Sains Malaysiana 47(11)(2018): 2789–2798

http://dx.doi.org/10.17576/jsm-2018-4711-22

 

Development of Nerve Conduit using Decellularized Human Umbilical Cord Artery Seeded with Centella asiatica Induced-Neurodifferentiated Human Mesenchymal Stem Cell

(Perkembangan Konduit Saraf menggunakan Arteri Tali Pusat Manusia Dinyahsel yang Disemai dengan Centella asiatica Teraruh-Sel Saraf yang Berbeza daripada Sel Stem Mesenkima Manusia)

 

HANITA MOHD HUSSIN¹, RUSZYMAH HAJI IDRUS^1,2 & YOGESWARAN LOKANATHAN^1*

 

¹Tissue Engineering Centre, UKM Medical Centre, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

²Department of Physiology, UKM Medical Centre, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

Received: 29 March 2018/Accepted: 6 July 2018

 

ABSTRACT

Various natural biological conduits have been investigated to bridge peripheral nerve injury especially in critical gap (greater than 3 cm in human). Autograft, the current gold standard, has several drawbacks including limited availability of donor graft, donor-site morbidity and mismatch in size in clinical practices. The aim of this study was to analyze the development of nerve conduit using decellularized human umbilical cord (HUC) artery seeded with neurodifferentiated human MSCs (ndMSCs) in bridging peripheral nerve gap. Artery conduits obtained from HUC were decellularized to remove native cells (n=3), then characterized by Hematoxylin and Eosin (H&E) staining and nuclei counterstaining with DAPI. The decellularized artery conduit was measured for every 2 weeks until 12 weeks. Next, mesenchymal stem cells (MSCs) were differentiated into neural lineage using 400 μg/mL of Centella asiatica. Then, 1.5×10⁶ of MSCs or ndMSCs were seeded into decellularized artery conduit to study cell attachment. H&E staining and nuclei counterstaining with DAPI showed that all cellular components were removed from the HUC arteries. The decellularized artery conduit did not collapse and the lumen remained rigid for 12 weeks. Immunocytochemistry analysis with neural markers namely S100β, P75 NGFR, MBP and GFAP showed that MSCs had differentiated into neural lineage cells. H&E staining showed that the seeded MSCs and ndMSCs attached to the lumen of the conduits as early as 2 days. In conclusion, this study showed that nerve conduit using decellularized HUC artery seeded with neurodifferentiated human MSCs was successfully developed and have the potential to bridge critical nerve gap.

 

Keywords: C. asiatica; mesenchymal stem cells; nerve conduit; nerve injury; umbilical cord artery

 

ABSTRAK

Pelbagai konduit biologi semula jadi telah dikaji untuk menyambungkan semula kecederaan saraf periferi terutamanya yang bersaiz kritikal (lebih daripada 3 cm pada manusia). Rawatan piawai pada masa kini adalah pemindahan autograf, tetapi ia mempunyai beberapa kelemahan dalam amalan klinikal seperti kekurangan tisu penderma, morbiditi pada tapak penderma dan saiz tisu yang tidak sepadan. Tujuan kajian ini dijalankan adalah untuk mengkaji perkembangan konduit saraf menggunakan arteri tali pusat manusia yang telah dinyahsel dan disemai dengan sel saraf yang terbeza daripada sel stem mesenkima. Konduit arteri tali pusat manusia yang telah dinyahsel (n=3) seterusnya dikaji keberkesanannya menggunakan pewarnaan Hematoxylin dan Eosin (H&E) dan pewarnaan nukleus oleh DAPI. Konduit arteri itu kemudiannya diukur kepanjangan pada setiap 2 minggu sehingga 12 minggu. Aruhan pembezaan sel saraf daripada sel stem mesenkima telah dilakukan melalui penggunaan 400 μg/mL C. asiatica. Kemudian, 1.5×10⁶ sel stem mesenkima atau sel saraf disemai ke dalam konduit arteri untuk mengkaji perlekatan sel. Pewarnaan Hematoxylin dan Eosin (H&E) dan DAPI pada nukleus menunjukkan bahawa semua komponen sel telah berjaya dinyahkan daripada arteri tali pusat manusia. Konduit arteri itu tidak menguncup dan lumennya kekal tegar selama 12 minggu. Analisis immunositokimia menggunakan penanda saraf iaitu S100β, P75 NGFR, MBP dan GFAP mendedahkan bahawa sel stem mesenkima telah terbeza kepada leluhur sel saraf. Pewarnaan H&E menunjukkan bahawa sel stem mesenkima dan sel saraf yang telah disemai melekat pada dinding lumen konduit tersebut seawal hari ke-dua. Secara kesimpulannya, kajian ini menunjukkan bahawa konduit saraf menggunakan arteri tali pusat manusia dan disemai dengan sel saraf yang terbeza daripada sel stem mesenkima telah berjaya dihasilkan dan berpotensi untuk diimplan pada kecederaan saraf periferi bersaiz kritikal.

 

Kata kunci: Arteri tali pusat; C. asiatica; kecederaan saraf; konduit saraf; sel stem mesenkima

 

REFERENCES

 

Badylak, S.F., Freytes, D.O. & Gilbert, T.W. 2009. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomaterialia 5(1): 1-13.

Crouzier, T., McClendon, T., Tosun, Z. & McFetridge, P.S. 2009. Inverted human umbilical arteries with tunable wall thicknesses for nerve regeneration. Journal of Biomedical Materials Research Part A 89A(3): 818-828.

Deal, N.D., Griffin, J.W. & Hogan, M.V. 2012. Nerve conduits for nerve repair or reconstruction. JAAOS-Journal of the American Academy of Orthopaedic Surgeons 20(2): 63-68.

Deng, J., Petersen, B.E., Steindler, D.A., Jorgensen, M.L. & Laywell, E.D. 2006. Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation. Stem Cells 24(4): 1054-1064.

Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D.J. & Horwitz, E. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for cellular therapy position statement. Cytotherapy 8(4): 315-317.

Gilpin, A. & Yang, Y. 2017. Decellularization strategies for regenerative medicine: From processing techniques to applications. BioMed Research International 2017: Article ID. 9831534.

Gordon, T., Sulaiman, O. & Boyd, J.G. 2003. Experimental strategies to promote functional recovery after peripheral nerve injuries. Journal of the Peripheral Nervous System 8(4): 236-250.

Gui, L., Muto, A., Chan, S.A., Breuer, C.K. & Niklason, L.E. 2009. Development of decellularized human umbilical arteries as small-diameter vascular grafts. Tissue Engineering Part A 15(9): 2665-2676.

Hassan, N.H., Sulong, A.F., Ng, M..H., Htwe, O., Idrus, R.B.H., Roohi, S., Naicker, A.S. & Abdullah, S. 2012. Neural-differentiated mesenchymal stem cells incorporated into muscle stuffed vein scaffold forms a stable living nerve conduit. Journal of Orthopaedic Research 30(10): 1674- 1681.

Hidayah, N.H., Fadzli, A., Ng, M.H., Ruszymah, B.H.I., Naicker, A.S. & Shalimar, A. 2012. Porous PLGA sheet and acellularized muscle stuffed vein seeded with neural-differentiated MSCs are potential scaffolds for nerve regeneration. Regen. Res. 1: 1-7.

Höke, A. 2006. Mechanisms of disease: What factors limit the success of peripheral nerve regeneration in humans? Nature Reviews Neurology 2(8): 448-454.

Jadalannagari, S. & Aljitawi, O.S. 2015. Ectodermal differentiation of Wharton’s jelly mesenchymal stem cells for tissue engineering and regenerative medicine applications. Tissue Engineering Part B: Reviews 21(3): 314-322.

Jamil, S.S., Nizami, Q. & Mehboobus, S. 2007. Centella asiatica (Linn.) Urban: A review. Natural Product Radiance 6(2): 158-170.

Jiang, H., Zheng, G., Junwei, L.V., Chen, H., Jinjin, L., Li, Y., Fan, G. & Ding, X. 2016. Identification of Centella asiatica’s effective ingredients for inducing the neuronal differentiation. Evid. Based Complement. Alternat. Med. 2016: Article ID. 9634750.

Kaizawa, Y., Kakinoki, R., Ikeguchi, R., Ohta, S., Noguchi, T., Oda, H. & Matsuda, S. 2016. Bridging a 30 mm defect in the canine ulnar nerve using vessel-containing conduits with implantation of bone marrow stromal cells. Microsurgery 36(4): 316-324.

Kaizawa, Y., Kakinoki, R., Ikeguchi, R., Ohta, S., Noguchi, T., Takeuchi, H., Oda, H., Yurie, H. & Matsuda, S. 2017. A nerve conduit containing a vascular bundle and implanted with bone marrow stromal cells and decellularized allogenic nerve matrix. Cell Transplantation 26(2): 215-228.

Kehoe, S., Zhang, X. & Boyd, D. 2012. FDA approved guidance conduits and wraps for peripheral nerve injury: A review of materials and efficacy. Injury 43(5): 553-572.

Liao, I.C., Wan, H., Qi, S., Cui, C., Patel, P., Sun, W. & Xu, H. 2013. Preclinical evaluations of acellular biological conduits for peripheral nerve regeneration. Journal of Tissue Engineering 4: 2041731413481036.

Lim, J., Razi, Z.R.M., Law, J., Azmawati, Mohammed Nawi., Haji Idrus, Ruszymah. & Ng. Min Hwei. 2016. MSCs can be differentially isolated from maternal, middle and fetal segments of the human umbilical cord. Cytotherapy 18(12): 1493-1502.

Liu, Z., Jin, Y.Q., Chen, L., Wang, Y., Yang, X., Cheng, J., Wu, W., Qi, Z. & Shen, Z. 2015. Specific marker expression and cell state of Schwann cells during culture in vitro. PLoS One 10(4): e0123278.

Lokanathan, Y., Ng, M.H., Shariful Hasan, Anuar Ali, Mazzre Mahmod, Ohnmar Htwe, Sharifah Ahmad Roohi, Ruszymah, Bt Hj Idrus, Shalimar Abdullah & Amaramalar Selvi Naicker 2014. Olfactory ensheathing cells seeded muscle-stuffed vein as nerve conduit for peripheral nerve repair: A nerve conduction study. Journal of Bioscience and Bioengineering 118(2): 231-234.

Lokanathan, Y., Omar, N., Ahmad Puzi, N.N., Saim, A. & Hj Idrus, R. 2016. Recent updates in neuroprotective and neuroregenerative potential of Centella asiatica. Malays J. Med. Sci. 23(1): 4-14.

Mallis, P., Gontika, I., Poulogiannopoulos, T., Zoidakis, J., Vlahou, A., Michalopoulos, E., Chatzistamatiou, T., Papassavas, A. & Stavropoulos-Giokasa, C. 2014. Evaluation of decellularization in umbilical cord artery. Transplantation Proceedings 46(9): 3232-3239.

Michetti, F., Corvino, V., Geloso, M.C., Lattanzi, W., Bernardini, C., Serpero, L. & Gazzolo, D. 2012. The S100B protein in biological fluids: More than a lifelong biomarker of brain distress. Journal of Neurochemistry 120(5): 644-659.

Mohandas R.K., Muddanna, R.S. & Rao, S.G. 2009. Enhancement of amygdaloid neuronal dendritic arborization by fresh leaf juice of Centella asiatica (Linn) during growth spurt period in rats. Evidence-Based Complementary and Alternative Medicine 6(2): 203-210.

Mohd Heikal, M., Siti Mariam, H., Mohd Ilham, A., Mee Fong, C., Aminuddin, B.S. & Ruszymah, B.H.I. 2014. Anti-proliferative activities of Centella asiatica extracts on human respiratory epithelial cells in vitro. Journal of Medicinal Plants Research 8(24): 864-869.

Neuhuber, B., Swanger, S.A., Howard, L., Mackay, A. & Fischera, I. 2008. Effects of plating density and culture time on bone marrow stromal cell characteristics. Experimental Hematology 36(9): 1176-1185.

Noor, M.M. & Ali, N.M. 2004. Kesan in vivo ekstrak daun Centella asiatica ke atas histologi testis dan kualiti sperma mencit. Sains Malaysiana 33(2): 97-103.

Norazzila, O., Yogeswaran, L. & Ruszymah, B.H.I. 2017. Comparison of cytotoxicity measurements of Centella asiatica (L.) on human wharton’s jelly-derived mesenchymal stem cells in vitro via mtt and prestoblue assay. Regenerative Research 5(2): 10-18.

Norazzila, O., Yogeswaran, L., Mohd Razi Zainul Rashid & Ruszymah Idrus. 2018. The effects of Centella asiatica (L.) Urban on neural differentiation of human mesenchymal stem cells in vitro. Frontiers in Cellular Neuroscience doi: 10.3389/conf.fncel.2016.36.00183.

Nurlaily, A., Ar, N.B. & Musalmah, M. 2012. Comparative antioxidant and anti-inflammatory activity of different extracts of Centella asiatica (L.) Urban and its active compounds, asiaticoside and madecassoside. Medicine and Health 7(2): 62-72.

Omar, N.S., Zakaria, Z.A.C., Mian Then Sue, Wan Ngah Wan Zurinah & Musalmah Mazlan. 2011. Centella asiatica modulates neuron cell survival by altering caspase-9 pathway. Journal of Medicinal Plants Research 5(11): 2201-2209.

Puttarak, P., Brantner, A. & Panichayupakaranant, P. 2016. Biological activities and stability of a standardized pentacyclic triterpene enriched Centella asiatica extract. Natural Product Sciences 22(1): 20-24.

Quint, C., Kondo, Y., Manson, R.J., Lawson, J.H., Dardik, A. & Niklason, L.E. 2011. Decellularized tissue-engineered blood vessel as an arterial conduit. Proceedings of the National Academy of Sciences 108(22): 9214-9219.

Ray, W.Z. & Mackinnon, S.E. 2010. Management of nerve gaps: Autografts, allografts, nerve transfers, and end-to-side neurorrhaphy. Experimental Neurology 223(1): 77.

Rodríguez-Rodríguez, V.E., Martínez-González, B., Quiroga- Garza, A., Reyes-Hernández, C.G., de la Fuente-Villarreal, D., de la Garza-Castro, O., Guzmán-López, S. & Elizondo- Omaña, R.E. 2017. Human umbilical vessels: Choosing the optimal decellularization method. ASAIO Journal (American Society for Artificial Internal Organs: 1992) 64(5): 575-580.

Roy, S., Silacci, P. & Stergiopulos, N. 2005. Biomechanical proprieties of decellularized porcine common carotid arteries. American Journal of Physiology-Heart and Circulatory Physiology 289(4): H1567-H1576.

Ruszymah, B.H.I., Chowdhury, S.R., Nur Azeanty Bt Abdul Manan, Ooi Sew Fong, Mohd Ilham Adenan & Aminuddin Bin Saim. 2012. Aqueous extract of Centella asiatica promotes corneal epithelium wound healing in vitro. Journal of Ethnopharmacology 140(2): 333-338.

Safwani, W.K.Z.W., Makpol, S., Sathapan, S. & Chua, H.K. 2012. The impact of long-term in vitro expansion on the senescence-associated markers of human adipose-derived stem cells. Applied Biochemistry and Biotechnology 166(8): 2101-2113.

Said, M. 2009. Traditional Malaysian salads (ulam) as a source of antioxidants. Prosiding Seminar Kimia Bersama UKM-ITB. pp. 9-11.

Schaner, P.J., Martin, N.D., Tulenko, T.N., Shapiro, I.M., Tarola, N.A., Leichter, R.F., Carabasi, R.A. & DiMuzio, P.J. 2004. Decellularized vein as a potential scaffold for vascular tissue engineering. Journal of Vascular Surgery 40(1): 146-153.

Shalaby, S.M., Amal, S., Ahmed, F.E., Shaban, S.F., Wahdan, R.A., Kandel, W.A. & Mohammed, S.S. 2017. Combined Wharton’s jelly derived mesenchymal stem cells and nerve guidance conduit: A potential promising therapy for peripheral nerve injuries. The International Journal of Biochemistry & Cell Biology 86: 67-76.

Somboonwong, J., Kankaisre, M., Tantisira, B. & Tantisira, M.H. 2012. Wound healing activities of different extracts of Centella asiatica in incision and burn wound models: An experimental animal study. BMC Complementary and Alternative Medicine 12(1): 103.

Soumyanath, A., Zhong, Y.P., Gold, S.A., Yu, X., Koop, D.R., Bourdette, D. & Gold, B.G. 2005. Centella asiatica accelerates nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in-vitro. J. Pharm. Pharmacol. 57(9): 1221-1229.

Sulong, A.F. & Hassan, N.H. 2014. Collagen-coated polylactic-glycolic acid (PLGA) seeded with neural-differentiated human mesenchymal stem cells as a potential nerve conduit. Adv. Clin. Exp. Med. 23: 353-362.

Sun, F., Zhou, K., Mi, W-J. & Qiu, J-H. 2011. Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats. Biomaterials 32(32): 8118-8128.

Tiwari, S., Gehlot, S. & Gambhir, I.S. 2011. Centella asiatica: A concise drug review with probable clinical uses. Journal of Stress Physiology & Biochemistry 7(1): 38-44.

Tuan-Mu, H.Y., Yu, C.H. & Hu, J.J. 2014. On the decellularization of fresh or frozen human umbilical arteries: Implications for small-diameter tissue engineered vascular grafts. Annals of Biomedical Engineering 42(6): 1305-1318.

Wakao, S., Matsuse, D. & Dezawa, M. 2014. Mesenchymal stem cells as a source of Schwann cells: Their anticipated use in peripheral nerve regeneration. Cells Tissues Organs 200(1): 31-41.

Wanakhachornkrai, O., Pongrakhananon, V., Chunhacha, P., Wanasuntronwong, A., Vattanajun, A., Tantisira, B., Chanvorachote, P. & Tantisira, M.H. 2013. Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells. BMC Complement. Altern. Med. 13: 204.

Xu, Y., Zhang, Z., Chen, X., Li, R., Li, D. & Feng, S. 2016. A silk fibroin/collagen nerve scaffold seeded with a co-culture of schwann cells and adipose-derived stem cells for sciatic nerve regeneration. PloS One 11(1): e0147184.

Zhan, X., Gao, M., Jiang, Y., Zhang, W.W., Wong, W.M., Yuan, Q., Su, H., Kang, X., Dai, X., Zhang, W., Guo, J. & Wu, W. 2013. Nanofiber scaffolds facilitate functional regeneration of peripheral nerve injury. Nanomedicine: Nanotechnology, Biology and Medicine 9(3): 305-315.

Zhuang, Y., Li, D., Fu, J., Shi, Q., Lu, Y. & Ju, X. 2015. Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages: Immunomodulatory ability is enhanced in aged cells. Molecular Medicine Reports 11(1): 166-174.

 

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