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Minipig-BMSCs Combined with a Self-Setting Calcium Phosphate Paste for Bone Tissue Engineering

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Abstract

Calcium phosphate cements (CPCs) are a new generation of bone repair materials with good biocompatibility for various stem cells. The minipig is a recommended large animal model for bone engineering research. This study aimed to evaluate the feasibility of utilizing CPC scaffolds for the adhesion, proliferation, and osteogenic differentiation of minipig’s bone marrow mesenchymal stem cells (pBMSCs). Passage 3 pBMSCs were seeded on the CPC scaffold and cultured with osteogenic culture medium (osteogenic group) or normal medium (control group). The density of viable cells increased in both groups, and pBMSCs firmly attached and spread well on the CPC scaffold. The alkaline phosphatase (ALP) activity in the osteogenic group had significantly increased on day 7 (D7) and peaked on D14. qRT-PCR revealed that mRNA levels of ALP and three osteogenic marker genes were significantly higher on D4, D7, and D14 in the osteogenic group. Alizarin Red S staining showed a significantly higher degree of bone mineralization from D7, D14 to D21 in the osteogenic group. These results indicated that pBMSCs can attach, proliferate well on CPC scaffold, and be successfully induced to differentiate into osteogenic cells. Our findings may be helpful for bone tissue engineering and the studies of bone regeneration.

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References

  1. Agarwal, R., & García, A. J. (2015). Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair. Advanced Drug Delivery Reviews, 94, 53–62.

    Article  CAS  Google Scholar 

  2. Gamie, Z., Tran, G. T., Vyzas, G., Korres, N., Heliotis, M., Mantalaris, A., et al. (2012). Stem cells combined with bone graft substitutes in skeletal tissue engineering. Expert Opinion on Biological Therapy, 12, 713–729.

    Article  CAS  Google Scholar 

  3. Marino, J. T., & Ziran, B. H. (2010). Use of Solid and Cancellous Autologous Bone Graft for Fractures and Nonunions. Orthopedic Clinics of North America, 41, 15–26.

    Article  Google Scholar 

  4. Low, K. L., Tan, S. H., Zein, S. H., Roether, J. A., Mouriño, V., & Boccaccini, A. R. (2010). Calcium phosphate-based composites as injectable bone substitute materials. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 94, 273–286.

    Google Scholar 

  5. Zhao, J., Zhang, Z., Wang, S., Sun, X., Zhang, X., Chen, J., et al. (2009). Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines. Bone, 45, 517–527.

    Article  CAS  Google Scholar 

  6. Park, J. H., Lee, E. J., Knowles, J. C., & Kim, H. W. (2014). Preparation of in situ hardening composite microcarriers: calcium phosphate cement combined with alginate for bone regeneration. Journal of Biomaterials Applications, 28, 1079–1084.

    Article  Google Scholar 

  7. Barrère, F., van Blitterswijk, C. A., & de Groot, K. (2006). Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics. International Journal of Nanomedicine, 1, 317–332.

    Google Scholar 

  8. Laurencin, C. T., Ambrosio, A. M., Borden, M. D., & Cooper, J. A. (1999). Tissue engineering: orthopedic applications. Annual Review of Biomedical Engineering, 1, 19–46.

    Article  CAS  Google Scholar 

  9. Yokoyama, A., Yamamoto, S., Kawasaki, T., Kohgo, T., & Nakasu, M. (2002). Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials. Biomaterials, 23, 1091–1101.

    Article  CAS  Google Scholar 

  10. Kinoshita, Y., & Maeda, H. (2013). Recent developments of functional scaffolds for craniomaxillofacial bone tissue engineering applications. Scientific World Journal, 2013, 863157.

    Article  Google Scholar 

  11. He, F., Li, J., & Ye, J. (2013). Improvement of cell response of the poly(lactic-co-glycolic acid)/calcium phosphate cement composite scaffold with unidirectional pore structure by the surface immobilization of collagen via plasma treatment. Colloids Surfactants B Biointerfaces, 103, 209–216.

    Article  CAS  Google Scholar 

  12. Vater, C., Lode, A., Bernhardt, A., Reinstorf, A., Heinemann, C., & Gelinsky, M. (2010). Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells. Journal of Biomedical Materials Research Part A, 92, 1452–1460.

    Google Scholar 

  13. Chen, W., Liu, J., Manuchehrabadi, N., Weir, M. D., Zhu, Z., & Xu, H. H. K. (2013). Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects. Biomaterials, 34, 9917–9925.

    Article  CAS  Google Scholar 

  14. Chen, W., Zhou, H., Weir, M. D., Tang, M., Bao, C., & Xu, H. H. (2013). Human embryonic stem cell-derived mesenchymal stem cell seeding on calcium phosphate cement-chitosan-RGD scaffold for bone repair. Tissue Engineering Part A, 19, 915–927.

    Article  CAS  Google Scholar 

  15. Xia, L., Zhang, M., Chang, Q., Wang, L., Zeng, D., Zhang, X., et al. (2013). Enhanced dentin-like mineralized tissue formation by AdShh-transfected human dental pulp cells and porous calcium phosphate cement. PLoS One, 8, e62645.

    Article  CAS  Google Scholar 

  16. Friedman, C. D., Costantino, P. D., Takagi, S., & Chow, L. C. (1998). Bonesource(TM) hydroxyapatite cement: A novel biomaterial for craniofacial skeletal tissue engineering and reconstruction. Journal of Biomedical Materials Research, 43, 428–432.

    Article  CAS  Google Scholar 

  17. Zhao, L., Burguera, E. F., Xu, H. H., Amin, N., Ryou, H., & Arola, D. D. (2010). Fatigue and human umbilical cord stem cell seeding characteristics of calcium phosphate-chitosan-biodegradable fiber scaffolds. Biomaterials, 31, 840–847.

    Article  CAS  Google Scholar 

  18. Zhao, L., Weir, M. D., & Xu, H. H. (2010). Human umbilical cord stem cell encapsulation in calcium phosphate scaffolds for bone engineering. Biomaterials, 31, 3848–3857.

    Article  CAS  Google Scholar 

  19. Pearce, A. I., Richards, R. G., Milz, S., Schneider, E., & Pearce, S. G. (2007). Animal models for implant biomaterial research in bone: a review. Eur. Cell. Mater., 13, 1–10.

    CAS  Google Scholar 

  20. Aerssens, J., Boonen, S., Lowet, G., & Dequeker, J. (1998). Interspecies differences in bone composition, density, and quality: Potential implications for in vivo bone research. Endocrinology, 139, 663–670.

    CAS  Google Scholar 

  21. Martiniaková, M., Grosskopf, B., Omelka, R., Vondráková, M., & Bauerová, M. (2006). Differences among species in compact bone tissue microstructure of mammalian skeleton: Use of a discriminant function analysis for species identification. Journal of Forensic Sciences, 51, 1235–1239.

    Article  Google Scholar 

  22. Martínez-González, J. M., Cano-Sánchez, J., Campo-Trapero, J., Gonzalo-Lafuente, J. C., Díaz-Regañón, J., & Vázquez-Piñeiro, M. T. (2005). Evaluation of minipigs as an animal model for alveolar distraction. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics, 99, 11–16.

    Article  Google Scholar 

  23. Horner, E. A., Kirkham, J., Wood, D., Curran, S., Smith, M., Thomson, B., et al. (2010). Long bone defect models for tissue engineering applications: criteria for choice. Tissue Engineering Part B, 16, 263–271.

    Article  Google Scholar 

  24. Hakimi, M., Jungbluth, P., Sager, M., Betsch, M., Herten, J., Becker, M., et al. (2010). Combined use of platelet-rich plasma and autologous bone grafts in the treatment of long bone defects in mini-pigs. Injury, 41, 717–723.

    Article  CAS  Google Scholar 

  25. Pieri, F., Lucarelli, E., Corinaldesi, G., Fini, M., Aldini, N. N., Giardino, R., et al. (2009). Effect of mesenchymal stem cells and platelet-rich plasma on the healing of standardized bone defects in the alveolar ridge: a comparative histomorphometric study in minipigs. Journal of Oral and Maxillofacial Surgery, 67, 265–272.

    Article  Google Scholar 

  26. Okabe, K., Yamada, Y., Ito, K., Kohgo, T., Yoshimi, R., & Ueda, M. (2009). Injectable soft-tissue augmentation by tissue engineering and regenerative medicine with human mesenchymal stromal cells, platelet-rich plasma and hyaluronic acid scaffolds. Cytotherapy, 11, 307–316.

    Article  CAS  Google Scholar 

  27. Wang, Y. H., Liu, Y., Maye, P., & Rowe, D. W. (2006). Examination of Mineralized Nodule Formation in Living Osteoblastic Cultures Using Fluorescent Dyes. Biotechnology Progress, 22, 1697–1701.

    Article  CAS  Google Scholar 

  28. Silva, G. A., Coutinho, O. P., Ducheyne, P., & Reis, R. L. (2007). Materials in particulate form for tissue engineering. 2. Applications in bone. Journal of Tissue Engineering Regenerative Medicine, 1, 97–109.

    Article  CAS  Google Scholar 

  29. Kretlow, J. D., Young, S., Klouda, L., Wong, M., & Mikos, A. G. (2009). Injectable biomaterials for regenerating complex craniofacial tissues. Advanced Materials, 21, 3368–3393.

    Article  CAS  Google Scholar 

  30. Hesaraki, S., & Nezafati, N. (2014). In vitro biocompatibility of chitosan/hyaluronic acid-containing calcium phosphate bone cements. Bioprocess and Biosystems Engineering, 37, 1507–1516.

    Article  CAS  Google Scholar 

  31. Wang, J., de Boer, J., & de Groot, K. (2008). Proliferation and Differentiation of MC3T3-E1 Cells on Calcium Phosphate/Chitosan Coatings. Journal of Dental Research, 87, 650–654.

    Article  CAS  Google Scholar 

  32. Kim, K., Dean, D., Mikos, A. G., & Fisher, J. P. (2009). Effect of initial cell seeding density on early osteogenic signal expression of rat bone marrow stromal cells cultured on cross-linked poly(propylene fumarate) disks. Biomacromolecules, 10, 1810–1817.

    Article  CAS  Google Scholar 

  33. Moreau, J. L., & Xu, H. H. K. (2009). Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate-chitosan composite scaffold. Biomaterials, 30, 2675–2682.

    Article  CAS  Google Scholar 

  34. Hofmann, S., Hagenmüller, H., Koch, A. M., Müller, R., Vunjak-Novakovic, G., Kaplan, D. L., et al. (2007). Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials, 28, 1152–1162.

    Article  CAS  Google Scholar 

  35. Leach, J. K., Kaigler, D., Wang, Z., Krebsbach, P. H., & Mooney, D. J. (2006). Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration. Biomaterials, 27, 3249–3255.

    Article  CAS  Google Scholar 

  36. Tang, M., Weir, M. D., & Xu, H. H. (2012). Mannitol-containing macroporous calcium phosphate cement encapsulating human umbilical cord stem cells. J. Tissue. Eng. Regen. Med., 6, 214–224.

    Article  CAS  Google Scholar 

  37. Zhou, H., & Xu, H. H. K. (2011). The fast release of stem cells from alginate-fibrin microbeads in injectable scaffolds for bone tissue engineering. Biomaterials, 32, 7503–7513.

    Article  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge Prof. Zhengliang Zhao at the Southern Medical University for SEM assistance and useful discussions.

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    Authors and Affiliations

    1. Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China

      Gengtao Qiu, Zhanjun Shi & Liang Zhao

    2. Department of Orthopaedic Surgery, Shunde First People Hospital, Shunde, 528300, Guangdong, China

      Gengtao Qiu

    3. Biomaterials and Tissue Engineering Division, Department of Endodontics, Periodontics and Prosthodontics, University of Maryland Dental School, Baltimore, MD, 21201, USA

      Ping Wang, Michael D. Weir, Jinyu Sun, Yang Song, Huakun H. Xu & Liang Zhao

    4. Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China

      Guangjun Li & Jixing Wang

    5. Department of Orthopaedic Surgery, Deqing Hospital, Huzhou, 313200, Zhejiang, China

      Guangjun Li

    6. Center for Stem Cell Biology and Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

      Huakun H. Xu

    7. University of Maryland Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

      Huakun H. Xu

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    1. Gengtao Qiu
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    Correspondence to Liang Zhao.

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    Funding

    This study was supported by National Natural Science Foundation of China 31328008 (LZ), Natural Science Foundation of Guangdong s20130010014253 (LZ) and 2014A03031327 (LZ), Guangdong Provincial Science and Technology Project 2012B010200024 (LZ), and Guangzhou Science and Technology Project 2012027 (LZ).

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    G. Qiu, P. Wang and G. Li have contributed equally to this work.

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    Qiu, G., Wang, P., Li, G. et al. Minipig-BMSCs Combined with a Self-Setting Calcium Phosphate Paste for Bone Tissue Engineering. Mol Biotechnol 58, 748–756 (2016). https://doi.org/10.1007/s12033-016-9974-6

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