Human mesenchymal stem cell morphology, migration, and differentiation on micro and nano-textured titanium

Abstract

Orthopedic implants rely on facilitating a robust interaction between the implant material surface and the surrounding bone tissue. Ideally, the interface will encourage osseointegration with the host bone, resulting in strong fixation and implant stability. However, implant failure can occur due to the lack of integration with bone tissue or bacterial infection. The chosen material and surface topography of orthopedic implants are key factors that influence the early events following implantation and may ultimately define the success of a device. Early attachment, rapid migration and improved differentiation of stem cells to osteoblasts are necessary to populate the surface of biomedical implants, potentially preventing biofilm formation and implant-associated infection. This article explores these early stem cell specific events by seeding human mesenchymal stem cells (MSCs) on four clinically relevant materials: polyether ether ketone (PEEK), Ti6Al4V (smooth Ti), macro-micro rough Ti6Al4V (Endoskeleton®), and macro-micro-nano rough Ti6Al4V (nanoLOCK®). The results demonstrate the incorporation of a hierarchical macro-micro-nano roughness on titanium produces a stellate morphology typical of mature osteoblasts/osteocytes, rapid and random migration, and improved osteogenic differentiation in seeded MSCs. Literature suggests rapid coverage of a surface by stem cells coupled with stimulation of bone differentiation minimizes the opportunity for biofilm formation while increasing the rate of device integration with the surrounding bone tissue.

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Figures

Graphical abstract

Fig. 1

Surfaces were imaged with SEM to demonstrate the unique nanostructures present on the nanoLOCK® surface relative to Endoskeleton®. A. – D. are 1,000X images for PEEK, Smooth Ti, Endoskeleton® and nanoLOCK® respectively. E. – H. are 10,000X images for PEEK, Smooth Ti, Endoskeleton® and nanoLOCK® respectively.

Fig. 2

MSC morphology was determined on each of the four unique surfaces by measuring Aspect Ratio, A., Circularity, B., and Roundness, C. Significance (p 

Fig. 3

MSC adhesion kinetics were assessed over the course of 24 h by counting the number of cells attached to each surface at 2hr, 12hr, and 24hr. Significance (p 

Fig. 4

Representative 40X immunofluorescence images of MSCs on each surface at 24hrs actin stress fibers (red), the focal adhesion protein vinculin (green), and the nuclei (blue).

Fig. 5

MSC migration velocity, A., and directionality, B., were examined on each of the four surfaces. Significance (p 

Fig. 6

Circular histograms demonstrating the migration direction of all cells measured on each surface, demonstrating that Endoskeleton® and nanoLOCK® did indeed present true random migration, whereas, PEEK demonstrated migration in two opposite directions.

Fig. 7

Assessment of hMSC differentiation through the early marker alkaline phosphatase (ALP), A., and mid marker osterix (OSX), B., at 3 and 10 days. Significance (p 

Figs1

Representative 10X immunofluorescence images of MSCs on each surface at 24hrs actin stress fibers (red), the focal adhesion protein vinculin (green), and the nuclei (blue). The vinculin stain on PEEK was not detected at low magnification due to the autofluorescence of PEEK overwhelming the vinculin signal

Figs2

Proliferation of MSCs on each surface was measured by quantitative analysis of DNA. In general, MSC proliferation was highest on the smooth Ti surfaces, followed by PEEK, with Endoskeleton® and nanoLOCK® presenting the lowest levels of proliferation. Significance (p