Cell and Tissue Research

, Volume 322, Issue 2, pp 289–298 | Cite as

In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel

  • Akiko Yokoyama
  • Ichiro Sekiya
  • Kyosuke Miyazaki
  • Shizuko Ichinose
  • Yuiro Hata
  • Takeshi Muneta
Regular Article


Graft implantation is one of the more popular procedures for repairing cartilage defects; however, sacrifices of the donor site have been an issue. Mesenchymal stem cells (MSCs) are a fascinating source for regenerative medicine because they can be harvested in a less invasive manner and are easily isolated and expanded, with multipotentiality including chondrogenesis. MSCs can be isolated from various adult mesenchymal tissues including synovium. Here, we attempted to form cartilage from the composites of synovium-derived MSCs with collagen gel in vitro. After 21 days of culture, the composites had increased their cartilage matrix, as demonstrated by toluidine blue staining and immunohistochemistry for type II collagen. The composites consisting of 5×107 and 108 cells/ml in gel were richer in proteoglycans than those consisting of lower cell densities. After 1 day, MSCs/gel composites contracted and the diameter decreased by 30%; however, they were stable thereafter. Round cells with short processes producing collagen fibrils showing a similar morphology to that of chondrocytes were seen in the composites by transmission electron microscopy. During composite culture, chondroitin sulfate and mRNA expression for cartilage-related genes increased, demonstrating cartilage maturation. Using an optimized method, we obtained cartilage discs with a diameter of 7 mm and a thickness of 500 μm. Our procedure should thus make it possible to produce a large cartilage matrix in vitro. The tissue engineering of autologous cartilage from the composites of synovium-derived MSCs with collagen gel in vitro for transplantation may be a future alternative to graft implantation for patients with cartilage defects.


Mesenchymal stem cells (MSCs) Synovium Collagen gel In vitro chondrogenesis Tissue engineering Human 



We thank Kenichi Shinomiya for supporting our studies, Yusuke Sakaguchi for help with the characterization of MSCs, Hideya Yoshimura for help with real-time PCR, Izumi Nakagawa for excellent technical assistance, Miyoko Ojima for expert help with histology, and Kelly Johanson for proofreading. Recombinant human BMP2 was from the Yamanouchi Pharmaceutical Company.


  1. Barry F, Boynton R, Murphy M, Haynesworth S, Zaia J (2001) The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 289:519–524PubMedCrossRefGoogle Scholar
  2. Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A 76:1274–1278PubMedCrossRefGoogle Scholar
  3. Brent B (1992) Auricular repair with autogenous rib cartilage grafts: two decades of experience with 600 cases. Plast Reconstr Surg 90:355–374PubMedCrossRefGoogle Scholar
  4. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889–895PubMedCrossRefGoogle Scholar
  5. Colter DC, Sekiya I, Prockop DJ (2001) Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 98:7841–7845CrossRefPubMedGoogle Scholar
  6. De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP (2001) Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 44:1928–1942CrossRefPubMedGoogle Scholar
  7. Ichinose S, Muneta T, Sekiya I, Itoh S, Aoki H, Tagami M (2003) The study of metal ion release and cytotoxicity in Co-Cr-Mo and Ti-Al-V alloy in total knee prosthesis - scanning electron microscopic observation. J Mater Sci Mater Med 14:79–86CrossRefPubMedGoogle Scholar
  8. Liu H, Lee YW, Dean MF (1998) Re-expression of differentiated proteoglycan phenotype by dedifferentiated human chondrocytes during culture in alginate beads. Biochim Biophys Acta 1425:505–515PubMedGoogle Scholar
  9. Maeda S, Miyabayashi T, Yamamoto JK, Roberts GD, Lepine AJ, Clemmons RM (2003) Temporal dynamic changes in synthesis of chondroitin sulfate isomers in canine articular chondrocyte culture. J Vet Med Sci 65:1373–1376PubMedCrossRefGoogle Scholar
  10. Maurice H, Crone M, Watt I (1988) Synovial chondromatosis. J Bone Joint Surg Br 70:807–811PubMedGoogle Scholar
  11. Mourao PA (1988) Distribution of chondroitin 4-sulfate and chondroitin 6-sulfate in human articular and growth cartilage. Arthritis Rheum 31:1028–1033PubMedCrossRefGoogle Scholar
  12. Nishimura K, Solchaga LA, Caplan AI, Yoo JU, Goldberg VM, Johnstone B (1999) Chondroprogenitor cells of synovial tissue. Arthritis Rheum 42:2631–2637PubMedCrossRefGoogle Scholar
  13. Ochi M, Uchio Y, Tobita M, Kuriwaka M (2001) Current concepts in tissue engineering technique for repair of cartilage defect. Artif Organs 25:172–179CrossRefPubMedGoogle Scholar
  14. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefPubMedGoogle Scholar
  15. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74CrossRefPubMedGoogle Scholar
  16. Sakaguchi Y, Sekiya I, Yagishita K, Ichinose S, Shinomiya K, Muneta T (2004) Suspended cells from trabecular bone by collagenase digestion become virtually identical to mesenchymal stem cells obtained from marrow aspirates. Blood 104:2728–2735CrossRefPubMedGoogle Scholar
  17. Sekiya I, Colter DC, Prockop DJ (2001) BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 284:411–418CrossRefPubMedGoogle Scholar
  18. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ (2002a) Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 20:530–541CrossRefPubMedGoogle Scholar
  19. Sekiya I, Vuoristo JT, Larson BL, Prockop DJ (2002b) In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 99:4397–4402CrossRefPubMedGoogle Scholar
  20. Sekiya I, Larson BL, Vuoristo JT, Reger RL, Prockop DJ (2005) Comparison of effect of BMP−2, −4, and −6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res 320:269–276PubMedCrossRefGoogle Scholar
  21. Shinmei M, Miyauchi S, Machida A, Miyazaki K (1992) Quantitation of chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic joint fluid. Arthritis Rheum 35:1304–1308PubMedGoogle Scholar
  22. Tanzer RC (1959) Total reconstruction of the external ear. Plast Reconstr Surg 23:1–15PubMedCrossRefGoogle Scholar
  23. Tsutsumi K, Shimakawa H, Kitagawa H, Sugahara K (1998) Functional expression and genomic structure of human chondroitin 6-sulfotransferase. FEBS Lett 441:235–241PubMedCrossRefGoogle Scholar
  24. Uchimura K, Kadomatsu K, Nishimura H, Muramatsu H, Nakamura E, Kurosawa N, Habuchi O, El-Fasakhany FM, Yoshikai Y, Muramatsu T (2002) Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates. J Biol Chem 277:1443–1450CrossRefPubMedGoogle Scholar
  25. Yamauchi S, Mita S, Matsubara T, Fukuta M, Habuchi H, Kimata K, Habuchi O (2000) Molecular cloning and expression of chondroitin 4-sulfotransferase. J Biol Chem 275:8975–8981PubMedCrossRefGoogle Scholar
  26. Yoshida K, Miyauchi S, Kikuchi H, Tawada A, Tokuyasu K (1989) Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography. Anal Biochem 177:327–332CrossRefPubMedGoogle Scholar
  27. Zannettino AC, Harrison K, Joyner CJ, Triffitt JT, Simmons PJ (2003) Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody, HOP-26. J Cell Biochem 89:56–66CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Akiko Yokoyama
    • 1
    • 2
  • Ichiro Sekiya
    • 1
  • Kyosuke Miyazaki
    • 3
  • Shizuko Ichinose
    • 4
  • Yuiro Hata
    • 2
  • Takeshi Muneta
    • 1
    • 5
  1. 1.Section of Orthopaedic Surgery, Graduate SchoolTokyo Medical and Dental UniversityTokyoJapan
  2. 2.Department of Plastic and Reconstructive SurgeryTokyo Medical and Dental UniversityTokyoJapan
  3. 3.Seikagaku CorporationTokyoJapan
  4. 4.Instrumental Analysis Research CenterTokyo Medical and Dental UniversityTokyoJapan
  5. 5.Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and BoneTokyo Medical and Dental UniversityTokyoJapan

Personalised recommendations