Combination of Human Amniotic Fluid Derived-Mesenchymal Stem Cells and Nano-hydroxyapatite Scaffold Enhances Bone Regeneration

Eman E A Mohammed; Hanan H Beherei; Mohamed El-Zawahry; Abdel Razik H Farrag; Naglaa Kholoussi; Iman Helwa; Khaled Gaber; Mousa A Allam; Mostafa Mabrouk; Alice K Abdel Aleem

doi:10.3889/oamjms.2019.730

Combination of Human Amniotic Fluid Derived-Mesenchymal Stem Cells and Nano-hydroxyapatite Scaffold Enhances Bone Regeneration

Open Access Maced J Med Sci. 2019 Sep 14;7(17):2739-2750. doi: 10.3889/oamjms.2019.730. eCollection 2019 Sep 15.

Authors

Eman E A Mohammed^{1

2}, Hanan H Beherei³, Mohamed El-Zawahry⁴, Abdel Razik H Farrag⁵, Naglaa Kholoussi⁶, Iman Helwa⁶, Khaled Gaber⁷, Mousa A Allam⁸, Mostafa Mabrouk³, Alice K Abdel Aleem^{1

2

9}

Affiliations

¹ Medical Molecular Genetics Department, National Research Centre, Cairo, Egypt.
² Stem Cell Research Group, Medical Research Centre of Excellence, National Research Centre, Cairo, Egypt.
³ Ceramic Department (Biomaterials), National Research Centre, Cairo, Egypt.
⁴ Fixed and Removable Prosthodontics Department, National Research Centre, Cairo, Egypt.
⁵ Pathology Departments, National Research Centre, Cairo, Egypt.
⁶ Immunogenetics Department, National Research Centre, Cairo, Egypt.
⁷ Prenatal and Fetal medicine Department, National Research Centre, Cairo, Egypt.
⁸ Spectroscopy Department, National Research Centre, Cairo, Egypt.
⁹ Neurology and Neuroscience Department, Weill Cornell Medical College, Doha, Qatar.

Abstract

Background: Human amniotic fluid-derived stem cells (hAF-MSCs) have a high proliferative capacity and osteogenic differentiation potential in vitro. The combination of hAF-MSCs with three-dimensional (3D) scaffold has a promising therapeutic potential in bone tissue engineering and regenerative medicine. Selection of an appropriate scaffold material has a crucial role in a cell supporting and osteoinductivity to induce new bone formation in vivo.

Aim: This study aimed to investigate and evaluate the osteogenic potential of the 2nd-trimester hAF-MSCs in combination with the 3D scaffold, 30% Nano-hydroxyapatite chitosan, as a therapeutic application for bone healing in the induced tibia defect in the rabbit.

Subject and methods: hAF-MSCs proliferation and culture expansion was done in vitro, and osteogenic differentiation characterisation was performed by Alizarin Red staining after 14 & 28 days. Expression of the surface markers of hAF-MSCs was assessed using Flow Cytometer with the following fluorescein-labelled antibodies: CD34-PE, CD73-APC, CD90-FITC, and HLA-DR-FITC. Ten rabbits were used as an animal model with an induced defect in the tibia to evaluate the therapeutic potential of osteogenic differentiation of hAF-MSCs seeded on 3D scaffold, 30% Nano-hydroxyapatite chitosan. The osteogenic differentiated hAF-MSCs/scaffold composite system applied and fitted in the defect region and non-seeded scaffold was used as control. The histopathological investigation was performed at 2, 3, & 4 weeks post-transplantation and scanning electron microscope (SEM) was assessed at 2 & 4 weeks post-transplantation to evaluate the bone healing potential in the rabbit tibia defect.

Results: Culture and expansion of 2nd-trimester hAF-MSCs presented high proliferative and osteogenic potential in vitro. Histopathological examination for the transplanted hAF-MSCs seeded on the 3D scaffold, 30% Nano-hydroxyapatite chitosan, demonstrated new bone formation in the defect site at 2 & 3 weeks post-transplantation as compared to the control (non-seeded scaffold). Interestingly, the scaffold accelerated the osteogenic differentiation of AF-MSCs and showed complete bone healing of the defect site as compared to the control (non-seeded scaffold) at 4 weeks post-transplantation. Furthermore, the SEM analysis confirmed these findings.

Conclusion: The combination of the 2nd-trimester hAF-MSCs and 3D scaffold, 30% Nano-hydroxyapatite chitosan, have a therapeutic perspective for large bone defect and could be used effectively in bone tissue engineering and regenerative medicine.

Keywords: 3D Scaffolds; Amniotic Fluid Stem Cells; Bone Healing; Bone Regeneration; Nano-hydroxyapatite Chitosan.