It has been shown for many connective tissue types that substrate orientation can control collagen and ECM deposition. This has been used to engineer tissues with aligned fibril structures such as tendon. In cortical bone, collagen is organised into a lamellar structure with highly aligned collagen sheets packed into a “twisted plywood” configuration, however, reproducing this in vitro proves challenging. In a previous unpublished study within our laboratory, mature late-osteoblast-like cells, MLO-A5, cultured on aligned polyurethane fibres deposited collagen along the direction of the fibres. Above the scaffold, collagen was deposited in a direction offset to the initial layer, continuing in subsequent layers, creating a lamellae-like twisted-plywood structure. In this study we aimed to test whether human cells at an early stage of osteogenic differentiation would deposit collagen exhibiting a twisted plywood deposition behaviour, therefore providing a model system of laboratory-grown lamellae for use as a model of bone formation.
Aligned and non-aligned polycaprolactone (PCL) fibers were fabricated by electrospinning and treated with air plasma. hTERT Y201 immortalised human MSCs  were seeded directly onto the scaffolds and supplemented with osteogenic induction media. On days 14, 21 and 28 collagen was imaged using second harmonic generation (SHG). Collagen alignment was analysed using the ImageJ “Directionality'' Plug-in. Samples were also stained for calcium and collagen deposition using Alizarin Red stain (ARS) and Sirius Red stain (SRS).
Collagen was initially shown to orientate along the direction of the aligned PCL fibres. By D21 orientation patterns that varied with depth could be observed, with collagen layers having a twist in the anti-clockwise direction, compared with their substrate layer with an average change in orientation of 16.53° ± 13.22. By D28, the orientation had shifted from the initial direction of the PCL fibres by 53.1° ± 15.6. For non-aligned scaffolds there was no noticeable collagen directionality, or difference between layers. Mineral and collagen staining showed that there were no significant differences in overall quantity of matrix produced within either scaffold type.
Attempts to tissue engineer bone generally result in a disorganised matrix, comparable to that of woven bone or a healing fracture. In this work, it was shown a possibility to guide the collagen into a structure comparable to the cortical bone lamallae. Interestingly examinations of lamellae organisation have shown periodicities of about 5-7µm, and gradual change of fibril direction, varying from 10-60° from the orientation of the lamellae at the osteon’s centre. Within this work, the change in collagen direction also remained within this range, but occurred over larger depths of 35-45µm . Further work should explore the mechanisms, as this may provide an insight into how collagen organises into tissue-specific structures.
 S. James et al., Stem Cell Reports, vol. 4, no. 6. 1004–1015 (2015).
This project has received funding from the EU’s Horizon 2020 research and innovation programme H2020-MSCA-RISE (grant agreement No 777926 – NanoSurf”) and The EPSRC on a doctoral training account (EP/R513313/1) from the department of Materials Science and Engineering, University of Sheffield.