ICRS 2019 - Conference Calendar

Displaying One Session

Plaza A Free Paper Session
Session Type
Free Paper Session
Date
06.10.2019
Time
13:30 - 15:00
Location
Plaza A
Podium Presentation Stem Cells

10.4.1 - Over-Confluence of expanded mesenchymal stem cells ameliorates their chondrogenic capacity in 3D cartilage tissue engineering

Presentation Number
10.4.1
Presentation Topic
Stem Cells
Lecture Time
13:30 - 13:39
Session Type
Free Paper Session
Corresponding Author

Abstract

Purpose

Tissue engineering of high-quality articular cartilage constructs using bone marrow-derived mesenchymal stem cells (BM-MSCs) remains an elusive goal. During the limb bud mesenchyme stage of skeletal development, MSCs undergo a process of condensation prior to the initiation of chondrogenesis. We hypothesise that culturing BM-MSCs in vitro to over confluence can mimic this condensation phase, leading to robust chondrogenic cells for articular cartilage tissue engineering applications.

Methods and Materials

Eleven BM-MSC samples were cultured for 5 days (low confluence) and 24 days (over confluence) before being harvested, seeded onto polyglycolic scaffolds and stimulated with transforming growth factor-β3 (TGF-β3) for 35 days to drive chondrogenic differentiation. The mature constructs were digested and analysed by quantitative immunoassays and qPCR, to measure the biochemical composition of various ECM components and the expression of chondrogenic markers, respectively. Four samples were analysed by RNA Seq analysis to reveal major pathways implicated in the over-confluence process.

Results

Biochemical analysis of the generated cartilage constructs demonstrated a statistically significant increase in type II to type I collagen content ratio and a significant increase in the proteoglycan content, in over confluence constructs compared to paired low confluence constructs. Gene analysis showed a statistically significant increase in SOX9, type II collagen and aggrecan expression and a decrease in the expression of the hypertrophic marker, type X collagen, in over confluence constructs. KEGG enriched pathway analysis of RNA Seq data revealed significantly active pathways for focal adhesion and ECM-receptor interactions.

Conclusion

This study has demonstrated, for the first time, that BM-MSCs, permitted to proliferate past confluence in 2D culture, can produce biochemically superior 3D tissue-engineered cartilage. This simple adjustment to the common BM-MSC culture technique could impact most of cartilage tissue engineering modalities utilising these cells.

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Podium Presentation Biomaterials and Scaffolds

10.4.2 - Design of chondro-instructive hydrogels for high-throughput 3D-biofabrication of cartilage tissue modules

Presentation Number
10.4.2
Presentation Topic
Biomaterials and Scaffolds
Lecture Time
13:39 - 13:48
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

3D-bioassembly approaches combining high-throughput fabrication of tissue modules with automated extrusion-based 3D printing of reinforcing thermoplastic polymer scaffolds represents a promising strategy to gain translational relevance in the field of cartilage tissue engineering. However, a major bottleneck lies in designing cell-laden hydrogels that are compatible with 3D-biofabrication, tailorable and cell-instructive, all-in-one. This study thus aimed to develop versatile chondro-instructive hydrogels for high-throughput 3D-biofabrication of tissue modules. We focused on 1) formulating customizable and bioactive thiol-ene click hydrogels by photo-polymerising thiolated heparin (HepSH), a native cartilage glycosaminoglycan (GAG), with allylated gelatin (GelAGE), and 2) investigating if vitreous humor (VH), a highly hydrated tissue closely resembling the composition of cartilage, can be applied as unmodified, cell-instructive hydrogels; systematically studying biofabrication compatibility, micro-tissue self-assembly and capacity to facilitate chondrogenesis.

Methods and Materials

GelAGE-HepSH micro-spheres were biofabricated and photo-polymerised (450nm, 20wt.-%GelAGE, 0.5wt%HepSH) and equine VH was extracted. Chondrocyte-laden hydrogels (5-15x106 cells/ml) were cultured (3-5w, TGF-β1) and physico-chemical properties, HepSH retention, cellular health (Live/dead®, alamarBlue®) and tissue formation (GAG, DNA, IHC: Safranin-O, Agg/Cx43, Col I/II, PCR:Col I/II,Agg, Sox9) was characterized.

Results

GelAGE-HepSH was successfully biofabricated into Ø1mm micro-spheres with tailorable physico-chemical properties (7-98kPa) and good viability (>80%). GelAGE further allowed efficient conjugation of HepSH (>96%), yielding significantly greater differentiation (GAG/DNA=134g/g, PCR: collagen-type-II=1.4x, aggrecan:1.9x) compared to GelAGE alone (GAG/DNA=24g/g). Likewise was unmodified and intact VH successfully seeded with cells, subsequently self-assembling into Ø1mm micro-spheres displaying uniform distribution of GAGs and collagen type II with significantly greater expression of chondrogenic markers (collagen-type-II=2.2x, 3.3x, aggrecan=1.6x, 13.3x) as well as GAG/DNA (14.1±2.5g/g) compared to controls (micro-pellets=7.0±1.3g/g, collagen-type-1 hydrogels=1.6±0.6g/g).

Conclusion

This study demonstrated that developed GelAGE-HepSH and VH hydrogels can be favorably applied as multifunctional, customizable and cell-instructive biomaterials compatible with high-throughput 3D-bioassembly approaches for clinical relevance and practicality - concurrently demonstrating high shape fidelity, tailorable physico-chemical properties and improved bioactivity for functional cartilage repair.

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Podium Presentation Intervertebral Disc

10.4.3 - Design and evaluation of electrospun structured polycaprolactone biomaterials for annulus fibrosus repair

Abstract

Purpose

Extensive Annulus fibrosus (AF) radial tears lead to intervertebral disc (IVD) herniation. While unrepaired defects in the AF are associated with high IVD degeneration prevalence, current surgical strategies disregard the structural integrity of the AF. This study aims at i) designing polycaprolactone (PCL) electrospun implants that mimic the AF multi-lamellar fibrous structure and ii) assessing their ability to properly repair an AF defect in a sheep model.

Methods and Materials

Oriented and non-oriented PCL implants were produced by electrospinning. In vitro apposition of ovine annular explants was characterized by cell morphology (nucleus and F-actin staining, EdU proliferation assay) and extracellular matrix (ECM) deposition (collagen, aggrecan). In vivo study was carried out on 6 sheep in which 5 lumbar discs were exposed using a left retroperitoneal approach. Box-shape defects (2x5mm, 2mm depth) were created in the outer AF, with randomized conditions including 10-layer implants, untreated and healthy groups. X-ray and MRI examinations were performed at 1 month, followed by immuno-histological analysis and second harmonic generation microscopy (SHG).

Results

PCL implants with average fiber diameters of 1µm and a tensile modulus (55±1MPa) matching the one of a native human AF lamella (~47MPa) were obtained (Fig. 1). In vitro spontaneous colonization of PCL implants by ovine AF explants was demonstrated at 14 and 28 days. In sheep, successful implantations of PCL implants were achieved. While empty defects exhibited irregular fibrous reparative tissue with numerous vascular ingrowths, cell infiltration between and within the implants, and a continuous type I collagen tissue formation between the implants and the surrounding AF tissue were evidenced (Fig. 2). SHG quantitative analysis confirmed that neo-synthesized collagen fibers were aligned within each layer, replicating the native AF tissue organization.

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Conclusion

These results highlight that a cell-free multi-layer PCL electrospun implant is a promising biomaterial for AF repair.

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Podium Presentation New Cartilage Technology

10.4.4 - Magnetic levitational bioassembly of cartilage in Space

Presentation Number
10.4.4
Presentation Topic
New Cartilage Technology
Lecture Time
13:57 - 14:06
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Tissue engineering is a promising technique that could solve an acute clinical problem – the lack of human tissues and organs for transplantation. Traditional tissue engineering is based on using synthetic or natural scaffolds as temporal and removable support. The aim of this study was to develop the technology of magnetic levitational bioassembly of 3D tissue constructs, which represents a novel rapidly emerging scaffold-free and label-free approach and alternative conceptual advance in tissue engineering.

Methods and Materials

The magnetic levitational bioassembly of 3D tissue construct from tissue spheroids could be characterized as direct levitational assembly of tissue spheroids in magnetic field, followed by their fusion into the single construct in specific location. New magnetic bioprinter has been designed, developed and certified for life space research. Tissue spheroids made from primary human chondrocytes and magnetic bioprinter were delivered on the «Soyuz MS-11» ship to the Russian segment of the international space station in a temperature-sensitive non-adhesive hydrogel. Six constructs were fabricated in the new magnetic bioprinter from tissue spheroids. The constructs fused within 48 hours at 37°С. Then, the fused constructs were removed from the magnetic bioprinter and fixed with 4% formaldehyde. Finally, the cuvettes with the constructs were sent to Earth where histology and histochemistry were performed.

Results

Scaffold-free and label-free magnetic levitational bioassembly of 3D tissue constructs from chondrospheres has been performed at the first time under microgravity conditions. It has been demonstrated that thermo-reversible hydrogel enables delivery of viable tissue spheroids to the International Space Station and prevents their undesirable preliminary tissue fusion. The novel original magnetic bioprinter has been designed, implemented, certified and successfully tested for space life science research.

Conclusion

We developed the technology of magnetic levitational bioassembly of 3D tissue constructs on the International Space Station using tissue spheroids.

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Podium Presentation Biomechanics

10.4.5 - Cartilage-on-chip: mimicking the complex mechanical stimulation present in articular cartilage

Presentation Number
10.4.5
Presentation Topic
Biomechanics
Lecture Time
14:06 - 14:15
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Human cartilage-on-chip models that faithfully mimic the complex mechanical movements of the joint hold great promise for increasing our understanding of joint diseases and development of therapeutic interventions. Current models, however,fail to appropriately replicate the combined compression and shear strain present in articulations. The aim of this study is to develop an innovative organ-on-chip platform mimicking these complex movements in the knee joint.

Methods and Materials

The cartilage-on-a-chip platform was fabricated from polydimethylsiloxane (PDMS) using soft-lithography. The platform comprises three different sections as detailed in Fig. 1.

Compressive strain and shear strain were imposed on the hydrogel by deforming the thin membrane by applying independently pressure to the three chambers.

Human chondrocytes (hCHs) were cultured in an agarose matrix using proliferation medium. Microbeads (15 µm) were used to evaluate hydrogel deformation upon mechanical stimulation. Cell viability was evaluated using a live dead/assay with (800 mbar applied at 1Hz for 1.5hrs per day, starting from day 4) or without mechanical stimulation after 6 days.

Results

We quantified agarose deformation as a function of strain (fig. 2): With higher pressure, higher strain was generated. Importantly, both physiological healthy (5-20%) and pathophysiological (>20%) strains could be applied on the hydrogel. Moreover, shear strain was obtained by single chamber stimulation (not shown).

We next determined the decrease in chondrocytes surface area in a hydrogel subjected to a homogeneous compression (800 mbar at 1Hz for 1.5hrs). Cell volume decrease was inversely correlated with the cell’s proximity to the compression membrane. hCHs could be cultured in our platform for at least 6 days, without a noticeable effect on cell viability.

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Conclusion

This novel microfluidic platform can be used to explore the impact of various mechanical stimuli on responses of individual cells. We will extent this platform with units mimicking synovium, ligaments, bone and meniscus to engineer a joint-on-chip.

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Podium Presentation Osteoarthritis

10.4.6 - The mechanism of spontaneous calcium signaling in chondrocytes and its regulation in cartilaginous matrix metabolism

Presentation Number
10.4.6
Presentation Topic
Osteoarthritis
Lecture Time
14:15 - 14:24
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

To investigate the role of spontaneous [Ca2+]i signaling in cartilaginous ECM metabolism regulation, and its correlation with OA severity.

Methods and Materials

Cartilage explants and primary chondrocytes were isolated from porcine knee joints. In situ calcium imaging was performed in cartilage explants with pharmacological interventions. Primary chondrocytes were used for RT-qPCR, WB, live and dead assay, Alcian blue and immunofluorescence staining. By using Fluo-8 AM based calcium imaging system, cartilage samples at different OA stages collected from patients undergoing knee arthroscopic surgery were used to analyze the spatiotemporal features of spontaneous [Ca2+]i signaling of in situ chondrocytes.

Results

we found that spontaneous [Ca2+]i signaling of in situ porcine chondrocytes was tightly regulated by [Ca2+]o influx, InsP3Rs mediated [Ca2+]i store release, and Orais mediated CRAC activation. [Ca2+]o deprivation was associated with decreased cell viability, and expression levels of ECM deposition genes. Whereas blocking ER Ca2+ release with InsP3R inhibitors significantly up-regulated ECM degradation genes, down-regulated SOX11 expression level, and was accompanied by decreased proteoglycan and collagen type Ⅱintensity. Furthermore, our data showed zonal dependent spontaneous [Ca2+]i signaling in healthy cartilage samples under 4 mM calcium environment.This signal was significantly attenuated in healthy cartilage samples when cultured in calcium free environment. No significant difference was found in [Ca2+]i intensity oscillation in chondrocytes located in middle zones among ICRS 1-3 samples, but OA severity dependent spatiotemporal features of spontaneous [Ca2+]i oscillations of deep zone chondrocytes was observed.

Conclusion

Our data provided evidence that spontaneous [Ca2+]i signaling of in situ porcine chondrocytes was tightly regulatedby [Ca2+]o influx, InsP3Rs mediated [Ca2+]i store release, and Orais mediated CRAC activation. Both [Ca2+]o concentration and InsP3Rs mediated ER Ca2+release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.This might reflect the role of spontaneous [Ca2+]i signaling during OA progression and provide insight into articular cartilage degradation during OA progression.

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Podium Presentation Cartilage /Cell Transplantation

10.4.7 - The Natural Repair of Articular Cartilage in Humans: An Immunohistological Study

Presentation Number
10.4.7
Presentation Topic
Cartilage /Cell Transplantation
Lecture Time
14:24 - 14:33
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Evidence suggests articular cartilage is capable of natural regeneration in some individuals; despite the oft-stated belief of its inability for self-repair. We have examined repair tissue formed following surgically induced cartilage defects in humans as part of an autologous cell implantation (ACI) procedure.

Methods and Materials

Sixteen patients (12 males, 4 females) had macroscopically healthy cartilage harvested from the trochlea for ACI. The quality of repair was assessed on MRIs taken at 14.7±3.7months and during arthroscopy at 15±3.5 months post-harvest using the Oswestry Arthroscopy Score (O-AS) and the International Cartilage Repair Society Arthroscopy Score (ICRS-AS), maximum scores of 10 and 12 respectively (where higher is better). Core biopsies of the repair tissue were assessed histologically (scored using the ICRSII and OsScore histology scores) and collagen types I, II, III, and VI determined immunohistochemically and compared to healthy cartilage.

Results

The mean O-AS and ICRS-AS of the repaired defects were 7.2±3.2SD and 10.1±3.5SD respectively with a mean defect area fill of 80%±23SD. The quality of the repair tissue formed was variable; hyaline cartilage was present in 50% of the biopsies and was associated with a significantly higher ICRS-AS (median 11 vs 7.5, p=0.05). The OsScore, but not the ICRSII score, correlated significantly with both the O-AS (r=0.49, p=0.05) and ICRS-AS (r=0.52, p=0.04). Collagen type I was detected in 12/14 biopsies, type II in 10/13 biopsies and types III and VI in 15/15 biopsies with variable staining patterns.

Conclusion

These results demonstrate the ability for articular cartilage to heal naturally following an injury, albeit with variable morphologies. The harvest defects may have an advantage in their ability to heal compared to condylar cartilage defects typically found in osteoarthritis, due to the lower loads at their location and macroscopically healthy cartilage having been removed. The mechanism by which this repair process occurs remains unknown and warrants additional studies.

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Podium Presentation Osteoarthritis

10.4.8 - Identification of M9 high-mannose glycan regulating hypertrophy in articular cartilage

Presentation Number
10.4.8
Presentation Topic
Osteoarthritis
Lecture Time
14:33 - 14:42
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Ectopic initiation of chondrocyte hypertrophy in articular chondrocytes involves cartilage degeneration and osteoarthritis (OA) progression. However, the mechanism by which chondrocytes lead to hypertrophic differentiation has not been clarified. Glycans modulate many biological processes for cell differentiation, hence, we conducted a comprehensive analysis of the glycophenotype during the chondrocyte hypertrophy process.

Methods and Materials

The hypertrophic changes of primary mouse chondrocytes were induced by adding insulin (10 ug / mL). The alterations of glycans at day 0, 10 and 20 were comprehensively investigated by glycoblotting, total glycome, and cluster analysis. Based on the results of the total glycome and cluster analysis, we decided to target glycan on the list of upregulated glycan structures. We added glycosidase enzyme that cleaves a structure of target glycan to primary chondrocytes and cartilage explants ex vivo and evaluated them by qPCR, dye-binding assay of proteoglycan, and histological staining.

Results

A total of 151 glycan structures were significantly changed by insulin stimulation. All glycans were divided into four groups by cluster analysis. High-mannose type N-glycans (M5, 6, 7, 8, 9) were located at the top of the expression level by hierarchical cluster analysis (Fig. 1). Unlike other high-mannose type N-glycans, only the amount of M9 decreased with hypertrophy. Artificial enzymatic cleavage of M9 (α-mannosidase) in chondrocytes increased the mRNA expression of Adamts5, Col10a1, and Indian hedgehog. Moreover, enzymatic cleavage of M9 for the femoral head cartilage increased the numbers of type X collagen-positive cells in the surface layer and the release of proteoglycans (Fig. 2). 

図1.png

図2.png

Conclusion

Our results presented the dynamic alteration of glycans in the process of chondrocyte hypertrophy. Enzymatic cleavage of M9 whose expression was reduced in the chondrocyte glycome induced hypertrophy of chondrocytes, suggesting that the alterations of high-mannose type N-glycans would regulate chondrocyte hypertrophy.

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Podium Presentation Osteoarthritis

10.4.9 - The concentration-dependent effect of CoCr ions and particles on osteoarthritic chondrocytes

Presentation Number
10.4.9
Presentation Topic
Osteoarthritis
Lecture Time
14:42 - 14:51
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Cobalt (Co) and chromium (Cr) ions and particles are released into the joint after implantation of metal implants due to biotribocorrosion. Co and Cr can induce apoptosis and alter gene expression levels in various cell types. The objective of this study was to determine the effects of Co and Cr ions on human articular chondrocytes and to evaluate inflammatory responses.

Methods and Materials

Human articular chondrocytes were subjected to different concentrations of Co and Cr ions and particles. Cell viability and early/late apoptosis assessment were performed in-vitro(2D cell cultures) using XTT assay and flow cytometry, respectively. Changes in chondrocytes’ morphology were assessed using fluorescent cell imaging. In addition, the effects on the biosynthetic activity of chondrocytes were evaluated by quantitative polymerase chain reaction (qPCR). Inflammatory response to different conditions was determined by the release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-8).

Results

Cobalt and chromium ions and particles significantly reduce metabolic activity and induce early and late apoptosis (Annexin Vand 7-AAD) with increasing concentrations already after 24 hours in culture. After 72 hours the majority of chondrocytes (>90%) were apoptotic. SOX 9 expression was enhanced with increasing concentrations, whereas collagen type 2 showed a linear decrease after 24 hours. IL-8 release was enhanced with increasing Cobalt and chromium ions and particles levels, whereas IL-1β, IL-6, and TNF-α showed no significant differences between the applied conditions.

Conclusion

Co and Cr ions and particles show a dose-dependent and time-dependent effect on articular chondrocytes. With increasing concentrations, Co and Cr ions and particles induce apoptosis in articular chondrocytes, decrease metabolic activity and chondrocyte-specific gene expression, and induce an inflammatory response. Hence, these adverse effects on articular chondrocytes need consideration in cases of partial surface replacement and unicompartimental arthroplasty.

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Podium Presentation Cartilage and Meniscus

10.4.10 - The role of biaxial sliding and cyclic uniaxial compression on solute transport in articular cartilage

Presentation Number
10.4.10
Presentation Topic
Cartilage and Meniscus
Lecture Time
14:51 - 15:00
Session Type
Free Paper Session
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Articular cartilage relies on diffusion pathways to obtain essential nutrients and molecules for cellular activity. Understanding these transport pathways is essential to maintaining and improving the health of articular cartilage and ultimately synovial joints. Several studies have shown that joint articulation is associated with fluid and solute uptake although it remains unclear what role biaxial sliding and cyclic uniaxial compression independently play.

Methods and Materials

Cartilage-bone plugs (10mm) were obtained from porcine knee joints and sealed along the radial edge into purpose made diffusion chambers. The bone side of the chamber was filled with PBS to maintain tissue hydration while the cartilage side was filled with 0.01mg/ml fluorescein sodium salt (FNa) prepared using PBS. Samples were either subject to biaxial sliding (1.4 MPa at 5 mm/s) or cyclic uniaxial compression (0-1.4 MPa at 0.4MPa/s) using a 25 mm diameter spherical indenter for 0.5 or 1 hour. Both loading configurations were compared to unloaded control samples. After diffusion tests, samples were sectioned and imaged using an epifluorescent microscope with a filter for FNa. Intensity profiles were mapped and normalized from the articular surface to the subchondral bone. Fluorescent intensity represents solute uptake into the cartilage and is scaled from 0 – 100% of the dynamic range.

Results

Samples that had been subjected to sliding demonstrated accelerated penetration and solute accumulation compared to compression samples (figure 1). Both loading configurations exceeded solute penetration and accumulation compared to unloaded controlsannotation 2019-04-14 172157.png

Conclusion

Biaxial sliding and cyclic uniaxial compression play an important role in the uptake of solutes into the cartilage matrix. Maintaining joint motion both post injury and in the arthritic process is a critical component of cartilage nutrition. Samples that had been subject to biaxial sliding demonstrated accelerated solute penetration and accumulation in the cartilage matrix, exceeding concentrations achieved cyclic uniaxial compression and passive diffusion.

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