Polímeros: Ciência e Tecnologia
Polímeros: Ciência e Tecnologia
Original Article

In vitro and in vivo cell tracking of PKH26-labeled osteoblasts cultured on PLDLA scaffolds

Duek, Alice Rezende; Costa, Gabriel Ciambelli Dias da; Más, Bruna Antunes; Barbo, Maria Lourdes Peris ; Motta, Adriana Cristina; Duek, Eliana Aparecida de Rezende

Downloads: 0
Views: 728


The importance of monitoring in vivo interaction that occurs between cells /bio/tissue recipient in the understanding of tissue regeneration processes becomes ever greater. This study aims to monitor and evaluate the influence of scaffold implants of poly (L-co-D, L lactic acid) - PLDLA synthesized in the laboratory, previously cultured with primary osteoblastic cells heterologously stained with the fluorescent vital dye, PKH26, on the tissue regeneration process in 8 mm central critical defects of the Wistar rat calvaria. The results obtained by MTT assay and monitoring of cells stained with PKH26 dye over 14 days of culture showed that the dye was cytocompatible with osteoblastic cells and did not exert a negative influence on the growth of unstained cells. In the in vivo study, macroscopic observations made during deployment times corroborate the results in vitro, as no apparent signs of toxicity were observed in the implanted bone defect area. The use of mobile monitoring with the dye, PKH26 in vivo is an effective strategy for the understanding of cell behaviour in the presence of PLDLA polymer.


PKH26, PLDLA, scaffolds, tissue engineering


1. Dantas, T. S., Lelis, E. R., Naves, L. Z., Fernandes-Neto, A. J., & Magalhães, D. (2011). Bone graft materials and their application in dentistry. UNOPAR Científica Ciências Biológicas e da Saúde, 13(2), 131-135. Retrieved in 9 November 2015, from pgsskroton.com.br/seer/index.php/biologicas/article/viewFile/1248/1198

2. Kunz, F., Bergemann, C., Klinkenberg, E. D., Weidmann, A., Lange, R., Beck, U., & Nebe, J. B. (2010). A novel modular device for 3-D bone cell culture and nondestructive cell analysis. Acta Biomaterialia, 6(9), 3798-3807. PMid:20227531. http://dx.doi.org/10.1016/j.actbio.2010.03.015.

3. Ikeda, T., Ikeda, K., Yamamoto, K., Ishizaki, H., Yoshizawa, Y., Yanagiguchi, K., Yamada, S., & Hayashi, Y. (2014). Fabrication and characteristics of chitosan sponge as a tissue engineering scaffold. BioMed Research International, 6, 1-8. PMid:24804246. http://dx.doi.org/10.1155/2014/786892.

4. Kroeze, R. J., Helder, M. N., Govaert, L. E., & Smit, T. H. (2009). Biodegradable Polymers in Bone, Tissue Engineering. Materials (Basel), 2(3), 833-856. http://dx.doi.org/10.3390/ma2030833.

5. Alexander, J. T., Branch, C. L., Jr, Subach, B. R., & Haid, R. W., Jr. (2002). Applications of a resorbable interbody spacer via a posterior lumbar interbody fusion technique. Orthopedics, 25(10), S1185-S1189. PMid:12401030.

6. Moser, R. C., Mcmanus, A., Riley, S., & Thomas, K. (2005). Strength retention of 70:30 Poly(Llactide-co- D, L lactide) following real-time aging. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 75(1), 56-63. PMid:16001395. http://dx.doi.org/10.1002/jbm.b.30238.

7. Brazelton, T. R., & Blau, H. M. (2005). Optimizing techniques for tracking transplanted stem cells in vivo. Stem Cells, 23(9), 1251-1265. PMid:16109764. http://dx.doi.org/10.1634/stemcells.2005-0149.

8. Yoo, J. J., Bichara, D. A., Zhao, X., Randolph, M. A., & Gill, T. J. (2011). Implant-assisted meniscal repair in vivo using a chondrocyte-seeded flexible PLGA scaffold. Journal of Biomedical Materials Research. Part A, 99(1), 102-108. PMid:21800420. http://dx.doi.org/10.1002/jbm.a.33168.

9. Polzer, H., Volkmer, E., Saller, M. M., Prall, W. C., Haasters, F., Drosse, I., Anz, D., Mutschler, W., & Schieker, M. (2012). Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy. Tissue Engineering. Part C, Methods, 18(2), 156-165. PMid:21951128. http://dx.doi.org/10.1089/ten.tec.2011.0275.

10. Matz, R. L., Erickson, B., Vaidyanathan, S., KukowskaLatallo, J. F., Baker, J. R., Jr., Orr, B. G., & Banaszak Holl, M. M. (2013). Polyplex exposure inhibits cell cycle, increase inflammatory response, and can cause protein expression without cell division. Molecular Pharmaceutics, 10(4), 1306-1317. PMid:23458572. http://dx.doi.org/10.1021/mp300470d.

11. Wang, W.-J., Wu, S.-P., Liu, J.-B., Shi, Y.-S., Huang, X., Zhang, Q.-B., & Yao, K.-T. (2013). MYC regulation of CHK1 and CHK2 promotes radioresistance in a stem cell-like population of nasopharyngeal carcinoma cells. Cancer Research, 73(3), 1219-1231. PMid:23269272. http://dx.doi.org/10.1158/0008-5472.CAN-12-1408.

12. Canola, K., Angenieux, B., Tekaya, M., Quiambao, A., Naash, M. I., Munier, F. L., Schorderet, D. F., & Arsenijevic, Y. (2007). Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Investigative Ophthalmology & Visual Science, 48(1), 446-454. PMid:17197566. http://dx.doi.org/10.1167/iovs.06-0190.

13. Boomsma, R. A., Swaminathan, P. D., & Geenen, D. L. (2007). Intravenously injected mesenchymal stem cells home to viable myocardium after coronary occlusion and preserve systolic function without altering infarct size. International Journal of Cardiology, 122(1), 17-28. PMid:17187879. http://dx.doi.org/10.1016/j.ijcard.2006.11.022.

14. Motta, A. C., & Duek, E. A. R. (2007). Synthesis and characterization of poly (L-co-D,L acid lactic). Polímeros: Ciência e Tecnologia, 17(2), 123-129. http://dx.doi.org/10.1590/S0104-14282007000200011.

15. Declercq, H. A., Verbeeck, R. M. H., Ridder, L. I. F. J. M., Schacht, E. H., & Cornelissen, M. J. (2005). Calcification as an indicator of osteocoindutive capacity of biomaterials in osteoblastic cells cultures. Biomaterials, 26(24), 4964-4974. PMid:15769532. http://dx.doi.org/10.1016/j.biomaterials.2005.01.025.

16. Duffy, G. P., Mcfadden, T. M., Byrne, E. M., Gill, S. L., Farrell, E., & O’Brien, F. J. (2011). Towards in vitro vascularisation of collagen-GAG scaffolds. European Cells & Materials, 21(12), 15-30. PMid:21225592. http://dx.doi.org/10.22203/eCM.v021a02.

17. Ignatius, A. A., & Claes, L. E. (1996). In vitro biocompatibility of bioresorbable polymers: poly (L, DL-lactide) and poly(L-lactideco-glycolide). Biomaterials, 17(8), 831-839. PMid:8730968. http://dx.doi.org/10.1016/0142-9612(96)81421-9.

18. Thadavirul, N., Pavasant, P., & Supaphol, P. (2014). Development of polycaprolactone porous scaffolds by combining solvent casting, particulate leaching, and polymer leaching techniques for bone tissue engineering. Journal of Biomedical Materials Research. Part A, 102(10), 3379-3392. PMid:24132871. http://dx.doi.org/10.1002/jbm.a.35010.

19. Sabir, M. I., Xu, E. X., & Li, L. (2009). A review on biodegradable polymeric materials for bone tissue engineering applications. Journal of Materials Science, 44(21), 5713-5724. http://dx.doi.org/10.1007/s10853-009-3770-7.

20. Chen, J., Wang, C., Lu, S., Wu, J., Guo, X., Duan, C., Dong, L., Song, Y., Zhang, J., Jing, D., Wu, L., Ding, J., & Li, D. (2005). In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells. Cell and Tissue Research, 319(3), 429-438. PMid:15672263. http://dx.doi.org/10.1007/s00441-004-1025-0.

21. Christian, W., Johnson, T. S., & Gill, T. J. (2008). In vitro and in vivo cell tracking of chondrocytes of different origin by fluorescent PKH 26 and CMFDA. Journal of Biomedical Science and Engineering, 1(3), 163-169. http://dx.doi.org/10.4236/ jbise.2008.13027.

22. Kang, E. J., Byun, J. H., Choi, Y. J., Maeng, G. H., Lee, S. L., Kang, D. H., Lee, J. S., Rho, G. J., & Park, B. W. (2010). In vitro and in vivo osteogenesis of porcine skin-derived mesenchymal stem cell–like cells with a demineralized bone and fibrin glue scaffold. Tissue Engineering. Part A, 16(3), 815-827. PMid:19778183. http://dx.doi.org/10.1089/ten. tea.2009.0439.

23. Lee, J. Y., Choi, M. H., Shin, E. Y., & Kang, Y. K. (2011). Autologous mesenchymal stem cells loaded in Gelfoam for structural bone allograft healing in rabbits. Cell and Tissue Banking, 12(4), 299-309. PMid:20652421. http://dx.doi.org/10.1007/s10561-010-9194-4.

24. Li, P., Zhang, R., Sun, H., Chen, L., Liu, F., Yao, C., Du, M., & Jiang, X. (2013). PKH26 can transfer to host cells in vitro and vivo. Stem Cells and Development, 22(2), 340-344. PMid:22913652. http://dx.doi.org/10.1089/scd.2012.0357.

25. Park, B. W., Kang, E. J., Byun, J. H., Son, M. G., Kim, H. J., Hah, Y. S., Kim, T. H., Mohana Kumar, B., Ock, S. A., & Rho, G. J. (2012). In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues. Differentiation, 83(5), 249-259. PMid:22469856. http://dx.doi.org/10.1016/j.diff.2012.02.008.

26. Lemos, M. M. (2006). Experimental study on the effect of induced tendinitis in the gastrocnemius muscle: histopathology and raman spectroscopy. Franca: Universidade de Franca.

27. Más, B. A. (2011). Imobilização de colágeno em arcabouços de poli (L-co-D,L ácido lático) (Master’s dissertation). Faculdade de Engenharia Mecânica, Universidade Estadual de Campinas, Campinas.

28. Tavassol, F., Schumann, P., Lindhorst, D., Sinikovic, B., Voss, A., von See, C., Kampmann, A., Bormann, K. H., Carvalho, C., Mülhaupt, R., Harder, Y., Laschke, M. W., Menger, M. D., Gellrich, N. C., & Rücker, M. (2010). Accelerated angiogenic host tissue response to poly (L-lactide-co-glycolide) scaffolds by vitalization with osteoblast-like cells. Tissue Engineering. Part A, 16(7), 2265-2279. PMid:20184434. http://dx.doi.org/10.1089/ten.tea.2008.0457.

5b7afca50e88258c3a896e51 polimeros Articles
Links & Downloads

Polímeros: Ciência e Tecnologia

Share this page
Page Sections