J. Malda (Utrecht, NL)
University Medical Center Utrecht OrthopaedicsPresenter Of 3 Presentations
25.1.1 - Biomaterials & Hydrogels for Bioprinting of Cartilage
P033 - Microstructural Differences Between the Osteochondral Units of Terrestrial and Aquatic Mammals
Abstract
Purpose
The osteochondral unit is the pivotal element of the mammalian joint, with a fundamental clinical relevance. It is known that in terrestrial mammals, that the biochemical components of the tissues of the osteochondral unit are strongly preserved across a wide range of species. There is, however, very limited knowledge of specific adaptations of the architecture of the cartilage extracellular matrix in aquatic mammals.
The aim of this study was, therefore, to investigate if and how the structure of both the cartilage and the bone component of the osteochondral unit differs between mammals living on land or in water.
Methods and Materials
To investigate this we analyzed the microstructural composition and architectural features of the osteochondral units from the humeral head of six aquatic and nine terrestrial mammalian species. Osteochondral tissue samples were harvested post-mortem from the weight bearing central area of the humeral head. Histological assesment was performed, and included saf-O staining and polarized light microscopy to visualize the orientation of the collagen fibers. The mechanical properties were assessed by uniaxial unconfined compression and Micro Computed Tomography (micro-CT) was performed.
Results
Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. Moreover, patches of cartilage tissue are present throughout the subchondral bone.
Conclusion
This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of this microarchitecture-function relationship is pivotal for the development of durable functional regenerative approaches for treatment of joint damage.
P042 - Covalent Protein Immobilization on Melt Electrowritten Microfiber Scaffolds for Guided Cartilage Regeneration
Abstract
Purpose
Current tissue engineering treatments for end-stage articular cartilage fail to produce long-term functional cartilage tissue. Here, melt electrowriting (MEW) is used to fabricate 3D scaffolds with micro-resolution to mimic the properties of the native cartilage extracellular matrix (Castilho et al., 2019). These scaffolds are activated using atmospheric-pressure plasma jet (APPJ), allowing for covalent immobilization of transforming growth factor β1 (TGF), an important cytokine for the production and maintenance of cartilage (Wang, Rigueur & Lyons, 2014), onto the scaffold’s microfibers. It is hypothesized these biofunctionalized scaffolds will support differentiation of mesenchymal stromal cells (MSCs) into the chondrogenic lineage and subsequent neo-cartilage formation.
Methods and Materials
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (regenHU), then activated using a computer-controlled APPJ device (Alavi et al., 2020). TGF was subsequently immobilized onto the MEW scaffolds using solution submersion (1µg/mL). Characterization of protein immobilization was performed using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. In vitro experiments were performed by seeding equine MSCs into the scaffolds and were cultured for 28 days. Neo-cartilage formation was quantified with dimethyl methylene blue/picogreen assays for glycosaminoglycan (GAG) production and confirmed with histological analysis.
Results
ELISA results confirmed covalent TGF concentration on the biofunctionalized scaffolds while immunofluorescently-labelled TGF was detected visually in scaffolds. The APPJ treatment caused increased hydrophilicity of the scaffolds, resulting in efficient cellular infiltration. In vitro analysis demonstrated that GAG production was significantly enhanced in both the immobilized TGF (+APPJ+TGF) and TGF (-APPJ+TGF) in medium groups, compared to the control groups without TGF supplementation (-APPJ+/-TGF). This finding was further validated by the heightened production of GAGs and collagen type II, observed in histological sections.
Conclusion
We have demonstrated that APPJ-facilitated covalent immobilization of TGF retains bioactivity and stimulates differentiation of MSCs into the chondrogenic lineage. Our results also demonstrate that the new constructs with immobilized TGF support neo-cartilage formation.
Moderator Of 2 Sessions
- Participants should understand the mechanism and effects of digitalization and also critically analyze the advantages and problems of this global process - from artificial intelligence to fake news on the net.
Meeting Participant Of
- E. Kon (Milano, IT)
- D. Grande (Manhasset, US)
- C. Lattermann (Boston, US)
- J. Malda (Utrecht, NL)
- S. Marlovits (Vienna, AT)
- T. Minas (West Palm Beach, US)
- S. Görtz (Newton, US)
- A. Krych (Rochester, US)
- W. Kafienah (Bristol, GB)
- G. Filardo (Bologna, IT)
- I. Dallo (Sevilla, ES)
- R. Frank (Denver/Aurora, US)
- F. Sciarretta (Rome, IT)
- S. Chubinskaya (Chicago, US)
- D. Saris (Rochester, US)
- E. Papacostas (Doha, QA)
- M. McNicholas (Liverpool, GB)
Presenter Of 2 Presentations
P033 - Microstructural Differences Between the Osteochondral Units of Terrestrial and Aquatic Mammals
Abstract
Purpose
The osteochondral unit is the pivotal element of the mammalian joint, with a fundamental clinical relevance. It is known that in terrestrial mammals, that the biochemical components of the tissues of the osteochondral unit are strongly preserved across a wide range of species. There is, however, very limited knowledge of specific adaptations of the architecture of the cartilage extracellular matrix in aquatic mammals.
The aim of this study was, therefore, to investigate if and how the structure of both the cartilage and the bone component of the osteochondral unit differs between mammals living on land or in water.
Methods and Materials
To investigate this we analyzed the microstructural composition and architectural features of the osteochondral units from the humeral head of six aquatic and nine terrestrial mammalian species. Osteochondral tissue samples were harvested post-mortem from the weight bearing central area of the humeral head. Histological assesment was performed, and included saf-O staining and polarized light microscopy to visualize the orientation of the collagen fibers. The mechanical properties were assessed by uniaxial unconfined compression and Micro Computed Tomography (micro-CT) was performed.
Results
Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. Moreover, patches of cartilage tissue are present throughout the subchondral bone.
Conclusion
This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of this microarchitecture-function relationship is pivotal for the development of durable functional regenerative approaches for treatment of joint damage.
P042 - Covalent Protein Immobilization on Melt Electrowritten Microfiber Scaffolds for Guided Cartilage Regeneration
Abstract
Purpose
Current tissue engineering treatments for end-stage articular cartilage fail to produce long-term functional cartilage tissue. Here, melt electrowriting (MEW) is used to fabricate 3D scaffolds with micro-resolution to mimic the properties of the native cartilage extracellular matrix (Castilho et al., 2019). These scaffolds are activated using atmospheric-pressure plasma jet (APPJ), allowing for covalent immobilization of transforming growth factor β1 (TGF), an important cytokine for the production and maintenance of cartilage (Wang, Rigueur & Lyons, 2014), onto the scaffold’s microfibers. It is hypothesized these biofunctionalized scaffolds will support differentiation of mesenchymal stromal cells (MSCs) into the chondrogenic lineage and subsequent neo-cartilage formation.
Methods and Materials
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (regenHU), then activated using a computer-controlled APPJ device (Alavi et al., 2020). TGF was subsequently immobilized onto the MEW scaffolds using solution submersion (1µg/mL). Characterization of protein immobilization was performed using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. In vitro experiments were performed by seeding equine MSCs into the scaffolds and were cultured for 28 days. Neo-cartilage formation was quantified with dimethyl methylene blue/picogreen assays for glycosaminoglycan (GAG) production and confirmed with histological analysis.
Results
ELISA results confirmed covalent TGF concentration on the biofunctionalized scaffolds while immunofluorescently-labelled TGF was detected visually in scaffolds. The APPJ treatment caused increased hydrophilicity of the scaffolds, resulting in efficient cellular infiltration. In vitro analysis demonstrated that GAG production was significantly enhanced in both the immobilized TGF (+APPJ+TGF) and TGF (-APPJ+TGF) in medium groups, compared to the control groups without TGF supplementation (-APPJ+/-TGF). This finding was further validated by the heightened production of GAGs and collagen type II, observed in histological sections.
Conclusion
We have demonstrated that APPJ-facilitated covalent immobilization of TGF retains bioactivity and stimulates differentiation of MSCs into the chondrogenic lineage. Our results also demonstrate that the new constructs with immobilized TGF support neo-cartilage formation.