Interplay between stiffness and degradation of architectured gelatin hydrogels leads to differential modulation of chondrogenesis in vitro and in vivo

Sarem, Melika; Arya, Neha; Heizmann, Miriam; Neffe, Axel T.; Barbero, Andrea; Gebauer, Tim; Martin, Ivan; Lendlein, Andreas; Shastri, V. Prasad

doi:10.1016/j.actbio.2018.01.025

Cited by 49 publications

(34 citation statements)

References 49 publications

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“…These findings are consistent with previous work studying degradation in non-adaptable hydrogels, but also suggests that adaptable covalent bonds can be tuned to support matrix deposition without requiring irreversible degradation of crosslinks. [46]…”

Section: Resultsmentioning

confidence: 99%

Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

et al. 2019

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Cartilage tissue engineering strategies often rely on hydrogels with fixed covalent crosslinks for chondrocyte encapsulation; yet the resulting material properties are largely elastic and can impede matrix deposition. To address this limitation, hydrazone crosslinked poly(ethylene glycol) hydrogels were formulated to achieve tunable viscoelastic properties and to study how chondrocyte proliferation and matrix deposition vary with the time-dependent material properties of covalent adaptable networks. Hydrazone equilibrium differences were leveraged to produce average stress relaxation times from hours (4.01×103s) to months (2.78×106s) by varying the percentage of alkyl-hydrazone (aHz) to benzyl-hydrazone (bHz) crosslinks. Swelling behavior and degradation associated with adaptability was characterized to quantify temporal network changes that can influence the behavior of encapsulated chondrocytes. After four weeks, mass swelling ratios varied from 36±3 to 17±0.4 and polymer retention ranged from 46±4% to 92±5%, with higher aHz content leading to loss of network connectivity with time. Hydrogels were formulated near the Flory-Stockmayer bHz percolation threshold (17% bHz) to investigate chondrocyte response to distinct levels of covalent architecture adaptability. Four weeks post-encapsulation, formulations with average relaxation times of 3 days (2.6×105s) revealed increased cellularity and an interconnected articular cartilage-specific matrix. Chondrocytes embedded in this adaptable formulation (22% bHz) deposited 190±30% more collagen and 140±20% more sulfated glycosaminoglycans compared to the 100% bHz control, which constrained matrix deposition to pericellular space. Collectively, these findings indicate that incorporating highly adaptable aHz crosslinks enhanced regenerative outcomes. However, connected networks containing more stable bHz bonds were required to achieve the highest quality neocartilaginous tissue.

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Section: Resultsmentioning

confidence: 99%

Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

et al. 2019

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“…Additionally, we did not see significant differences in expression of hypoxia-associated genes such as hypoxia-inducible factors 1 and 3 (HIF1A, HIF3A), egl-9 family hypoxia-inducible factor 3 (EGLN3) [34], and hypoxia-inducible lipid droplet-associated (HILPDA) between MSCs from various ACNs. This was an interesting observation as it has been widely accepted that multicellular tumor spheroids experience hypoxia [35, 36] and the diffusion of nutrition and oxygen in multicellular aggregates is compromised in direct relation to the aggregates size [37].…”

Section: Resultsmentioning

confidence: 99%

Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis

et al. 2019

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BackgroundAlthough mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis.MethodsAggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-β1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence.ResultsReducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to β-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a β-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue.ConclusionsThis study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1103-y) contains supplementary material, which is available to authorized users.

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“…However, this study also demonstrated failure due to shear at the cartilage–calcium–polyphosphate interface, indicating the importance of a solid contact between different biomaterials applied in two- or multilayered constructs. Stiffer constructs seem superior to softer ones regarding cartilage regeneration in vivo [27,28], with good integration into the defects histologically.…”

Section: Discussionmentioning

confidence: 99%

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs

Bothe

Deubel

Hesse

et al. 2019

IJMS

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Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.

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Interplay between stiffness and degradation of architectured gelatin hydrogels leads to differential modulation of chondrogenesis in vitro and in vivo

Cited by 49 publications

References 49 publications

Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs

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