Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications.

The term macromolecular crowding is used to describe equilibria and kinetics of biochemical reactions and biological processes that occur via mutual volume exclusion of macromolecules in a highly crowded structureless medium. In vivo, the extracellular space is heavily crowded by a diverse range of macromolecules and thus, biological processes occur rapidly, whilst in vitro, in the absence of macromolecules, the same processes occur very slowly, if they are initiated at all (1-3). This talk will discuss the concept of macromolecular crowding, alone or in combination with other in vitro microenvironment modulators, in tendon engineering context.

Acknowledgements: This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement No. 866126. This publication has emanated from research supported by grants from Science Foundation Ireland (SFI) under grant number 19/FFP/6982.

The fibrocartilaginous enthesis displays a complex interface between two mechanically dissimilar tissues, namely tendon and bone. This graded transition zone consists of parallel collagen type I fibres arising from the tendon and inserting into bone across zones of fibrocartilage with aligned collagen type I and collagen type II fibres and mineralised fibrocartilage. Due the high stress concentrations arising at the interface, entheses are prone to traumatic and chronic overuse injuries such as rotator cuff and anterior cruciate ligament (ACL) tears. Treatment strategies range from surgical reattachment for complete tears and conservative treatments (physiotherapy, anti-inflammatory drugs) in chronic inflammatory conditions. Generally, the native tissue architecture is not re-established and mechanically inferior scar tissue is formed. Current interfacial tissue engineering approaches pose scaffold-associated drawbacks and limitations, such as foreign body response. Using a thermo-responsive electrospun scaffold that provides architectural signals similar to native tissues and can be removed prior to implantation, we aim to develop an ECM-rich, cell-based implant for tendon-enthesis regeneration. Alcian blue staining revealed highest sGAG deposition in cell (human adipose derived stem cells) sheets grown on random electrospun fibres and lowest sGAG deposition in collagen type I sponges. Cells did not show an equal distribution throughout the collagen type II scaffolds but tended to form localised aggregates. Thermo-responsive electrospun fibres with random and aligned fibre orientation provided an adequate three-dimensional environment for chondrogenic differentiation of multilayer hADSC-sheets shown by high ECM-production, especially high sGAG deposition. Chondrogenic cell sheets showed increased expression of SOX9, COL2A1, COL1A1, COMP and ACAN after 7 days of chondrogenic induction when compared to pellet culture. Anisotropic fibres enabled the generation of aligned chondrogenic cell sheets, shown by cell and collagen fibre alignment. Thermoresponsive electrospun fibres showed high chondro-inductivity due to their three-dimensionality and therefore pose a promising tool for the generation of scaffold-free multilayer constructs for tendon-enthesis repair within short culture periods. Aligned chondrogenic cell sheets mimic the zonal orientation of the native enthesis as the fibrocartilaginous zone exhibits high collagen alignment.

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Following injuries and surgical repair, the enthesis is often not reestablished and so far, traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. In this work, we hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the enthesis. A three-layer sponge composed of a tendon-like layer (collagen I), a cartilage-like layer (collagen II) and a bone-like layer (collagen I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold porosity and structural continuity at the interfaces were assessed through SEM analysis. Bone-marrow derived stem cells (BMSCs) were seeded by syringe vacuum assisted technique on the scaffold. Scaffolds were cultured in basal media for 3 days before switching to differentiation media (chondrogenic, tenogenic and osteogenic). BMSCs metabolic activity, proliferation and viability were assessed by alamarBlue, PicoGreen and Live/Dead assays. At D21 the scaffolds were fixed, cryosectioned and Alizarin Red and Alcian Blue stainings were performed in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I and tenascin in the scaffolds was evaluated by immunofluorescence staining at D21 in order to assess tenogenic differentiation of BMSCs. Subsequently, the cartilage-like layer was functionalized with IGF-1, seeded with BMSCs and cultured in basal media up to D21. Structural continuity at the interfaces of the scaffolds was confirmed by SEM and scaffold porosity was assessed as >98%. The scaffolds supported cell proliferation and infiltration homogeneously throughout all the layers up to D21. Osteogenic differentiation of BMSC selectively in the bone-like layer was confirmed by Alizarin red staining in scaffolds cultured in basal and osteogenic media. Alcian blue staining revealed the presence of proteoglycans selectively in the cartilage-like layer in scaffolds cultured in chondrogenic media but not in basal media. Increased expression of the tenogenic markers collagen I and tenascin were observed in the tendon-like layer of scaffolds cultured in tenogenic but not in basal media for 21 days. The presence of IGF-1 increased osteogenic and chondrogenic differentiation of BMSCs, whereas no difference was observed for tenogenic differentiation. In conclusion, a 3-layer collagen sponge was successfully fabricated with distinct but integrated layers; the different collagen composition of the non-functionalized 3-layer sponge was able to regulate BMSC differentiation in a localized manner within the scaffold. The scaffold functionalization with IGF-1 accelerated chondrogenic and osteogenic BMSC differentiation. Overall, functionalization of the 3-layer scaffolds holds promising potential in enthesis regeneration.

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 time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. 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 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 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 (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. 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.

The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration.

A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs.

Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers).

In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration.

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.

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. In vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). 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 through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with MMC at 2 % oxygen tension showed increased synthesis and deposition of collagen type I. 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, western blot analysis revealed increased expression of tendon-specific protein Scleraxis, while a detailed gene analysis illustrated upregulation of tendon-specific genes and downregulation of trans-differentiation genes again when cells cultured with MMC under hypoxic conditions. 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.

Tenocytes from several mammal species have been shown to be prone to phenotypic drift at early sub-culture passages. In the present study we compared allogenic and xenogenic serum supplementation suitability as a supplement for the in vitro expansion of equine tenocytes (eTCs), in combination with the presence or absence of crowding conditions. eTCs were isolated from superficial digital flexor tendon and expanded in normal growth medium containing DMEM, 10% appropriate serum, 1% penicillin/streptomycin solution. Isolation was performed by migration method in growth medium containing the selected serum. Silver staining, densitometry, zymography, immunofluorescence, metabolic activity, proliferation, viability and morphology were performed after 3, 5 and 7 days in culture with a seeding density of 10,000 cells/cm2. Treatment conditions were equine serum (ES) or foetal bovine serum (FBS), with or without 75 μg/mL of crowding agent carrageenan (CR). Viability and metabolic activity of eTCs were affected by FBS. eTCs in ES reached higher cell density than in FBS in day 7, especially with CR. Morphology of eTCs was maintained under different sera. Silver staining on pepsin digested cell layers shows that collagen type I deposition rate is remarkably enhanced in the presence of CR in all conditions. Immunofluorescence showed increased expression for collagen I, III, V and VI in both sera in the presence of CR. Deposition of all collagen types but type VI was increased by ES supplementation. We conclude that ES in combination with CR can represent a reliable choice for the ex vivo expansion of eTCs.

To repair soft tissue, it is vital to ensure that the biomaterial is able to mimic the complex elasticity of the native tissue. It has been demonstrated that substrate stiffness has a huge influence on cellular growth, differentiation, motility and phenotype maintenance. The goal of the present study is to characterize extensively a set of polymeric films with variable mechanical profiles. A range of synthetic biodegradable polymers was selected according to the physico-chemical intrinsic properties of aliphatic polymers. They have similar chemistry (absorbable polyesters made from lactic acid, glycolic acid, trimethylene carbonate, dioxanone & β-caprolactone), however show different mechanical and degradation properties. The films were manufactured by thermal presser and then characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). The mechanical properties of the films were assessed by uniaxial tensile tests in wet conditions and also by atomic force microscopy (AFM) to assess the material's stiffness at a micro-level. In vitro assays were performed to assess the cell cytocompatibility, proliferation and differentiation potential of the films. The mechanical properties of the materials are within the range intended for musculoskeletal tissue repair. Biological assays showed good cell adhesion, cell proliferation and cell viability. Stem cells were able to differentiate into adipogenic, osteogenic, chondrogenic and tenogenic lineages. Overall the selection of polymers gave good options for a potential tissue repair scaffold. In the future, the combined effect of stiffness and topography will be assessed on cell phenotype maintenance.

Collagen scaffolds are generally characterized by their random fibre distribution and weak mechanical properties, which makes them unsuitable as substitutes for highly anisotropic tissues such as cornea or tendon. Recently, we developed a technique to create collagen type I scaffolds with well-defined anisotropic micro-patterns. Porcine collagen was mixed with PBS10X, NaOH and one of the following cross-linkers: glutaraldehyde (GTA), genipin and 4-arm polyethylene glycol (4SP). The resulting mixture was casted on micro-grooved (2×2×2 μm) polydimethylsiloxane (PDMS) moulds and allowed to dry in a laminar flow hood to obtain 5mg/ml collagen films. Different pH, temperatures (Tº), and cross-linker concentrations were tested in the process. Collagen gelation kinetics was analysed with rheometry and surface topography was assessed with scanning electron microscopy (SEM). Human bone marrow stem cells (HBMSCs) were seeded on the films and cell alignment was analysed by rhodamine/phalloidin staining and imaged with fluorescence microscopy. From all three cross-linkers tested, only 4SP cross-linked scaffolds showed a well-defined micro-grooved pattern. Increasing pH and Tº on 4SP-treated collagen decreased gelation time, which resulted in complete inhibition of the pattern, suggesting that an initial low viscous solution is required for a correct PDMS pattern infiltration. A wide range of 4SP concentrations (0.5, 1, 1.5 mM) maintained the well-defined topography on the films, opening the door to future fine-tuning of the stiffness sensed by cells. hBMSCs seeded on top of the scaffolds aligned along the pattern for 14 days in culture. Collectively, this data highlights the potential of these collagen scaffolds as tendon substitutes.

Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type II collagen isolated from cartilaginous tissues has yet to be assessed. In addition, it has been shown from previous studies that sex steroid hormones affect the collagen content in male and female animals, herein, type II collagens from male and female porcine cartilage were assessed in order to investigate gender effects on the property of collagen scaffolds. Moreover, type II collagen has a supportive role in articular cartilage in the knee joint. Therefore, the aim is to assess the properties of type II collagen scaffolds as a function of species, tissue and gender for cartilage regeneration. Type II collagen was extracted from male and female porcine trachea, auricular, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was conducted to confirm the purity of extracted collagen. Collagen sponges were created via freeze-drying. Scaffold structure and pore size were evaluated by scanning electron microscopy (SEM). Thermal stability was assessed by differential scanning calorimetry (DSC). Sponges were seeded with human adipose derived stem cells to assess chondro-inductive potential of collagen sponges after 7, 14 and 21 days of culture. In conclusion, collagen sponges support the proliferation and differentiation of human adipose derived stem cells to different extents.

Tissue engineering by self-assembly offers the possibility to fabricate contiguous cell sheets that are stabilised by intact cell-cell contacts and endogenously produced extracellular matrix (ECM) However, these systems lack the possibility to introduce topographical cues, that are fundamental for the organisation of many types of tissues. Herein we venture to fabricate aligned electrospun thermoresponsive nanofibres to sustain growth and detachment of ECM-rich living substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used. 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 to estimate matrix deposition and composition. 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 revealed that hADSCs were able to grow on the scaffold. The cells aligned on the fibres after 3 days and they were able to detach as intact cell sheets in presence of MMC. Moreover, it was demonstrated that MMC, by a volume extrusion effect, enhances collagen type I deposition, one of the main components of the ECM. Collectively the pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich cell sheets.

Cell-based scaffold-free tissue equivalents present a limited clinical translation as consequence of the delayed extracellular matrix (ECM) deposition due to the prolonged production time in vitro. Different combinations of media supplements such as ascorbic acid, growth factors, oxygen tension among others can modulate the cell fate or the ECM synthesis. New research lines are focusing on the use of macromolecular crowders (MMCs) as media supplement for cell sheet production due to their ability to increase ECM deposition by volume exclusion effect, pro-collagenases alosteric regulation, matrix self-assembly by confinement and diffusion limitation (most probably, modulating the interaction between the ECM, MMPs and TIMPs). Herein, different molecular weights and concentrations of a natural potential MMC (Crowder-A) have been tested in equine adipose-derived stem cell (eADSC) and human dermal fibroblast (hADF) cultures in comparison with other commonly used crowders such as carrageenan and the Ficoll™ cocktail 70 KDa and 400 KDa. The eADSCs were characterized according to the current criteria for horse MSCs. Tri-lineage and FACS analysis showed eADSC osteogenic and adipogenic potentials and the presence of the markers CD29, CD44, CD90. The screening of the aforementioned Crowder-A was performed in cultures of 15,000 cells / cm² for the eADSCs and 25,000 cells / cm² for the hADFs during 3, 5, and 7 days. Non-MMC conditions were used as negative controls. Collagen type I was analysed by SDS-PAGE. Other collagen types were studied by immunocytochemistry assays. Significant increase of some ECM components was observed in some concentrations and molecular weights of the Crowder-A.

Mesenchymal stem cells (MSCs), characterised by their self-renewal and multidifferentiation potential, are a favoured cell population for future tissue engineering applications. Differentiation of MSCs towards a specific lineage has been extensively studied, mainly through the use of growth factors or conditioned media. However, growth factor supplementation is a mono-domain approach and considering the number of permutations, it is unlikely to find the optimal cocktail. Although PRPs are used extensively, its use is controversial, and standardization is impossible. Conditions media have various limitations, including how much, when and how effective it is at the time that it would be aspirated. Thus, co-culture systems are at forefront of scientific research and technological innovation. Co-culture system gives access to the complete cell secretome and offers the advantages of autologous therapy. However, several weeks of co-culture are necessary to observe stem cell differentiation. We hypothesize that, by using macromolecular crowding, which has been shown to recapitulate the dense in vivo microenvironment of the extracellular area and enhance matrix deposition in vitro with its excluded volume effect, it will accelerate stem cell differentiation towards tenogenic lineage. Further, we will assess if tendon specific extracellular matrix deposited by tenocytes is sufficient for stem cell differentiation without the necessity of cell contact between tenocytes and stem cells.

A suitable wound closure is an indispensable requirement for an uncomplicated and expedient recovery after an abdominal surgery. The closure technique will have a great impact on the healing process of the wound. Surgical complications, such as wound dehiscence (sometimes associated with evisceration), infection, hernia, nerve injury and incisional pain are very common in the postoperative period of an abdominal surgery. Besides, although their development can be promoted by other risk factors like age, sex, lifestyle, diet, health condition, the closure method can also influence the emergence of these undesirable complications. For this reason, and having the wellbeing and quality of life of the patients in mind, particularly high-risk patients, a closure system consisting of anchors applied on either side of the wound that aims to reduce the tension caused on the surrounding tissues of a wound and, consequently, decrease the risk of herniation was evaluated in a pilot animal study and compared with the traditional suture approach.

Phenotypic drift of stem cells and insufficient production of extracellular matrix (ECM) are frequently observed in tissue-engineered cartilage substitutes, posing major weaknesses of clinically relevant therapies targeting cartilage repair. Microenvironment plays an important role for stem cell maintenance and differentiation and therefore an optimal chondrogenic differentiation protocol is highly desirable. Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates biological processes by several orders of magnitude. MMC was recently shown to significantly increase ECM deposition and to promote chondrogenic differentiation of stem cells. We hypothesise that the addition of sulphated high-molecular weight polysaccharides (carrageenan) to the media positively affects stem cell maintenance and chondrogenic differentiation. Herein, we venture to assess the impact of MMC on the maintenance of stem cell phenotype and multipotency, and ECM deposition in xeno-free human bone marrow mesenchymal stem cell (BMSCs) cultures. We investigate different xeno- and serum-free stem cell media with MMC for expansion of BMSCs, assessing multipotency maintenance (FACS analysis), cell viability, metabolic activity, proliferative capacity and matrix deposition (SDS-PAGE, ICC) at day 4 and day 10. Experiments will be conducted at 2 different passages (p3, p7). Medium without MMC will be used as control. Based on these results, cells expanded with the best protocol will be subsequently investigated for chondrogenic differentiation comparing different xeno-/serum-free and serum containing differentiation media. Chondrogenic differentiation will be assessed via Alcian blue and Safranin O stainings, gene expression for chondrogenic marker genes and quantification of GAG content. Finally, these findings will pave the way for developing more effective strategies for cartilage tissue engineering.

Collagen materials are extensively used in regenerative medicine. However, they still present limitations such as a mono-domain composition and poor mechanical properties. On the other hand, tissue grafts overcome most of these limitations. In addition, the potential of tissue grafts in musculoskeletal tissue engineering has not been fully investigated. Herein, we ventured to assess the potential of a decellularised porcine peritoneum for musculoskeletal applications by comparing its characteristics with a commercial collagen scaffold employed in tendon. Results indicated that the porcine peritoneum had higher mechanical properties and a lower crosslinking ratio (p < 0.01). Furthermore, it presented a lower resistance to collagenase digestion, which suggests a faster remodelling in vivo of the tissue graft. Immunohistochemistry analysis showed a preserved and multicomponent structure in the porcine peritoneum contrary to the collagen matrix, confirming the multifunctional nature of the tissue graft. Regarding the cell-response assessment, tenocytes and ADSCs were able to grow on both materials, however, proliferation was enhanced by the porcine peritoneum (p<0.01). Immune response by THP-1 showed an acute inflammatory response by macrophages to the collagen matrix, contrary to that observed in the porcine peritoneum which triggered a mild reaction. The in-progress in vivo study in a rabbit tendon model will elucidate the potential of porcine peritoneum for tendon repair applications. The present study shows how the multifunctionality of the porcine peritoneum provides higher cytocompatibility than a mono-domain collagen matrix with human tenocytes and ADSC. Besides, its lower immune response in vitro suggests better remodelling after implantation.