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Effects of Seeding Density on Proteoglycan Assembly of Passaged Mesenchymal Stem Cells

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Abstract

Cartilage repair strategies have utilized a wide range of cell types at a similarly wide range of seeding densities, both of which may affect ECM assembly. In this study, we investigated the effects of cell type and seeding density on proteoglycan assembly and the associated mechanical properties of tissue engineered constructs. Bovine and equine articular chondrocytes (AC) and first (P1) and second passage (P2) equine mesenchymal stem cells (MSCs) were encapsulated into alginate disk gels at densities of 1-, 10-, 25-, and 50 × 106 cells/mL for chondrocytes, and 25 × 106 cells/mL for P1-MSCs and 1-, 10-, and 25 × 106 cells/mL for P2-MSCs. Glycosaminoglycan content of the gels and surrounding media, as well as the resulting aggregate modulus of the disks was quantified at times up to 6 weeks. GAG accumulation was found to depend on cell type and density, and was observed to change with time. P1-MSCs produced the most total GAG (gel + media). Retention of GAG in gels was highest for bovine AC gels (which were used as a gold standard comparison), with equine MSCs retaining the least GAG. Though P1-MSCs were able to produce the largest amount of GAG, their ability to retain the produced GAG was very limited. This deficiency in retention by MSCs may be related to the lack of accumulation of link protein in MSC seeded gels. This is consistent with the lower amounts of GAG found in stem cell seeded gels by several studies, but also begins to elucidate metabolic patterns of MSCs. Future studies in cartilage engineering utilizing MSCs should explore ways of enhancing GAG retention.

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References

  1. Almarza, A. J., and K. A. Athanasiou. Effects of initial cell seeding density for the tissue engineering of the temporomandibular joint disc. Ann. Biomed. Eng. 33(7):943–950, 2005.

    Article  Google Scholar 

  2. Buschmann, M. D., Y. A. Gluzband, A. J. Grodzinsky, and E. B. Hunziker. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J. Cell Sci. 108(Pt 4):1497–1508, 1995.

    Google Scholar 

  3. Caplan, A. I. Mesenchymal stem cells. J. Orthop. Res. 9(5):641–650, 1991.

    Article  Google Scholar 

  4. Chang, S. C., J. A. Rowley, G. Tobias, N. G. Genes, A. K. Roy, D. J. Mooney, C. A. Vacanti, and L. J. Bonassar. Injection molding of chondrocyte/alginate constructs in the shape of facial implants. J. Biomed. Mater. Res. 55(4):503–511, 2001.

    Article  Google Scholar 

  5. Darling, E. M., and K. A. Athanasiou. Rapid phenotypic changes in passaged articular chondrocyte subpopulations. J. Orthop. Res. 23(2):425–432, 2005.

    Article  Google Scholar 

  6. Enobakhare, B. O., D. L. Bader, and D. A. Lee. Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue. Anal. Biochem. 243(1):189–191, 1996.

    Article  Google Scholar 

  7. Fuchs, J. R., S. Terada, D. Hannouche, E. R. Ochoa, J. P. Vacanti, and D. O. Fauza. Engineered fetal cartilage: structural and functional analysis in vitro. J. Pediatr. Surg. 37(12):1720–1725, 2002.

    Article  Google Scholar 

  8. Genes, N. G., J. A. Rowley, D. J. Mooney, and L. J. Bonassar. Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces. Arch. Biochem. Biophys. 422(2):161–167, 2004.

    Article  Google Scholar 

  9. Giannoni, P., A. Crovace, M. Malpeli, E. Maggi, R. Arbico, R. Cancedda, and B. Dozin. Species variability in the differentiation potential of in vitro-expanded articular chondrocytes restricts predictive studies on cartilage repair using animal models. Tissue Eng. 11(1–2):237–248, 2005.

    Article  Google Scholar 

  10. Gleghorn, J. P., A. R. Jones, C. R. Flannery, and L. J. Bonassar. Boundary mode frictional properties of engineered cartilaginous tissues. Eur. Cell Mater. 14:20–28, 2007; discussion 28-9.

    Google Scholar 

  11. Gleghorn, J. P., C. S. Lee, M. Cabodi, A. D. Stroock, and L. J. Bonassar. Adhesive properties of laminated alginate gels for tissue engineering of layered structures. J. Biomed. Mater. Res. A 85(3):611–618, 2008.

    Google Scholar 

  12. Hunter, C. J., J. K. Mouw, and M. E. Levenston. Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness. Osteoarthritis Cartilage 12(2):117–130, 2004.

    Article  Google Scholar 

  13. Kim, Y. J., R. L. Sah, J. Y. Doong, and A. J. Grodzinsky. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal. Biochem. 174(1):168–176, 1988.

    Article  Google Scholar 

  14. Kobayashi, S., A. Meir, and J. Urban. Effect of cell density on the rate of glycosaminoglycan accumulation by disc and cartilage cells in vitro. J. Orthop. Res. 26(4):493–503, 2008.

    Article  Google Scholar 

  15. Langer, R., and J. P. Vacanti. Tissue engineering. Science 260(5110):920–926, 1993.

    Article  Google Scholar 

  16. Mackay, A. M., S. C. Beck, J. M. Murphy, F. P. Barry, C. O. Chichester, and M. F. Pittenger. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4(4):415–428, 1998.

    Article  Google Scholar 

  17. Mahmoudifar, N., and P. M. Doran. Effect of seeding and bioreactor culture conditions on the development of human tissue-engineered cartilage. Tissue Eng. 12(6):1675–1685, 2006.

    Article  Google Scholar 

  18. Mandl, E. W., S. W. van der Veen, J. A. Verhaar, and G. J. van Osch. Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity. Tissue Eng. 10(1–2):109–118, 2004.

    Article  Google Scholar 

  19. Mauck, R. L., B. A. Byers, X. Yuan, and R. S. Tuan. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech. Model. Mechanobiol. 6(1–2):113–125, 2007.

    Article  Google Scholar 

  20. Mauck, R. L., S. L. Seyhan, G. A. Ateshian, and C. T. Hung. Influence of seeding density and dynamic deformational loading on the developing structure/function relationships of chondrocyte-seeded agarose hydrogels. Ann. Biomed. Eng. 30(8):1046–1056, 2002.

    Article  Google Scholar 

  21. Mauck, R. L., M. A. Soltz, C. C. Wang, D. D. Wong, P. H. Chao, W. B. Valhmu, C. T. Hung, and G. A. Ateshian. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. 122(3):252–260, 2000.

    Article  Google Scholar 

  22. Mauck, R. L., X. Yuan, and R. S. Tuan. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage 14(2):179–189, 2006.

    Article  Google Scholar 

  23. Mayne, R., M. S. Vail, P. M. Mayne, and E. J. Miller. Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity. Proc. Natl Acad. Sci. USA 73(5):1674–1678, 1976.

    Article  Google Scholar 

  24. Mow, V. C., S. C. Kuei, W. M. Lai, and C. G. Armstrong. Biphasic creep and stress relaxation of articular cartilage in compression: theory and experiments. J. Biomech. Eng. 102(1):73–84, 1980.

    Article  Google Scholar 

  25. Nicodemus, G. D., S. M. Giunta, and S. J. Bryant. Rational design of 3D hydrogels to capture and retain ECM molecules within mechanically stimulated PRG gels. In: 2009 Transactions of the Orthopaedic Research Society, Las Vegas, NV, 2009.

  26. Panossian, A., S. Ashiku, C. H. Kirchhoff, M. A. Randolph, and M. J. Yaremchuk. Effects of cell concentration and growth period on articular and ear chondrocyte transplants for tissue engineering. Plast. Reconstr. Surg. 108(2):392–402, 2001.

    Article  Google Scholar 

  27. Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147, 1999.

    Article  Google Scholar 

  28. Platt, D., T. Wells, and M. T. Bayliss. Proteoglycan metabolism of equine articular chondrocytes cultured in alginate beads. Res. Vet. Sci. 62(1):39–47, 1997.

    Article  Google Scholar 

  29. Quinn, T. M., and A. J. Grodzinsky. Longitudinal modulus and hydraulic permeability of poly(methacrylic acid) gels—effects of charge-density and solvent content. Macromolecules 26(16):4332–4338, 1993.

    Article  Google Scholar 

  30. Saini, S., and T. M. Wick. Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development. Biotechnol. Prog. 19(2):510–521, 2003.

    Article  Google Scholar 

  31. Sharma, B., C. G. Williams, T. K. Kim, D. Sun, A. Malik, M. Khan, K. Leong, and J. H. Elisseeff. Designing zonal organization into tissue-engineered cartilage. Tissue Eng. 13(2):405–414, 2007.

    Article  Google Scholar 

  32. Sittinger, M., C. Perka, O. Schultz, T. Haupl, and G. R. Burmester. Joint cartilage regeneration by tissue engineering. Z. Rheumatol. 58(3):130–135, 1999.

    Article  Google Scholar 

  33. Tran-Khanh, N., C. D. Hoemann, M. D. McKee, J. E. Henderson, and M. D. Buschmann. Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix. J. Orthop. Res. 23(6):1354–1362, 2005.

    Article  Google Scholar 

  34. Tuan, R. S. Cellular signaling in developmental chondrogenesis: N-cadherin, Wnts, and BMP-2. J. Bone Joint Surg. Am. 85-A(Suppl 2):137–141, 2003.

    MathSciNet  Google Scholar 

  35. Veilleux, N. H., I. V. Yannas, and M. Spector. Effect of passage number and collagen type on the proliferative, biosynthetic, and contractile activity of adult canine articular chondrocytes in type I and II collagen-glycosaminoglycan matrices in vitro. Tissue Eng. 10(1–2):119–127, 2004.

    Article  Google Scholar 

  36. von der Mark, K., V. Gauss, H. von der Mark, and P. Muller. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267(5611):531–532, 1977.

    Article  Google Scholar 

  37. Wong, M., M. Siegrist, X. Wang, and E. Hunziker. Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J. Orthop. Res. 19(3):493–499, 2001.

    Article  Google Scholar 

  38. Worster, A. A., A. J. Nixon, B. D. Brower-Toland, and J. Williams. Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am. J. Vet. Res. 61(9):1003–1010, 2000.

    Article  Google Scholar 

  39. Xu, J. W., V. Zaporojan, G. M. Peretti, R. E. Roses, K. B. Morse, A. K. Roy, J. M. Mesa, M. A. Randolph, L. J. Bonassar, and M. J. Yaremchuk. Injectable tissue-engineered cartilage with different chondrocyte sources. Plast. Reconstr. Surg. 113(5):1361–1371, 2004.

    Article  Google Scholar 

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Acknowledgments

This work was funded by the Alfred P. Sloan foundation and Cornell University. The authors thank Drs. Alan Nixon and Lisa Fortier, as well as members of the Comparative Orthopaedics Laboratory at Cornell University’s College of Veterinary Medicine for their assistance in the acquisition of the equine chondrocytes and mesenchymal stem cells. Mary Lou Norman is acknowledged for her help with the processing of the histology samples.

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  1. Department of Biomedical Engineering, Cornell University, 149 Weill Hall, Ithaca, NY, 14853, USA

    Omotunde M. Babalola & Lawrence J. Bonassar

  2. Sibley School of Mechanical and Aerospace Engineering, Cornell University, 149 Weill Hall, Ithaca, NY, 14853, USA

    Lawrence J. Bonassar

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  1. Omotunde M. Babalola
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Correspondence to Lawrence J. Bonassar.

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Babalola, O.M., Bonassar, L.J. Effects of Seeding Density on Proteoglycan Assembly of Passaged Mesenchymal Stem Cells. Cel. Mol. Bioeng. 3, 197–206 (2010). https://doi.org/10.1007/s12195-010-0107-1

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