Sains Malaysiana 48(10)(2019): 2169–2176

http://dx.doi.org/10.17576/jsm-2019-4810-12

Human Platelet Lysate Promotes Proliferation but Fails to Maintain Chondrogenic Markers of Chondrocytes

(Lisat Platelet Manusia Mempromosikan Proliferasi tetapi Gagal Mengekalkan Penanda Kondrogen dalam Kondrosit)

PRANA HARDINATA BIN BUDI HARTO¹, MUHAMMAD HANIF BIN MAHMUD¹, AISYA HANIM BINTI OTHMAN¹, NURULAIN HANANI BT NGADENIN¹, NUR SAIHAH BT MOHD AZAHAR¹, MUHAMMAD NAJIB FATHI BIN HASSAN¹, NOR HAMDAN MOHD YAHAYA², RIZAL ABDUL RANI², CHOOI FUN LEONG³, MIN HWEI NG¹ & JIA XIAN LAW¹*

¹Tissue Engineering Centre, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, JalanYaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Orthopaedic Department, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, JalanYaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

³Pathology Department, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Received: 7 January 2019/Accepted: 23 September 2019

ABSTRACT

Traditionally, foetal bovine serum (FBS) is used as a serum supplement for stem cell expansion in vitro. However, it is associated with xenoimmunisation and the transmission of animal pathogens, which may cause harm to stem cell recipients. As a safer alternative, human platelet lysate (HPL) has been introduced for propagating stem cells. Chondrocytes are expanded in vitro for cartilage repair via autologous chondrocyte implantation (ACI). In this study, we compare the efficacy of HPL prepared from expired platelet concentrates with that of FBS for promoting the proliferation and maintenance of the chondrogenic markers of primary human chondrocytes expanded in vitro. Chondrocytes were cultured in F12:Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% HPL, 10% HPL and 10% FBS. The cell morphology, viability and growth rate were examined from passage 1 (P1) to P3. RNA was isolated from P3 cells for quantitative polymerase chain reaction (qPCR) to determine the gene expression level of the chondrogenic, dedifferentiation and hypertrophic markers. HPL promoted chondrocyte proliferation without compromising cell viability. In addition, the chondrocytes cultured with HPL were smaller. However, HPL failed to maintain the chondrogenic markers, except SOX 9 (SRY-box transcription factor 9), which was upregulated, but not significantly. Nonetheless, HPL also suppressed the expression of type X collagen (Col X), a chondrocyte hypertrophic marker. In summary, we demonstrate the benefits of HPL supplementation in human chondrocyte culture, where it enhances cell proliferation and suppresses chondrocyte hypertrophy. In the future, HPL can be used for the large-scale expansion of chondrocytes for ACI.

Keywords: Autologous chondrocyte implantation; cartilage; chondrocyte; osteoarthritis; platelet lysate

ABSTRAK

Secara tradisinya, serum anak lembu (FBS) digunakan untuk pengkulturan sel in vitro. Namun demikian, FBS dikaitkan dengan penolakan oleh sistem imun dan penularan patogen haiwan yang berkemungkinan membawa kemudaratan kepada penerima sel induk. Lisat platelet manusia (HPL) telah diperkenalkan sebagai alternatif yang lebih selamat untuk pengkulturan sel. Sel kondrosit telah dikultur secara in vitro untuk merawat rawan yang rosak melalui teknik implantasi kondrosit autologus (ACI). Pada kajian ini, kami membandingkan keberkesanan HPL yang disediakan daripada platelet pekat tamat tempoh dan FBS dalam merangsang proliferasi dan mengekalkan penanda kondrogenik sel kondrosit manusia yang dikulturkan secara in vitro. Sel kondrosit dikultur dalam medium F12:DMEM yang ditambah dengan 5% HPL, 10% HPL dan 10% FBS. Morfologi, kadar kehidupan dan kadar pertumbuhan sel daripada P1 hingga P3 dikenal pasti. RNA dipencil daripada sel pada P3 untuk qPCR untuk menentukan ekspresi gen penanda-penanda kondrogenik, penyahbezaan dan hipertrofi. Hasil kajian mendapati HPL merangsang proliferasi sel kondrosit tanpa memkompromikan kadar kehidupan sel. Selain itu, sel kondrosit yang dikultur dengan HPL menunjukkan saiz yang lebih kecil. Namun demikian, HPL gagal mengekalkan penanda-penanda kondrogenik kecuali SOX 9 yang meningkat secara tidak signifikan. Pada masa yang sama, HPL juga didapati menurunkan ekspresi kolagen jenis X (Col X), satu penanda hipertrofi sel kondrosit. Secara kesimpulan, kajian kami menunjukkan HPL bermanfaat untuk pengkulturan sel kondrosit dengan merangsang proliferasi dan menghalang perubahan hipertrofi sel kondrosit. Pada masa yang akan datang, HPL boleh digunakan untuk pengkulturan sel kondrosit berskala besar.

Kata kunci: Implantasi kondrosit autologus; lisat platelet; osteoarthritis; rawan; sel kondrosit

REFERENCES

Ahmad, S., Gantenbein, B., Evangelopoulos, D.S., Schär, M.O., Schwienbacher, S., Kohlhof, H., & Kohl, S. 2015. Arthroplasty-current strategies for the management of knee osteoarthritis. Swiss Medical Weekly 145: w14096.

Amoako, A.O. & Pujalte, G.G.A. 2014. Osteoarthritis in young, active, and athletic individuals. Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders 7: 27-32.

Cimino, M., Gonçalves, R.M., Barrias, C.C. & Martins, M.C.L. 2017. Xeno-free strategies for safe human mesenchymal stem/stromal cell expansion: Supplements and coatings. Stem Cells International 2017: 6597815.

Doucet, C., Ernou, I., Zhang, Y., Llense, J., Begot, L., Holy, X. & Lataillade, J. 2005. Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications. Journal of Cellular Physiology 205(2): 228-236.

Hamada, T., Sakai, T., Hiraiwa, H., Nakashima, M., Ono, Y., Mitsuyama, H. & Ishiguro, N. 2013. Surface markers and gene expression to characterize the differentiation of monolayer expanded human articular chondrocytes. Nagoya Journal of Medical Science 75(1-2): 101-111.

Hamoud, A.F., Bin Mohamad Yahya, N.H., Hui, C.C., Bin Saim, A. & Bt Hj Idrus, R. 2012. The potential of intro-articular injection of chondrogenic-induced bone marrow stem cells to retard the progression of osteoarthritis in a sheep model. Experimental Gerontology 47: 458-564.

Hardingham, T.E., Oldershaw, R.A. & Tew, S.R. 2006. Cartilage, SOX9 and Notch signals in chondrogenesis. Journal of Anatomy 209(4): 469-480.

Hemeda, H., Giebel, B. & Wagner, W. 2014. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy 16(2): 170-180.

Hildner, F., Eder, M.J., Hofer, K., Aberl, J., Redl, H., van Griensven, M., Gabriel, C. & Peterbaue-Scherb, A. 2015. Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: A comparison with articular chondrocytes. Journal of Tissue Engineering and Regenerative Medicine 9(7): 808-818.

Kaps, C., Loch, A., Haisch, A., Smolian, H., Burmester, G.R., Häupl, T. & Sittinger, M. 2002. Human platelet supernatant promotes proliferation but not differentiation of articular chondrocytes. Medical and Biological Engineering and Computing 40(4): 485-490.

Law, J.X., Chowdhury, S.R., Aminuddin, B.S. & Ruszymah, B.H.I. 2017a. Role of plasma-derived fibrin on keratinocyte and fibroblast wound healing. Cell and Tissue Banking 18(4): 585-595.

Law, J.X., Liau, L.L., Saim, A., Yang, Y. & Idrus, R. 2017b. Electrospun collagen nanofibers and their applications in skin tissue engineering. Tissue Engineering and Regenerative Medicine 14(6): 699-718.

Law, J.X., Musa, F., Ruszymah, B.H.I., El Haj, A.J. & Yang, Y. 2016. A comparative study of skin cell activities in collagen and fibrin constructs. Medical Engineering and Physics 38(9): 854-861.

Li, Y., Tew, S.R., Russell, A.M., Gonzalez, K.R., Hardingham, T.E. & Hawkins, R.E. 2004. Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Engineering 10(3-4): 575-584.

Lin, Z., Fitzgerald, J.B., Xu, J., Willers, C., Wood, D., Grodzinsky, A.J. & Zheng, M.H. 2008. Gene expression profiles of human chondrocytes during passaged monolayer cultivation. Journal of Orthopaedic Research 26(9): 1230-1237.

Luo, Y., Sinkeviciute, D., He, Y., Karsdal, M., Henrotin, Y., Mobasheri, A., Önnerfjord, P. & Bay-Jensen, A. 2017. The minor collagens in articular cartilage. Protein & Cell 8(8): 560-572.

Ma, B., Leijten, J.C.H., Wu, L., Kip, M., van Blitterswijk, C.A., Post, J.N. & Karperien, M. 2013. Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture. Osteoarthritis and Cartilage 21(4): 599-603.

Mistry, H., Connock, M., Pink, J., Shyangdan, D., Clar, C., Royle, P., Court, R.A., Biant, L.C., Metcalfe, A. & Waugh, N. 2017. Autologous chondrocyte implantation in the knee: Systematic review and economic evaluation. Health Technology Assessment 21(6): 1-294.

Pereira, R.C., Scaranari, M., Benelli, R., Strada, P., Reis, R.L., Cancedda, R. & Gentili, C. 2013. Dual effect of platelet lysate on human articular cartilage: A maintenance of chondrogenic potential and a transient proinflammatory activity followed by an inflammation resolution. Tissue Engineering Part A 19(11-12): 1476-1488.

Peterson, L., Vasiliadis, H.S., Brittberg, M. & Lindahl, A. 2010. Autologous chondrocyte implantation: A long-term follow-up. The American Journal of Sports Medicine 38(6): 1117-1124.

Schulze-Tanzil, G. 2009. Activation and dedifferentiation of chondrocytes: Implications in cartilage injury and repair. Annals of Anatomy-Anatomischer Anzeiger 191(4): 325-338.

Shen, G. 2005. The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage. Orthodontics & Craniofacial Research 8(1): 11-17.

Stiebel, M., Miller, L.E. & Block, J.E. 2014. Post-traumatic knee osteoarthritis in the young patient: Therapeutic dilemmas and emerging technologies. Open Access Journal of Sports Medicine 5: 73-79.

Sykes, J.G., Kuiper, J.H., Richardson, J.B., Roberts, S., Wright, K.T. & Kuiper, N.J. 2018. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy. European Cells & Materials 35: 255-267.

Tew, S.R., Li, Y., Pothacharoen, P., Tweats, L.M., Hawkins, R.E. & Hardingham, T.E. 2005. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis and Cartilage 13(1): 80-89.

Ude, C.C., Seet, W.T., Sharen Aini, S., Aminuddin, B.S. & Ruszymah, B.H.I. 2018. Shelf life evaluation of clinical grade chondrogenic induced aged adult stem cells for cartilage regeneration. Scientific Reports 8: 4345.

Viste, A., Piperno, M., Desmarchelier, R., Grosclaude, S., Moyen, B. & Fessy, M.H. 2012. Autologous chondrocyte implantation for traumatic full-thickness cartilage defects of the knee in 14 patients: 6-year functional outcomes. Orthopaedics & Traumatology: Surgery & Research 98(7): 737-743.

Yao, T. & Asayama, Y. 2017. Animal-cell culture media: History, characteristics, and current issues. Reproductive Medicine and Biology 16(2): 99-117.

*Corresponding author; email: lawjx@ppukm.ukm.edu.my