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Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_4 | Pages 64 - 64
1 Mar 2021
Korntner S Pieri A Pugliese ZWE Zeugolis D
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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.


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 56 - 56
1 Jul 2020
Tsiapalis D De Pieri A Sallent I Galway N Zeugolis D Galway N Korntner S
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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.


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 82 - 82
1 Jul 2020
De Pieri A Korntner S Rochev Y Zeugolis D Galway N
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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.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 105 - 105
1 Nov 2018
Wu Z Korntner S Mullen A Skoufos I Tzora A Zeugolis D
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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.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 97 - 97
1 Nov 2018
Pugliese E Korntner S Zeugolis DI
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The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_16 | Pages 120 - 120
1 Nov 2018
Korntner S Gaspar D Zeugolis D
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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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 40 - 40
1 Jan 2017
Korntner S Lehner C Kunkel N Traweger A Tempfer H
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Metabolic disorders are frequently associated with tendon degeneration and impaired healing after acute injury. However, the underlying cellular and molecular mechanisms remain largely unclear. We have previously shown that human and rat tendon cells responde to glucose stimulation in vitro by secretion of insulin. Therefore, we now hypothesize that nutritional glucose uptake affects tendon healing in a rat model.

In female rats (n=30/group), unilateral full-thickness Achilles tendon defects were created. Immediately after surgery animals were either fed a glucose rich- or a control diet for up to 4 weeks. Gait analysis (Catwalk, Noldus) was performed at three time points. In addition, tendon thickness measurements, biomechanical testing and immunohistochemical analysis were conducted. Subsequently, gene expression analysis, comparing cDNA pools (n=5) prepared from repair tissues of both groups was performed.

The repair tissues of the high glucose group were significantly thicker compared to the control group (p<0.001). The intermediate toe spread, an indicator of pain, were significantly improved in the high glucose group one and two weeks post surgery. Biomechanical analysis revealed that the repair tissues of the high glucose group were significantly stiffer (p<0.05) compared to the control group, no significant difference was detected for maximum tensile load…. The proportion of Ki67+ cells in the repair tissue was 3.3% in the control diet group and 9,8% in the high glucose group, indicating increased cell proliferation (p<0.001). Finally, gene expression analysis revealed the chondrogenic marker genes Collagen II, Aggrecan, COMP and SOX9 to be upregulated and genes involved in lipid metabolism like PPARgamma and Fabp2 to be downregulated in the glucose diet group.

Here we show fort he first time that a high-glucose diet affects gait pattern and tendon biomechanics, influences tendon thickness and cell proliferation. Gene expression analysis reveals a regulation of chondrogenic as well as adipogenic marker genes. The molecular mechanisms underlying these effects on cells and extracellular matrix are currently under investigation, potentially revealing targets for developing a dietary intervention scheme to support tendon regeneration after trauma or tendon disease.


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_11 | Pages 1 - 1
1 Oct 2015
Korntner S Kunkel N Lehner C Gehwolf R Wagner A Augat P Resch H Bauer H Traweger A Tempfer H
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Introduction

Metabolic disorders are among known risk factors for tendinopathies or spontaneous tendon ruptures. However, the underlying cellular and molecular mechanisms remain unclear. We have previously shown that human and rat tendon cells produce and secrete insulin upon glucose stimulation. Therefore, we hypothesize that nutritional glucose uptake affects tendon healing in a rat model.

Materials and Methods

Unilateral full-thickness Achilles tendon defects were created in 60 female rats. Animals were randomly assigned to three groups receiving different diets for 2 weeks (high glucose diet, low glucose/high fat diet, control diet). Gait analysis was performed at three time points (n=20/group). In addition, tendon thickness, biomechanical (n=14/group), and histological and immunohistochemical analysis was conducted. Subsequently, a subtractive-suppression-hybridization (SSH) screen comparing cDNA pools (n=5) prepared from repair tissues of the high glucose and the control diet group was conducted to identify differentially expressed genes.


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_11 | Pages 7 - 7
1 Oct 2015
Lehner C Gehwolf R Ek CJ Korntner S Bauer H Bauer HC Traweger A Tempfer H
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Introduction

Tendon cells originate from yet poorly described precursor cells and develop in a particular “niche” close to vascular walls. Several factors have been described to determine this niche such as mechanical stimuli, oxygen tension, composition and structure of the extracellular matrix (ECM). Also, the vasculature is considered to play a crucial role for tendon cell development, yet evidence of how this is accomplished is lacking. In this study we therefore focussed on the endothelium of tendon vessels postulating the existence of a paracellular barrier.

Materials and Methods

By electron microscopy, immunohistochemistry, and RT-PCR we investigated the presence of constituents making up such an endothelial barrier which we subsequently tested for its functionality by tracer injection. Moreover, we performed differentiation experiments into the adipogenic, chondrogenic and osteogenic lineage on tendon derived cells in the presence and absence of serum. Expression levels and activity of matrixmetalloproteinases (MMPs) were assessed by western blot and zymography.