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

 

Diserahkan: 29 Mac 2018/Diterima: 6 Julai 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

 

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*Pengarang untuk surat-menyurat; email: lyoges@ppukm.ukm.edu.my