Osteogenic differentiation of CD271(+) cells from rabbit bone marrow cultured on three phase PCL/TZ-HA bioactive scaffolds: comparative study with mesenchymal stem cells (MSCs)

Abstract

Tissue engineering is one of the major challenges of orthopedics and trauma surgery for bone regeneration. Biomaterials filled with mesenchymal stem cells (MSCs) are considered the most promising approach in bone tissue engineering. Furthermore, our previous study showed that the multi-phase poly [ε-caprolactone]/thermoplastic zein-hydroxyapatite (PCL/TZ-HA) biomaterials improved rabbit (r) MSCs adhesion and osteoblast differentiation, thus demonstrating high potential of this bioengineered scaffold for bone regeneration. In the recent past, CD271 has been applied as a specific selective marker for the enrichment of MSCs from bone marrow (BM-MSCs). In the present study, we aimed at establishing whether CD271-based enrichment could be an efficient method for the selection of rBM-MSCs, displaying higher ability in osteogenic differentiation than non-selected rBM-MSCs in an in vitro system. CD271(+) cells were isolated from rabbit bone marrow and were compared with rMSCs in their proliferation rate and osteogenic differentiation capability. Furthermore, rCD271(+) cells were tested in their ability to adhere, proliferate and differentiate into osteogenic lineage, while growing on PCL/TZ-HA scaffolds, in comparison to rMSCs. Our result demonstrate that rCD271(+) cells were able to adhere, proliferate and differentiate into osteoblasts when cultured on PCL/TZ-HA scaffolds in significantly higher levels as compared to rMSCs. Based on these findings, CD271 marker might serve as an optimal alternative MSCs selection method for the potential preclinical and clinical application of these cells in bone tissue regeneration.

Keywords: CD271; Rabbit bone marrow; biomaterials; bone tissue engineering; mesenchymal stem cells; osteoblast.

Figure 1

A. Representative flow cytometry plots showing the presence of rCD271⁺ cells from rabbit bone marrow mononuclear cells; B. Left panels shows cultures of rMSCs that have been isolated by plastic adherence ability and right panels shows cultures of rCD271⁺ cells isolated by antibody selection (14 days cultures, respectively). Untreated cells are expanded in vitro. Treated cells show nodular aggregates that are typical of osteogenesis. Alizarin Red S staining (ARS) show extracellular calcium deposits; C. Alkaline phosphatase activity measured after 21 days post induction. Original magnification × 100. Data are presented as means ± SD.

Figure 2

SEM micrographs of rMSCs and rCD271⁺ cells seeded on three-phases PCL/TZ-HA: (A) rMSCs and (B) rCD271⁺ cells on PCL/TZ-HA surfaces, (C) rMSCs and (D) rCD271⁺ cells on PCL/TZ-HA after 24 h of culture; SEM micrographs after 21 days of culture of the (E, G, I) rMSCs and (F, H, J) rCD271⁺ cells on PCL/TZ-HA. The SEM images illustrate rCD271⁺ cells and rMSCs populations after 24 h that were attached on the scaffold surface; after 21 days the cells proliferated on the entire surface of the scaffold. Bars = 100 µm, 20 µm, 500 µm.

Figure 3

Quantitative assays. rMSCs and rCD271⁺ cells on three-phase PCL/TZ-HA scaffolds A. DNA content; B. ALP activity; C. Total calcium content, ARS; D. RT-PCR analysis of osteogenic marker osteopontin (OPN). Data are presented as means ± SD.