Periodic Reporting for period 1 - MACROS (Magnetically Tunable Chondrocyte Cell Sheet Engineering for Osteoarthritis Therapy)
Periodo di rendicontazione: 2023-03-01 al 2025-08-31
Sintesi del contesto e degli obiettivi generali del progetto
Background. Osteoarthritis (OA) is a major cause of disability and socioeconomic burden. It progressively destroys articular cartilage and reduces mobility and quality of life across all ages, including a large working-age population, while driving substantial direct (healthcare) and indirect (productivity) costs. Current regenerative options are limited. Cell injection (the benchmark) can help early OA but demands high cell numbers, suffers low retention/viability, and is expensive (~€15,000 per treatment). Cell-sheet therapy is emerging as a safer, more effective format because it preserves cell–cell and cell–matrix interactions, yet the standard temperature-responsive pNIPAM platform is far too stiff (2–3 GPa) and non-tunable, biasing chondrocytes toward fibrocartilage (type I collagen) rather than hyaline cartilage (type II collagen) needed for durable joint function.
The overarching goal of this project is to deliver an innovative, magnetically tunable stiffness cell-sheet engineering platform that yields hyaline-like chondrocyte sheets with superior regenerative potential for early-stage OA, without adding exogenous bio-factors.
Objectives (mapped to WPs):
1. WP1 – Magnetic-responsive substrate: Engineer a non-covalent based magnetic hydrogel enabling (i) robust monolayer formation and on-demand, gentle detachment into intact cell sheets; and (ii) in situ stiffness modulation by external magnetic fields.
2. WP2 – Dynamic stiffness control: Establish proof-of-concept that stiffening–softening cycles alter cell-sheet behavior in predictable ways (using stiffness-sensitive cells) and quantify cytoskeletal and mechano-transduction markers.
3. WP3 – Cartilage-mimicking chondrocyte sheets: Apply the dynamic protocol (stiffen for proliferation → soften for hyaline matrix deposition) and benchmark against pNIPAM sheets using immunostaining, gene expression, and CAG’s condyle chip assay for functional efficacy.
Expected impacts of the project are:
• Technical bottleneck removed: Replaces fixed, ultra-stiff pNIPAM with a tunable platform that matches chondrocyte mechanobiology, increasing the type II:type I collagen ratio and preserving a chondrogenic phenotype.
• Clinical value: Cell sheets require approximately 10× fewer cells than injections for similar defect areas, enabling a 30–40% cost reduction (to around €10,000) and potentially broadening clinical accessibility.
• Translational readiness: The approach eliminates the need for exogenous growth factors, simplifying safety and regulatory assessment; the gentle, stimulus-controlled detachment preserves cell viability and extracellular matrix (ECM) continuity.
Pathway to impact is separate into three period dependents on the project period:
Short term (project end):
• Used the non-covalent cell sheet fabrication hydrogel platform with quantitative evidence of hyaline-like matrix enrichment and superior function in the condyle chip assay.
Medium term (12–36 months post-project):
• Exploitation with TUM/CAG: IP filing on dynamic stiffness and cell-sheet process; integration into CO.DON AG.s preclinical pipeline (including donor variability, safety, animal reduction via chip-based assays); early regulatory dialogue (EMA scientific advice) on ATMP classification, potency assays, and comparability.
• Manufacturing & QA: Transfer of cell sheet fabrication platform and substrate fabrication to GMP-amenable workflows; definition of critical quality attributes (CQA) for stiffness, detachment yield, and ECM composition.
Long term (3–6 years):
• Clinical translation in early OA cohorts where joint-replacement is inappropriate: first-in-human feasibility, then controlled trials versus cell injection.
• Economic and societal impact: Lower procedure costs, improved durability (targeting benefits beyond 5 years), reduced absenteeism, and productivity gains for Europe’s working-age population.
The success on the impact of the project will be measured by the following criteria:
• Scientific: First physical, factor-free dynamic-stiffness strategy for cell-sheet engineering; new insights into mechanical memory in chondrocytes; wider adoption of chip-based efficacy assays, accelerating OA research while reducing animal use.
• Clinical: More durable symptom relief and function vs. cell injection, with better ECM quality and sheet integrity.
• Economic: Projected 30–40% reduction in cell-related costs; pathway for a European SME/industry (CAG) to access the large OA market with an ATMP-compatible product, strengthening EU competitiveness.
The success will be measured by the following criteria:
• Benchmarks: ≥2× increase in type II:I collagen ratio vs. pNIPAM; ≥90% sheet viability post-detachment; reproducible stiffness switching (<0.4 T culture modulation; >0.5 T detachment).
• Translation: The adoption of experimental results and protocol by CO.DON AG; in particular in the context of chondrocyte expansion, and transformation into cell sheet.
The overarching goal of this project is to deliver an innovative, magnetically tunable stiffness cell-sheet engineering platform that yields hyaline-like chondrocyte sheets with superior regenerative potential for early-stage OA, without adding exogenous bio-factors.
Objectives (mapped to WPs):
1. WP1 – Magnetic-responsive substrate: Engineer a non-covalent based magnetic hydrogel enabling (i) robust monolayer formation and on-demand, gentle detachment into intact cell sheets; and (ii) in situ stiffness modulation by external magnetic fields.
2. WP2 – Dynamic stiffness control: Establish proof-of-concept that stiffening–softening cycles alter cell-sheet behavior in predictable ways (using stiffness-sensitive cells) and quantify cytoskeletal and mechano-transduction markers.
3. WP3 – Cartilage-mimicking chondrocyte sheets: Apply the dynamic protocol (stiffen for proliferation → soften for hyaline matrix deposition) and benchmark against pNIPAM sheets using immunostaining, gene expression, and CAG’s condyle chip assay for functional efficacy.
Expected impacts of the project are:
• Technical bottleneck removed: Replaces fixed, ultra-stiff pNIPAM with a tunable platform that matches chondrocyte mechanobiology, increasing the type II:type I collagen ratio and preserving a chondrogenic phenotype.
• Clinical value: Cell sheets require approximately 10× fewer cells than injections for similar defect areas, enabling a 30–40% cost reduction (to around €10,000) and potentially broadening clinical accessibility.
• Translational readiness: The approach eliminates the need for exogenous growth factors, simplifying safety and regulatory assessment; the gentle, stimulus-controlled detachment preserves cell viability and extracellular matrix (ECM) continuity.
Pathway to impact is separate into three period dependents on the project period:
Short term (project end):
• Used the non-covalent cell sheet fabrication hydrogel platform with quantitative evidence of hyaline-like matrix enrichment and superior function in the condyle chip assay.
Medium term (12–36 months post-project):
• Exploitation with TUM/CAG: IP filing on dynamic stiffness and cell-sheet process; integration into CO.DON AG.s preclinical pipeline (including donor variability, safety, animal reduction via chip-based assays); early regulatory dialogue (EMA scientific advice) on ATMP classification, potency assays, and comparability.
• Manufacturing & QA: Transfer of cell sheet fabrication platform and substrate fabrication to GMP-amenable workflows; definition of critical quality attributes (CQA) for stiffness, detachment yield, and ECM composition.
Long term (3–6 years):
• Clinical translation in early OA cohorts where joint-replacement is inappropriate: first-in-human feasibility, then controlled trials versus cell injection.
• Economic and societal impact: Lower procedure costs, improved durability (targeting benefits beyond 5 years), reduced absenteeism, and productivity gains for Europe’s working-age population.
The success on the impact of the project will be measured by the following criteria:
• Scientific: First physical, factor-free dynamic-stiffness strategy for cell-sheet engineering; new insights into mechanical memory in chondrocytes; wider adoption of chip-based efficacy assays, accelerating OA research while reducing animal use.
• Clinical: More durable symptom relief and function vs. cell injection, with better ECM quality and sheet integrity.
• Economic: Projected 30–40% reduction in cell-related costs; pathway for a European SME/industry (CAG) to access the large OA market with an ATMP-compatible product, strengthening EU competitiveness.
The success will be measured by the following criteria:
• Benchmarks: ≥2× increase in type II:I collagen ratio vs. pNIPAM; ≥90% sheet viability post-detachment; reproducible stiffness switching (<0.4 T culture modulation; >0.5 T detachment).
• Translation: The adoption of experimental results and protocol by CO.DON AG; in particular in the context of chondrocyte expansion, and transformation into cell sheet.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
We developed a cell sheet fabrication system using a poly(acrylamide-co-acrylic acid) (PAc) hydrogel as a base platform. The PAc hydrogel was selected for its tunable stiffness within the physiological range (1–100 kPa) and ease of surface chemical modification. To enable reversible cell attachment and detachment, we implemented a desthiobiotin–streptavidin (DSB–Sav) system on the hydrogel surface. Streptavidin, possessing cell-binding RGD analogues, binds non-covalently to four DSB molecules (0.2 kDa each), which can subsequently be displaced by stronger-binding biotin. The surface modification was achieved through carbodiimide coupling chemistry, successfully grafting the DSB–Sav system onto the PAc hydrogel. Confocal microscopy confirmed the functionalization and biotin-induced detachment of streptavidin from the hydrogel surface. The modified hydrogel exhibited enhanced cell adhesion and spreading relative to the unmodified surface, though slightly lower compared to RGD-modified systems. Importantly, rapid and controllable detachment of cells was demonstrated, up to 90% of attached cells could be released within 5 minutes upon exchange with biotin-containing medium. Notably, we also showed that the detachment can be induced by exchanging with optimized culture medium containing no biotin (details could not be disclosed due to confidentiality). Next, we optimized culture protocol supported monolayer formation of fibroblast cells on the hydrogel, leading to the fabrication of continuous and intact cell sheets. These sheets retained their extracellular matrix proteins (fibronectin) and cell–cell junctions, as verified by immunostaining. The sheets could be mechanically lifted and transferred using tweezers without structural damage, confirming sufficient cohesion and integrity. The main technical achievement was the successful fabrication of functional fibroblast cell sheets on a hydrogel with physiologically relevant stiffness, enabling mechanical sensing by cells while preserving the potential for on-demand detachment.
To introduce mechanical stimulation capability, a magnetic hydrogel system was developed by dispersing carbonyl iron (CI) particles within the PAc hydrogel matrix. The CI particles were surface-modified with (3-aminopropyl)triethoxysilane (APTES) to improve dispersibility and corrosion resistance. This amine functionalization also accelerated gelation, reducing the curing time to less than two minutes and enabling homogeneous in situ formation directly within well plates. Rheological analysis confirmed that, at 25 wt%, the storage modulus increased from approximately 1 kPa (without magnetic field) to 7 kPa under a 250 mT magnetic field, demonstrating successful fabrication of a magnetoelastic PAc hydrogel.
Despite this proof of concept, long-term culture stability was compromised by particle corrosion after seven days of incubation, likely caused by residual acrylic acid within the hydrogel. Pre-incubation in Ca/Mg-PBS reduced corrosion during the first 72 hours but degradation continued beyond this period, leading to cell death. Since cell sheet maturation typically requires at least seven days, extended magnetic hydrogel culture was not feasible under current conditions.
Nevertheless, the dynamic stiffness capability of the system was evaluated using a custom magnetic setup, where a neodymium magnet positioned beneath the well plate applied a magnetic field of approximately 200 mT. Fibroblast cells were cultured on the magnetic hydrogel for 72 hours under three treatment conditions:
1. Constant magnetic field: magnet applied continuously for 72 hours.
2. Dynamic magnetic field: magnet applied for 48 hours followed by 24 hours without the field.
3. No magnetic field: magnet not applied throughout the experiment.
The constant magnetic field promoted fibroblast spreading, whereas cells cultured without the magnetic field remained rounded. Interestingly, the dynamic field condition also produced rounded cells, suggesting that fibroblasts are sensitive to changes in stiffness induced by magnetic modulation.
This study yielded two major technical outcomes:
1. Identification of the catalytic role of APTES in accelerating PAc gelation through amine-mediated reaction pathways.
2. Establishment of a reproducible protocol for in situ formation of magnetic PAc hydrogels in multiwell plates.
As a contingency, efforts were directed toward forming chondrocyte-derived cell sheets on non-magnetic PAc hydrogels. Early trials using the DSB–Sav interface resulted in premature detachment, attributed to weak non-specific adhesion. Substituting streptavidin with avidin markedly improved cell attachment.
We further optimized the biotin grafting strategy by comparing three biotin linkers with varying poly(ethylene glycol) (PEG) spacer lengths (0, 11, and 30 units). While PEG length did not influence initial attachment, longer spacers inhibited complete monolayer formation over extended culture. We then screened hydrogel stiffness values of 1.3 3.5 and 6.5 kPa to evaluate cell spreading and YAP localization. After 7 days, monolayer integrity and fibroblast morphology were consistently achieved only at 6.5 kPa, confirming the optimal stiffness for stable sheet formation.
Using the optimized 6.5 kPa hydrogel, we established a two-phase chondrocyte culture protocol:
• Days 1–4: Serum-containing medium to promote proliferation.
• Days 5–7: Serum-free medium to induce differentiation.
Early-passage chondrocytes (PD 1.5) successfully formed type II collagen-positive cell sheets, retaining cartilage-specific phenotype. However, due to scalability constraints, late-passage cells (PD 8) were used for comparison with pNIPAM well plpate (commercial benchmark). The PD 8 chondrocytes exhibited dedifferentiation, with loss of type II collagen and upregulation of type I collagen. Gene expression analysis showed increased COL2A1 and COL1A1 transcription on PAc compared to pNIPAM, but only type I collagen protein was detected by immunostaining in both conditions.
To introduce mechanical stimulation capability, a magnetic hydrogel system was developed by dispersing carbonyl iron (CI) particles within the PAc hydrogel matrix. The CI particles were surface-modified with (3-aminopropyl)triethoxysilane (APTES) to improve dispersibility and corrosion resistance. This amine functionalization also accelerated gelation, reducing the curing time to less than two minutes and enabling homogeneous in situ formation directly within well plates. Rheological analysis confirmed that, at 25 wt%, the storage modulus increased from approximately 1 kPa (without magnetic field) to 7 kPa under a 250 mT magnetic field, demonstrating successful fabrication of a magnetoelastic PAc hydrogel.
Despite this proof of concept, long-term culture stability was compromised by particle corrosion after seven days of incubation, likely caused by residual acrylic acid within the hydrogel. Pre-incubation in Ca/Mg-PBS reduced corrosion during the first 72 hours but degradation continued beyond this period, leading to cell death. Since cell sheet maturation typically requires at least seven days, extended magnetic hydrogel culture was not feasible under current conditions.
Nevertheless, the dynamic stiffness capability of the system was evaluated using a custom magnetic setup, where a neodymium magnet positioned beneath the well plate applied a magnetic field of approximately 200 mT. Fibroblast cells were cultured on the magnetic hydrogel for 72 hours under three treatment conditions:
1. Constant magnetic field: magnet applied continuously for 72 hours.
2. Dynamic magnetic field: magnet applied for 48 hours followed by 24 hours without the field.
3. No magnetic field: magnet not applied throughout the experiment.
The constant magnetic field promoted fibroblast spreading, whereas cells cultured without the magnetic field remained rounded. Interestingly, the dynamic field condition also produced rounded cells, suggesting that fibroblasts are sensitive to changes in stiffness induced by magnetic modulation.
This study yielded two major technical outcomes:
1. Identification of the catalytic role of APTES in accelerating PAc gelation through amine-mediated reaction pathways.
2. Establishment of a reproducible protocol for in situ formation of magnetic PAc hydrogels in multiwell plates.
As a contingency, efforts were directed toward forming chondrocyte-derived cell sheets on non-magnetic PAc hydrogels. Early trials using the DSB–Sav interface resulted in premature detachment, attributed to weak non-specific adhesion. Substituting streptavidin with avidin markedly improved cell attachment.
We further optimized the biotin grafting strategy by comparing three biotin linkers with varying poly(ethylene glycol) (PEG) spacer lengths (0, 11, and 30 units). While PEG length did not influence initial attachment, longer spacers inhibited complete monolayer formation over extended culture. We then screened hydrogel stiffness values of 1.3 3.5 and 6.5 kPa to evaluate cell spreading and YAP localization. After 7 days, monolayer integrity and fibroblast morphology were consistently achieved only at 6.5 kPa, confirming the optimal stiffness for stable sheet formation.
Using the optimized 6.5 kPa hydrogel, we established a two-phase chondrocyte culture protocol:
• Days 1–4: Serum-containing medium to promote proliferation.
• Days 5–7: Serum-free medium to induce differentiation.
Early-passage chondrocytes (PD 1.5) successfully formed type II collagen-positive cell sheets, retaining cartilage-specific phenotype. However, due to scalability constraints, late-passage cells (PD 8) were used for comparison with pNIPAM well plpate (commercial benchmark). The PD 8 chondrocytes exhibited dedifferentiation, with loss of type II collagen and upregulation of type I collagen. Gene expression analysis showed increased COL2A1 and COL1A1 transcription on PAc compared to pNIPAM, but only type I collagen protein was detected by immunostaining in both conditions.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
This project established a new hydrogel-based cell sheet platform that advances the state of the art in tissue engineering by enabling fabrication under physiological stiffness conditions, a capability not demonstrated with conventional thermoresponsive pNIPAm systems. The key results are summarized below:
1. Development of physiological-stiffness PAc hydrogel
A novel PAc-based hydrogel (non-pNIPAM) was developed with stiffness within the physiological range, allowing fabrication of cell sheets from primary foreskin fibroblasts and chondrocytes. This platform simplifies laboratory implementation and may serve as a superior alternative to current pNIPAm-based systems, particularly for stiffness-sensitive cells such as MSCs that has been used in regenerative clinical applications.MSCs regenerative outcome is attributed to its capacity to secrete various growth factors, and this capacity is negatively impaired by the high stiffness beyond physiological range. Future studies are required to investigate the potential of PAc hydrogel in retaining or promoting the regenerative capacity of MSCs cell sheet compared to the thermoresponsive benchmark.
2. Medium-optimized cell detachment phenomenon
We discovered that adjusting the composition of the cell culture medium can trigger spontaneous detachment of single cells and monolayers from the avidin–polyacrylamide hydrogel surface. This novel finding suggests a previously unreported mechanism in cell–material interactions and opens new possibilities for developing stimuli-responsive approaches in tissue engineering.
3. Magnetic PAc hydrogel fabrication protocol
A reproducible protocol was established to prepare homogeneous magnetic PAc hydrogel within standard well plates. This integration and the magnetic set-up facilitates stiffness control; however, optimization is required to prevent particle corrosion and improve cell adhesion.
4. Retention of chondrogenic phenotype
Early data suggest that PAc-based hydrogels can support type II collagen secretion by early differentiated chondrocytes, indicating potential for cartilage repair applications. Further development is needed to extend culture duration and confirm long-term phenotypic stability. However, in practical applications, early differentiated chondrocytes are of limited use because their low cell numbers are insufficient to meet therapeutic requirements. Cell sheets prepared from late differentiated chondrocytes showed no improvement in re-differentiation at either the gene or protein level. We hypothesize that this may be due to the relatively short re-differentiation period applied in this study (4 days), compared with the 14-day protocols commonly reported in the literature. Currently, the main limitation of the PAc-based hydrogel is its ability to support cell culture for only up to seven days, after which the monolayer cells spontaneously detach. Further optimization is required to enhance the stability of the system and extend the culture duration.
1. Development of physiological-stiffness PAc hydrogel
A novel PAc-based hydrogel (non-pNIPAM) was developed with stiffness within the physiological range, allowing fabrication of cell sheets from primary foreskin fibroblasts and chondrocytes. This platform simplifies laboratory implementation and may serve as a superior alternative to current pNIPAm-based systems, particularly for stiffness-sensitive cells such as MSCs that has been used in regenerative clinical applications.MSCs regenerative outcome is attributed to its capacity to secrete various growth factors, and this capacity is negatively impaired by the high stiffness beyond physiological range. Future studies are required to investigate the potential of PAc hydrogel in retaining or promoting the regenerative capacity of MSCs cell sheet compared to the thermoresponsive benchmark.
2. Medium-optimized cell detachment phenomenon
We discovered that adjusting the composition of the cell culture medium can trigger spontaneous detachment of single cells and monolayers from the avidin–polyacrylamide hydrogel surface. This novel finding suggests a previously unreported mechanism in cell–material interactions and opens new possibilities for developing stimuli-responsive approaches in tissue engineering.
3. Magnetic PAc hydrogel fabrication protocol
A reproducible protocol was established to prepare homogeneous magnetic PAc hydrogel within standard well plates. This integration and the magnetic set-up facilitates stiffness control; however, optimization is required to prevent particle corrosion and improve cell adhesion.
4. Retention of chondrogenic phenotype
Early data suggest that PAc-based hydrogels can support type II collagen secretion by early differentiated chondrocytes, indicating potential for cartilage repair applications. Further development is needed to extend culture duration and confirm long-term phenotypic stability. However, in practical applications, early differentiated chondrocytes are of limited use because their low cell numbers are insufficient to meet therapeutic requirements. Cell sheets prepared from late differentiated chondrocytes showed no improvement in re-differentiation at either the gene or protein level. We hypothesize that this may be due to the relatively short re-differentiation period applied in this study (4 days), compared with the 14-day protocols commonly reported in the literature. Currently, the main limitation of the PAc-based hydrogel is its ability to support cell culture for only up to seven days, after which the monolayer cells spontaneously detach. Further optimization is required to enhance the stability of the system and extend the culture duration.