Skip to main content
SpringerLink
Log in

A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study

  • Original Article
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

The present study investigates the impact of biomolecules (biotin, glucose, chondroitin sulphate, proline) as supplement, (individual and in combination) on primary human meniscus cell proliferation. Primary human meniscus cells isolated from patients undergoing meniscectomy were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM). The isolated cells were treated with above mentioned biomolecules as individual (0–100 µg/ml) and in combinations, as a supplement to DMEM. Based on the individual biomolecule study, a unique combination of biomolecules (UCM) was finalized using one way ANOVA analysis. With the addition of UCM as supplement to DMEM, meniscal cells reached 100 % confluency within 4 days in 60 mm culture plate; whereas the cells in medium devoid of UCM, required 36 days for reaching confluency. The impact of UCM on cell viability, doubling time, histology, gene expression, biomarkers expression, extra cellular matrix synthesis, meniscus cell proliferation with respect to passages and donor’s age were investigated. The gene expression studies for E-cadherin and peroxisome proliferator-activated receptor (PPAR∆) using RT-qPCR and immunohistochemical analysis for Ki67, CD34 and Vimentin confirmed that UCM has significant impact on cell proliferation. The extracellular collagen and glycosaminoglycan secretion in cells supplemented with UCM were found to increase by 31 and 37 fold respectively, when compared to control on the 4th day. The cell doubling time was reduced significantly when supplemented with UCM. The addition of UCM showed positive influence on different passages and age groups. Hence, this optimized UCM can be used as an effective supplement for meniscal tissue engineering.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Aswani DV, Ramya KV, Dhananjay P, Ashim K (2013) Biotin uptake by T47D breast cancer cells: functional and molecular evidence of sodium-dependent multivitamin transporter (SMVT). Int J Pharm 441:535–543

    Article  Google Scholar 

  • Ayo SH, Radnik RA, Garoni JA, Glass WF, Kreisberg JI (1990) High glucose causes an increase in extracellular matrix proteins in cultured mesangial cells. Am J Pathol 136:1339–1348

    CAS  Google Scholar 

  • Baker BM, Ashwin SN, Russell GH, Robert LM (2009) Tissue engineering with meniscus cells derived from surgical debris. Osteoarthr Cartil 17:336–345

    Article  CAS  Google Scholar 

  • Barbul A (2008) Proline precursors to sustain Mammalian collagen synthesis. J Nutr 138:2021S–2024S

    CAS  Google Scholar 

  • Brink HE, Bernstein J, Nicoll JB (2009) Fetal dermal fibroblasts exhibit enhanced growth and collagen production in two- and three-dimensional culture in comparison to adult fibroblasts. J Tissue Eng Regen Med 3:623–633

    Article  CAS  Google Scholar 

  • Brown JR, DiGiovanna MP, Killelea B, Lannin DR, Rimm DL (2014) Quantitative assessment Ki-67 score for prediction of response to neoadjuvant chemotherapy in breast cancer. Lab Invest 94:98–106

    Article  CAS  Google Scholar 

  • Chalisova NI, Kontsevaya EA, Zhekalov AN, Sinyachkin DA (2013) Effects of combinations of amino acids on cell proliferation in tissue culture of old rats. Adv Gerontol 3:77–79

    Article  Google Scholar 

  • Chen S, Lewallen M, Xie T (2013) Adhesion in the stem cell niche: biological roles and regulation. Development 140:255–265

    Article  CAS  Google Scholar 

  • Demoor M, David O, Gomez-Leduc MTB, Hervieu M, Fabre H, Lafont J, Denoix JM, Fabrice A, Frederic MG, Florence L, Philippe G (2014) Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction. Biochim Biophys Acta 1840:2414–2440

    Article  CAS  Google Scholar 

  • Fraser D, Brunskill N, Ito T, Phillips A (2003) Long-term exposure of proximal tubular epithelial cells to glucose induces transforming growth factor-β1 synthesis via an autocrine PDGF loop. Am J Pathol 163:2565–2574

    Article  CAS  Google Scholar 

  • Freymann U, Endres M, Neumann K, Scholman HJ, Morawietz L, Kaps C (2012) Expanded human meniscus-derived cells in 3-D polymer–hyaluronan scaffolds for meniscus repair. Acta Biomater 8:677–685

    Article  CAS  Google Scholar 

  • Fruchtl M, Sakon J, Beitle R (2015) Expression of a collagen-binding domain fusion protein: effect of amino acid supplementation, inducer type, and culture conditions. Biotechnol Prog 3:503–509

    Article  Google Scholar 

  • Ghasemi-Mobarakeh L, Morshed Karbalaie K, Fesharaki Nasr-Esfahani MH, Baharvand H (2008) Electrospun poly (ε-caprolactone) nanofiber mat as extracellular matrix. Yakhteh Med J 10:179–184

    CAS  Google Scholar 

  • Gopinathan J, Steffie M, Elakkiya V, Mamatha MP, Santosh SK, Dinakar Rai BK, Selvakumar R, Amitava B (2015) Biomolecule incorporated poly-ε-caprolactone nanofibrous scaffolds for enhanced human meniscal cell attachment and proliferation. RSC Adv 5:73552–73561

    Article  CAS  Google Scholar 

  • Haudenschild DR, Chen J, Pang N, Steklov N, Grogan SP, Lotz MK, Lima DD (2011) Vimentin contributes to changes in chondrocyte stiffness in osteoarthritis. J Orthop Res 29:20–25

    Article  Google Scholar 

  • Heidari M, Tahmasebi MT, Etemad S, Salehkhou S, Heidari-Vala H, Akhondi MM (2011) In vitro human chondrocyte culture; a modified protocol. Middle East J Sci Res 9:102–109

    CAS  Google Scholar 

  • Jerosch J (2011) Effects of glucosamine and chondroitin sulfate on cartilage metabolism in OA: outlook on other nutrient partners especially omega-3 fatty acids. Int J Rheumatol 2011:17

  • Kreuz PCG, Samans B, Martinelli D, Kruger JP, Mittelmeier W, Endres M M, Cancedda M, Kaps C (2013) Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage. Osteoarthr Cartil 21:1997–2005

    Article  CAS  Google Scholar 

  • Kubo M, Kosei A, Tomohiro M, Matsusue Y, Mori K (2009) Chondroitin sulfate for the treatment of hip and knee osteoarthritis: current status and future trends. Life Sci 85:477–483

    Article  CAS  Google Scholar 

  • Latt SA, Stetten G (1976) Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J Histochem Cytochem 24:24–33

    Article  CAS  Google Scholar 

  • Lee P, Tran K, Chang W, Shelke NB, Kumbar SG, Yu X (2014) Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration. J Biomed Nanotechnol 10:1469–1479

    Article  CAS  Google Scholar 

  • Levett PA, Melchels FP, Schrobback K, Hutmacher DW, Malda J, Klein TJ (2014) A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. Acta Biomater 10:214–223

    Article  CAS  Google Scholar 

  • Little CJ, Kulyk WM, Chen X (2014) The effect of chondroitin sulphate and hyaluronic acid on chondrocytes cultured within a fibrin-alginate hydrogel. J Funct Biomater 5:197–210

    Article  Google Scholar 

  • Maitre JL, Heisenberg CP (2013) Three functions of cadherins in cell adhesion. Curr Biol 23:R626–R633

    Article  CAS  Google Scholar 

  • Mason SS (2013) Exploring tissue engineering: vitamin d3 influences on the proliferation and differentiation of an engineered osteoblast precursor cell line during early bone tissue development. Dissertation, Portland State University

  • Matsiko A, Levingstone T, Brien FO (2013) Advanced strategies for articular cartilage defect repair. Materials 6:637–668

    Article  CAS  Google Scholar 

  • McNulty AL, Guilak F (2008) Integrative repair of the meniscus: lessons from in vitro studies. Biorheology 45:487

    Google Scholar 

  • Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein BE (2013) Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions. Nat Biotechnol 31:1133–1136

    Article  CAS  Google Scholar 

  • Muzzarelli RA, Greco F, Busilacchi A, Sollazzo V, Gigante A (2012) Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: a review. Carbohydr Polym 89:723–739

    Article  CAS  Google Scholar 

  • Nakata K, Shino K, Hamada M, Mae T, Miyama T, Shinjo H, Horibe S, Tada K, Ochi T, Yoshikawa H (2001) Human meniscus cell: characterization of the primary culture and use for tissue engineering. Clin Orthop 391:S208–S218

    Article  Google Scholar 

  • Niehrs C (2012) The complex world of WNT receptor signaling. Nat Rev Mol Cell Biol 13:767–779

    Article  CAS  Google Scholar 

  • Oda S, Otsuki S, Kurokawa Y, Hoshiyama Y, Nakajima M, Neo M (2015) A new method for meniscus repair using type I collagen scaffold and infrapatellar fat pad. J Biomater Appl 29:1439–1448

    Article  CAS  Google Scholar 

  • Perrier-Groult E, Pasdeloup M, Malbouyres M, Galéra P, Mallein-Gerin F (2013) Control of collagen production in mouse chondrocytes by using a combination of bone morphogenetic protein-2 and small interfering RNA targeting Col1a1 for hydrogel-based tissue-engineered cartilage. Tissue Eng C Methods 19:652–664

    Article  CAS  Google Scholar 

  • Reinert TH, Jundt G (1999) In situ measurement of collagen synthesis by human bone cells with a Sirius red based colorimetric microassay: effects of transforming growth factor beta 2 and ascorbic acid 2-phosphate. Histochem Cell Biol 112:271–276

    Article  Google Scholar 

  • Ruohola JK, Viitanen TP, Valve EM, Seppanen JA, Loponen NT, Keskitalo JJ, Päivi TL, Pirkko LH (2001) Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing MCF-7 human breast cancer cells. Am J Cancer Res 61:4229–4237

    CAS  Google Scholar 

  • Scholtysek C, Julia K, He Fu, Stefan U, Natacha I, Cornelia S, Mario M, Michael S, Laura D, Christina B, Arnd K, Andreas H, Klaus E, Jean-Pierre D, Farida D, Peter JT, Beatrice D, Georg S, Gerhard K (2013) PPAR β/∆ governs Wnt signaling and bone turnover. Nat Med 19:608–613

    Article  CAS  Google Scholar 

  • Shapiro HM (1988) Practical flow cytometry, 2nd edn. Wiley, New York, p 129

    Google Scholar 

  • Stalling SS, Nicoll SB (2008) Fetal ACL fibroblasts exhibit enhanced cellular properties compared with adults. Clin Orthop Relat Res 466:3130–3137

    Article  Google Scholar 

  • Stanley JS, Griffin JB, Zempleni J (2001) Biotinylation of histones in human cells. Effects of cell proliferation. Eur J Biochem 268:5424–5429

    Article  CAS  Google Scholar 

  • Takechi R, Taniguchi A, Ebara S, Fukui T, Watanabe T (2008) Biotin deficiency affects the proliferation of human embryonic palatal mesenchymal cells in culture. J Nutr 138:680–684

    CAS  Google Scholar 

  • Tran-Khanh N, Hoemann CD, McKee MD, Henderson JE, Buschmann MD (2005) Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix. J Orthop Res 23:1354–1362

    Article  CAS  Google Scholar 

  • Van Der Bracht H, Verdonk H, Verbruggen R, Elewaut D, Verdonk P (2007) Cell-based meniscus tissue engineering. In: Ashammakhi N, Reis R, Chiellini E (eds) Topics in tissue engineering, vol 3. Biomaterials and tissue engineering group, Oulu, pp 1–13

  • Verdonk PC, Forsyth RG, Wang J, Almqvist KF, Verdonk R, Veys EM, Verbruggen G (2005) Characterisation of human knee meniscus cell phenotype. Osteoarthr Cartil 13:548–560

    Article  CAS  Google Scholar 

  • Washington JM, Rathjen J, Felquer F, Lonic A, Bettess MD, Hamra N, Ljiljana S, Boon SNT, Julie-Anne L, Rebecca AK, Michael BM, Peter DR (2010) L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture. Am J Physiol Cell Physiol 298:C982–C992

    Article  CAS  Google Scholar 

  • Whitley CB, Ridnour MD, Draper KA, Dutton CM, Neglia JP (1989) Diagnostic test for mucopolysaccharidosis: direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem 35:374–379

    CAS  Google Scholar 

  • Yano N, Suzuki D, Endoh M, So-Youn L, Young-Suk K, Seung-Yun S, Eun-Cheol K (2009) High ambient glucose induces angiotensin-independent AT-1 receptor activation, leading to increases in proliferation and extracellular matrix accumulation in MES-13 mesangial cells. Biochem J 423:29–43

    Article  Google Scholar 

  • Zempleni J, Mock DM (2001) Biotin homeostasis during the cell cycle. Nutr Res Rev 14:45–64

    Article  CAS  Google Scholar 

  • Zhang H, Leng P, He T, Wang Y (2015) Comparison of hIGF-1 gene transfection to the hBMSCs and human meniscal fibrochondrocytes. Med Sci Monit 21:681–688

    Article  Google Scholar 

Download references

Acknowledgments

The authors like to express their deep gratitude to the management of PSG Institutions and Tamil Nadu State Council for Science and Technology, Govt. of Tamil Nadu for their financial and other shapes of support to carry out this work. We appreciate the support, guidance and contribution from Dr. P. Radhakrishnan, Director and Dr. T. Lazar Mathew, Advisor, PSG Institute of Advanced Studies, Dr. David V. Rajan, Ortho One Orthopaedic Specialty Centre and Dr. S. Ramalingam, PSG Institute of Medical Sciences and Research, Coimbatore. Authors also acknowledge the support from Mr. Darshit, PSG Institute of Medical Sciences and Research, Coimbatore for q-PCR studies.

Author information

Authors and Affiliations

  1. Tissue Engineering Laboratory, PSG Institute of Advanced Studies, Coimbatore, 641004, India

    Mamatha M. Pillai, V. Elakkiya, J. Gopinathan, Amitava Bhattacharyya & R. Selvakumar

  2. Department of Biotechnology, PSG College of Technology, Coimbatore, 641004, India

    C. Sabarinath

  3. Department of Pathology, PSG Institute of Medical Sciences and Research, Coimbatore, 641004, India

    S. Shanthakumari

  4. Arthroscopy and Sports Medicine, Ortho One-Orthopaedic and Specialty Centre, Coimbatore, 641005, India

    K. Santosh Sahanand

  5. Department of Orthopaedics, PSG Institute of Medical Sciences and Research, Coimbatore, 641004, India

    B. K. Dinakar Rai

Authors
  1. Mamatha M. Pillai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. Elakkiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Gopinathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Sabarinath
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Shanthakumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K. Santosh Sahanand
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. B. K. Dinakar Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Amitava Bhattacharyya
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R. Selvakumar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Selvakumar.

Ethics declarations

Conflict of interest

None of the authors have a conflict of interests including direct or indirect financial relations with any of the trademarks and companies mentioned in this paper.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pillai, M.M., Elakkiya, V., Gopinathan, J. et al. A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study. Cytotechnology 68, 1747–1761 (2016). https://doi.org/10.1007/s10616-015-9926-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-015-9926-1

Keywords

Search

Navigation