Osteogenic Differentiation Potential of Human Bone Marrow and Amniotic Fluid-Derived Mesenchymal Stem Cells in Vitro & in Vivo

Abstract

Background: Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering.

Aim: This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies.

Subjects and methods: Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters.

Results: The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potential.

Conclusions: AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.

Keywords: AF-MSCs; Amniotic fluid; BM-MSCs; Bone marrow; Mesenchymal stem cells; Osteogenic differentiation.

Figure 1

Proliferation and culture expansion of BM-MSCs, 2nd trimester AF- MSCs & 3rd trimester AF-MSCs; A-D) represented Bone marrow (BM) Proliferation & culture expansion at different passage; A) P0, B) P1, C) P2, D) P3 by x10 magnification; E-H) showed 2nd trimester AF-MSCs Proliferation & culture expansion at different passage; E) P0; F) P1; G) P2; H) P3 by x 10 magnification; I-L) represented 3rd trimester AF-MSCs Proliferation & culture expansion at different passage; I) P0; J) P1; K) P2; L) P3 by x 10 magnification

Figure 2

Osteogenic differentiation of BM-MSCs, 2nd trimester AF-MSCs and 3rd trimester AF-MSCs after 14 days; Osteogenic differentiation of BM cells on day 14; A) Control; B) Osteogenic differentiation stained by Alizarin; Osteogenic differentiation of 2nd trimester AF cells; C) Control; D) Osteogenic differentiation stained by Alizarin after 14 days; Osteogenic differentiation of 3rd trimester AF cells; E) Control; F) Osteogenic differentiation stained by Alizarin after 14 days

Figure 3

Osteogenic differentiation of BM-MSCs, 2nd AF-MSCs & 3rd AF-MSCs at 28 days; Osteogenic differentiation of BM cells on day 28th; A) Control; B) Osteogenic differentiation stained by Alizarin; Osteogenic differentiation of 2nd trimester AF cells; C) Control; D) Osteogenic differentiation stained by Alizarin; Osteogenic differentiation of 3rd AF cells; E) Control; F) osteogenic differentiation stained by Alizarin

Figure 4

X-ray for AF-MSCs and BM-MSCs transplantation in rats. AF- and BM-derived undifferentiated MSCs, and differentiated osteogenic cells transplanted on decorticated lumber rat-spine beds. Among the BM group, after 4 months, the control rat, transplanted with undifferentiated MSCs showed no change at the lumbar spine. While, one of the two osteogenic rats transplanted with osteogenic differentiated MSCs, showed bone mottling, a shadow area at the lumbar spine. For the AF– group, after 3 months, the control rat (undifferentiated MSCs) showed no bone changes. However, the two osteogenic rats (transplanted with osteoblastic 2nd trimester AF- derived MSCs) showed bone mottling

Figure 5

Histopathological examination of sections of experimental and control rats, 4 months after transplantation with (BM-MSCs) & 3 months after transplantation with (AF-MSCs) stained with hematoxylin and eosin dye. (A & B) Are sections of rat spinal defect transplanted with BM-MSCs, A) section of rat spinal defect transplanted with BM- MSCs differentiated into osteogenic progenitors shows cartilage area (short arrow), calcified cartilage (arrowhead) and formed new bone (long arrow) and host bone (asterisk). B) Section of rat spinal defect transplanted with undifferentiated BM-MSCs shows irregular calcified areas (short arrows) that associated with few osteocytes (arrowhead) and blood vessels (asterisk). (C & D) Represented sections of rat spinal injury transplanted with AF-MSCs, C) Section of rat spinal defect transplanted with AF-MSCs differentiated into osteogenic progenitors shows calcified cartilage (short arrow), newly formed bone (long arrow), host bone (arrowhead) and bone marrow (asterisk). D) Section of rat spinal defect transplanted with undifferentiated AF-MSCs shows calcified area with few osteogenic (short arrow) on the top of the host bone (arrowhead) (H&E, Scale bar: 20 µm)