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In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering

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Abstract

Tissue engineering of an anterior cruciate ligament (ACL) implant with functional enthesis requires site-directed seeding of different cell types on the same scaffold. Therefore, we studied the suitability of self-assembled three-dimensional spheroids generated by lapine ACL ligament fibroblasts for directed scaffold colonization. The spheroids were characterized in vitro during 14 days in static and 7 days in dynamic culture. Size maintenance of self-assembled spheroids, the vitality, the morphology and the expression pattern of the cells were monitored. Additionally, we analyzed the total sulfated glycosaminoglycan, collagen contents and the expression of the ligament components type I collagen, decorin and tenascin C on protein and for COL1A1, DCN and TNMD on gene level in the spheroids. Subsequently, the cell colonization of polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) polymer scaffolds was assessed in response to a directed, spheroid-based seeding technique. ACL cells were able to self-assemble spheroids and survive over 14 days. The spheroids decreased in size but not in cellularity depending on the culture time and maintained or even increased their differentiation state. The area of P[LA-CL] scaffolds, colonized after 14 days by the cells of one spheroid, was in average 4.57 ± 2.3 mm2. Scaffolds consisting of the polymer P[LA-CL] were more suitable for colonization by spheroids than PDS embroideries. We conclude that ACL cell spheroids are suitable as site-directed seeding strategy for scaffolds in ACL tissue engineering approaches and recommend the use of freshly assembled spheroids for scaffold colonization, due to their balanced proliferation and differentiation.

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Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

ACL:

Anterior cruciate ligament

COL1A1 :

Gene coding for type I collagen

DAPI:

4′,6-Diamidino-2-phenylindole

DCN :

Gene coding for decorin

DMEM:

Dulbecco’s modified Eagle’s medium

DMMB:

Dimethyl methylene blue

ECM:

Extracellular matrix

EtBr:

Ethidium bromide

FCS:

Fetal calf serum

FDA:

Fluorescein diacetate

HE:

Hematoxylin and eosin staining

l:

Lapine

MFI:

Mean fluorescence intensity

MMP:

Matrix metalloproteinase

P(LA-CL):

Polylactide-co-caprolactone

PBS:

Phosphate-buffered saline

PDS:

Polydioxanone

PFA:

Paraformaldehyde

PVA:

Polyvinyl alcohol

RT:

Room temperature

SD:

Standard deviation

SEM:

Scanning electron microscopy

TBS:

Tris-buffered saline

TNMD :

Gene coding for tenomodulin

References

  • Adams CL, Chen YT, Smith SJ, Nelson WJ (1998) Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of e-cadherin-green fluorescent protein. J Cell Biol 142:1105–1119

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Anada T, Fukuda J, Sai Y, Suzuki O (2012) An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids. Biomaterials 33:8430–8441

    Article  CAS  PubMed  Google Scholar 

  • Asthana A, Kisaalita WS (2012) Microtissue size and hypoxia in hts with 3d cultures. Drug Discov Today 17:810–817

    Article  PubMed  Google Scholar 

  • Benjamin M, Ralphs JR (2000) The cell and developmental biology of tendons and ligaments. Int Rev Cytol 196:85–130

    Article  CAS  PubMed  Google Scholar 

  • Berahim Z, Moharamzadeh K, Rawlinson A, Jowett AK (2011) Biologic interaction of three-dimensional periodontal fibroblast spheroids with collagen-based and synthetic membranes. J Periodontol 82:790–797

    Article  PubMed  Google Scholar 

  • Bobacz K, Ullrich R, Amoyo L, Erlacher L, Smolen JS, Graninger WB (2006) Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts. Ann Rheum Dis 65:169–177

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chiquet-Ehrismann R, Tucker RP (2011) Tenascins and the importance of adhesion modulation. Cold Spring Harb Perspect Biol 3(5). doi:10.1101/cshperspect.a004960

  • de Wreede R, Ralphs JR (2009) Deposition of collagenous matrices by tendon fibroblasts in vitro: a comparison of fibroblast behavior in pellet cultures and a novel three-dimensional long-term scaffoldless culture system. Tissue Eng Part A 15:2707–2715

    Article  PubMed  Google Scholar 

  • Dhandayuthapani B, Yoshida Y, Maekawa T, Kumar DS (2011) Polymeric scaffolds in tissue engineering application: A review. Int J Polym Sci 2011:19

    Article  Google Scholar 

  • Ekdahl M, Wang JH, Ronga M, Fu FH (2008) Graft healing in anterior cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc 16:935–947

    Article  PubMed  Google Scholar 

  • Gliesche K, Breier A, Schmack G (2011) Herstellung textiler Scaffolds mittels Sticktechnik. Biomaterialien 6:155–159

    Google Scholar 

  • Griffith LG, Swartz MA (2006) Capturing complex 3d tissue physiology in vitro. Nat Rev Mol Cell Biol 7:211–224

    Article  CAS  PubMed  Google Scholar 

  • Haddad-Weber M et al (2010) Bmp12 and bmp13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells. Cytotherapy 12:505–513

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hakkinen L, Strassburger S, Kahari VM, Scott PG, Eichstetter I, Lozzo RV, Larjava H (2000) A role for decorin in the structural organization of periodontal ligament. Lab Invest 80:1869–1880

    Article  CAS  PubMed  Google Scholar 

  • Ho WJ, Pham EA, Kim JW, Ng CW, Kim JH, Kamei DT, Wu BM (2010) Incorporation of multicellular spheroids into 3-d polymeric scaffolds provides an improved tumor model for screening anticancer drugs. Cancer Sci 101:2637–2643

    Article  CAS  PubMed  Google Scholar 

  • Homicz MR, McGowan KB, Lottman LM, Beh G, Sah RL, Watson D (2003) A compositional analysis of human nasal septal cartilage. Arch Facial Plast Surg 5:53–58

    Article  PubMed  Google Scholar 

  • Juneja SC (2013) Cellular distribution and gene expression profile during flexor tendon graft repair: a novel tissue engineering approach. J Tissue Eng 4:2041731413492741

    Article  PubMed Central  PubMed  Google Scholar 

  • Juneja SC, Veillette C (2013) Defects in tendon, ligament, and enthesis in response to genetic alterations in key proteoglycans and glycoproteins: a review. Arthritis 2013:154812

    Article  PubMed Central  PubMed  Google Scholar 

  • Kardon G (1998) Muscle and tendon morphogenesis in the avian hind limb. Development 125:4019–4032

    CAS  PubMed  Google Scholar 

  • Kato S, Saito M, Funasaki H, Marumo K (2013) Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro. Knee Surg Sports Traumatol Arthrosc

  • Komiyama Y et al (2013) Tenomodulin expression in the periodontal ligament enhances cellular adhesion. PLoS one 8:e60203

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lehmann M, Martin F, Mannigel K, Kaltschmidt K, Sack U, Anderer U (2013) Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes. Eur J Histochem 57:e31

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Lu HH, Subramony SD, Boushell MK, Zhang X (2010) Tissue engineering strategies for the regeneration of orthopedic interfaces. Ann Biomed Eng 38:2142–2154

    Article  PubMed Central  PubMed  Google Scholar 

  • Mackie EJ (1997) Molecules in focus: tenascin-c. Int J Biochem Cell Biol 29:1133–1137

    Article  CAS  PubMed  Google Scholar 

  • Morellini F, Schachner M (2006) Enhanced novelty-induced activity, reduced anxiety, delayed resynchronization to daylight reversal and weaker muscle strength in tenascin-c-deficient mice. Eur J Neurosci 23:1255–1268

    Article  PubMed  Google Scholar 

  • Ray JA, Doddy N, Regula D, Williams JA, Melveger A (1981) Polydioxanone (pds), a novel monofilament synthetic absorbable suture. Surg Gynecol Obstet 153:497–507

    CAS  PubMed  Google Scholar 

  • Schellings MW, Pinto YM, Heymans S (2004) Matricellular proteins in the heart: possible role during stress and remodeling. Cardiovasc Res 64:24–31

    Article  CAS  PubMed  Google Scholar 

  • Schulze-Tanzil G, Mobasheri A, Clegg PD, Sendzik J, John T, Shakibaei M (2004) Cultivation of human tenocytes in high-density culture. Histochem Cell Biol 122:219–228

    Article  CAS  PubMed  Google Scholar 

  • Smith L, Xia Y, Galatz LM, Genin GM, Thomopoulos S (2012) Tissue-engineering strategies for the tendon/ligament-to-bone insertion. Connect Tissue Res 53:95–105

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stoll C et al (2010) Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and plga cultures compared with tendon tissue: implications for tendon tissue engineering. J Orthop Res 28:1170–1177

    Article  CAS  PubMed  Google Scholar 

  • Takezawa T, Mori Y, Yonaha T, Yoshizato K (1993) Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts. Exp Cell Res 208:430–441

    Article  CAS  PubMed  Google Scholar 

  • Thorpe CT, Birch HL, Clegg PD, Screen HR (2013) The role of the non-collagenous matrix in tendon function. Int J Exp Pathol 94:248–259

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful for technical assistance of Benjamin Kohl and Marion Lemke. This study was funded by the German Research Foundation (DFG-SCHU1979/9-1) and equipment was provided by the Sonnenfeld Foundation.

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Authors and Affiliations

  1. Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, FEM, Garystrasse 5, 14195, Berlin, Germany

    M. Hoyer, C. Meier, W. Ertel, A. Lohan & G. Schulze-Tanzil

  2. Department of Bioanalytics, Technische Universität Berlin, Berlin, Germany

    M. Hoyer & L.-A. Garbe

  3. Department of Materials and Processing, Leibniz-Institut für Polymerforschung Dresden e.V., Dresden, Germany

    A. Breier, J. Hahner & G. Heinrich

  4. Institute of Material Science, Technische Universität Dresden, Dresden, Germany

    G. Heinrich

  5. Research Institute of Leather and Plastic Sheeting—FILK, Freiberg, Germany

    N. Drechsel & M. Meyer

  6. Department of Trauma and Reconstructive Surgery, Universitätsklinikum Carl Gustav Carus, Dresden, Germany

    C. Rentsch

  7. Institute of Anatomy, Paracelsus Medical University, Nuremberg and Salzburg, Prof. Ernst Nathan Strasse 1, 90340, Nuremberg, Germany

    G. Schulze-Tanzil

Authors
  1. M. Hoyer
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  2. C. Meier
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  3. A. Breier
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  4. J. Hahner
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  5. G. Heinrich
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  6. N. Drechsel
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  7. M. Meyer
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  8. C. Rentsch
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  9. L.-A. Garbe
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  10. W. Ertel
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  11. A. Lohan
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  12. G. Schulze-Tanzil
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Corresponding author

Correspondence to G. Schulze-Tanzil.

Additional information

A. Lohan and G. Schulze-Tanzil have shared last authorship.

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Hoyer, M., Meier, C., Breier, A. et al. In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering. Histochem Cell Biol 143, 289–300 (2015). https://doi.org/10.1007/s00418-014-1280-4

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  • DOI: https://doi.org/10.1007/s00418-014-1280-4

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