5 µg/mL EPO for 1 week compared to the control group (A). Western blot analysis confirmed an increase in Fibronectin (FN) matrix protein and Runx2 osteoblast differentiation factor expression in the EPO group compared to the control (B). For pathway analysis studies, cultured hPDL cells were treated with control media, 0.5 µg/mL EPO, 10 µm VEGF inhibitor and VEGF inhibitor with EPO addition for 24 h (C–H) and the effect of treatment conditions on collagen I, III, fibronectin, and vinculin was assessed. In these studies, EPO treatment resulted in a significant upregulation of Collagen III (D) and FN (E) compared to the control confirming the capability of EPO to promote new mineralization and matrix formation. Inhibiting VEGF while adding EPO further upregulated the expression levels of collagen III (D), fibronectin (E) and for vinculin (F) in a significant fashion. The effect of EPO, VEGF inhibitor treatment, and EPO rescue on fibronectin protein expression was confirmed by Western blot (G). The statistically significant difference of p < 0.05 between an experimental group versus the control is indicated by an * and a statistically significant difference of p < 0.05 between two experimental groups is indicated by a #.
Fig 2: We hypothesize that EPO promotes new bone formation in an alveolar defect model by depositing a strong collagen I/III and fibronectin-rich extracellular matrix which upon VEGF induced new blood vessel invasion facilitates new calcium phosphate precipitation and apatite crystal growth.
Fig 3: Picrosirius red staining (PSR) revealed well organized bundles of collagen fibers at the extraction socket site of the control and EPO treated groups of rats at 4 weeks (A and C) and at 8 weeks (B and D) post extraction. Immunohistochemical labeling demonstrating high levels of fibronectin protein expression in the newly formed extracellular matrix in the Bio-Oss group (E) and in sheath-like layers at the new bone formation sites in EPO treatment group (F). There was a significant increase in key extracellular matrix proteins collagen I, collagen III, E-cadherin 1, and fibronectin four weeks after surgery (G) and compared to BioOss and collagen membrane controls, while matrix protein production subsides eight weeks post-surgery (H). The significance value of p < 0.01 is indicated by ** and p < 0.001 is indicated by ***.
Fig 4: (A–F) X-Rays of 4 weeks (A–C) and 8 weeks (D–F) post-surgery rat maxillae following treatment with collagen membrane alone (A,D), BioOss (B,E) and EPO impregnated collagen sponge (C,F). Note the high radio-opacity in the EPO treatment groups 4 and 8 weeks after surgery (C,F) compared to the BioOss (B,E) and the collagen membrane control group (A,D). The graph in (G) revealed the significant difference in the mean grey value at 4 weeks indicated by * (p < 0.05) between the control and the Bio-Oss group and between the control and the EPO treated group (p < 0.01) indicated by **. At 8 weeks, the mean grey value was significantly lower in the Bio-Oss group compared to the control and EPO treated group (p < 0.05) indicated by *. The position of the rat maxillary molars is indicated as m1, m2, and m3, and the extraction site is demarked with a double arrow. The radio-opacity indicative of mineralization in the collagen/EPO treatment group was significantly higher than in the collagen membrane control group and in the Bio-Oss group 4 weeks after surgery.
Fig 5: (A,B) Effect of collagen membrane, BioOss, and collagen/EPO scaffolds on the expression of key angiogenesis/osteogenesis genes four weeks after surgery (A) and 8 weeks after surgery (B). Note the significant effect of the collagen/EPO scaffold on Vegf and on the osteoblast transcription factors Osx and Runx2 (A). The significance value of p < 0.05 is indicated by *, p < 0.01 is indicated by ** and p < 0.001 is indicated by ***. There was a distinct presence of newly formed capillaries in the collagen/EPO treatment group when compared to the BioOss treatment group (C,D). E and F document a close proximity of the newly mineralized bone and blood vessels (small arrows) in the EPO treatment group as illustrated by trichrome stain (E) and von Kossa staining (F).
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