Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM.

Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted

SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes.

Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration.

Tissue engineering by self-assembly is a technique that consists of growing cells on surfaces made of thermoresponsive polymers, that allow the production of contiguous cell sheets by simply lowering the temperature below the polymer's low critical solution temperature. In this approach cell-cell junctions and deposited extracellular matrix (ECM) remain intact, which provides a better cell localisation at the site of injury. However, these systems lack the possibility to fabricate multi-layered and three-dimensional cell sheets that would better recapitulate native tissues. Moreover, the fabrication of ECM-rich cell sheets would be highly desirable. This limitation could be overcome by inducing macromolecular crowding (MMC) conditions. Herein we venture to fabricate electrospun thermoresponsive nanofibres to sustain the growth and detachment of ECM-rich tissue substitutes in the presence of a MMC microenvironment.

A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used for all experiments. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted in order to estimate matrix deposition and composition. Adipogenic, osteogenic and chondrogenic assays were performed both with and without the presence of MMC. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes.

The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability, proliferation and metabolic activity revealed that hADSCs were able to grow on the thermoresponsive scaffold. The cells were able to detach as an intact cell sheet in presence of MMC. Moreover, it was demonstated that MMC, by a volume extrusion effect, enhances Collagen type I deposition, which is one of the main components of the ECM. Histological analysis revealed that in the presence of MMC the cells were able to self-assembled into three dimensional multi-layers. The cells were able to differentiate towards the osteogenic and adipogenic lineage in the presence of MMC. Interestingly we were able to fabricate three-dimensional chondrogenic cell sheet both with and without MMC. Collectively the pNIPAm/NTBA thermoresponsive fibres were able to sustain the growth and the detachment of ECM-rich multi-layered cell sheets.

The pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich tissue equivalents. We believe that replacement, repair and restoration of tissue function can be accomplished best using cells that create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency still unmatched by man-made devices.

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength.

Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry.

Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment.

Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix.