Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture

Smith, Lachlan J.; Chiaro, Joseph A.; Nerurkar, Nandan L.; Cortés, Daniel H.; Horava, Sarena D.; Hebela, Nader M.; Mauck, Robert L.; Dodge, George R.; Elliott, Dawn M.

doi:10.22203/ecm.v022a22

Cited by 60 publications

(73 citation statements)

References 46 publications

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“…The concentration of IL-1Ra was determined from our own pilot studies and the value is comparable to that used for a similar purpose in another study 56,57 . Levels of LDH and gene expression of MMP13 and ADAMTS4 were measured as described above after seven days of culture.…”

Section: Methodsmentioning

confidence: 89%

Mechanical confinement regulates cartilage matrix formation by chondrocytes

et al. 2017

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Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for 3D culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

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Section: Methodsmentioning

confidence: 89%

Mechanical confinement regulates cartilage matrix formation by chondrocytes

et al. 2017

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“…Confined compression was performed according to previously described methods. 20 The testing system consisted of an acrylic chamber fixed above a porous, stainless steel platen within a testing bath filled with phosphate-buffered saline (PBS). Compression was applied using an impermeable ceramic indenter sized matched to the compression chamber, and attached to a mechanical testing system fitted with a 5-N load cell (Instron 5542, Norwood, MA).…”

Section: Mechanical Evaluation Of Crosslinking Kineticsmentioning

confidence: 99%

“…20 Briefly, NP tissue was excised from four intervertebral discs from each of eight bovine caudal spines purchased from a slaughterhouse according to institutional guidelines. This tissue was then incubated overnight at 37°C in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 2% penicillin/streptomycin/fugizone (PSF).…”

Section: Mechanical Evaluation Of Crosslinking Kineticsmentioning

confidence: 99%

“…The complete media formulation has been published previously. 20 Constructs (n = 3) cultured for 42 days in CM + were qualitatively examined for cell viability using live-dead staining. Constructs (n = 5) cultured for 0, 14, or 42 days in CM…”

Section: Msc Survival Differentiation and Functional Maturationmentioning

confidence: 99%

“…29 Primer sequences for all genes were published previously. 20 Results are presented as mean -standard deviation. Effects of culture time and media condition on measured properties (Young's modulus, GAG and DNA contents, and mRNA expression) were determined using two-way ANOVAs.…”

Section: Msc Survival Differentiation and Functional Maturationmentioning

confidence: 99%

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In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

Smith

Gorth

Showalter

et al. 2014

Tissue Engineering Part A

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Intervertebral disc degeneration is implicated as a major cause of low-back pain. There is a pressing need for new regenerative therapies for disc degeneration that restore native tissue structure and mechanical function. To that end we investigated the therapeutic potential of an injectable, triple-interpenetrating-network hydrogel comprised of dextran, chitosan, and teleostean, for functional regeneration of the nucleus pulposus (NP) of the intervertebral disc in a series of biomechanical, cytotoxicity, and tissue engineering studies. Biomechanical properties were evaluated as a function of gelation time, with the hydrogel reaching *90% of steady-state aggregate modulus within 10 h. Hydrogel mechanical properties evaluated in confined and unconfined compression were comparable to native human NP properties. To confirm containment within the disc under physiological loading, toluidine-blue-labeled hydrogel was injected into human cadaveric spine segments after creation of a nucleotomy defect, and the segments were subjected to 10,000 cycles of loading. Gross analysis demonstrated no implant extrusion, and further, that the hydrogel interdigitated well with native NP. Constructs were next surface-seeded with NP cells and cultured for 14 days, confirming lack of hydrogel cytotoxicity, with the hydrogel maintaining NP cell viability and promoting proliferation. Next, to evaluate the potential of the hydrogel to support cell-mediated matrix production, constructs were seeded with mesenchymal stem cells (MSCs) and cultured under prochondrogenic conditions for up to 42 days. Importantly, the hydrogel maintained MSC viability and promoted proliferation, as evidenced by increasing DNA content with culture duration. MSCs differentiated along a chondrogenic lineage, evidenced by upregulation of aggrecan and collagen II mRNA, and increased GAG and collagen content, and mechanical properties with increasing culture duration. Collectively, these results establish the therapeutic potential of this novel hydrogel for functional regeneration of the NP. Future work will confirm the ability of this hydrogel to normalize the mechanical stability of cadaveric human motion segments, and advance the material toward human translation using preclinical largeanimal models.

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Articular Repair/Regeneration in Healthy and Inflammatory Conditions: From Advanced In Vitro to In Vivo Models

Teixeira

Pereira

Almeida

et al. 2020

Adv Funct Materials

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The burden of chronic inflammatory diseases of joints and the spine is increasing with population ageing and unhealthy lifestyles. Articular cartilage and intervertebral discs (IVDs) are avascular and aneural tissues with abundant extracellular matrix, low cell density, and reduced regenerative capacity after damage or degeneration. The most advanced in vitro and ex vivo cell-/tissue-/biomaterial-based models and technologies to improve physiological mimicry are discussed, focusing on the impact of inflammation on articular repair/regeneration. In addition, in vivo models, developed to study cartilage and IVD repair/regeneration are addressed. While animal models continue to provide crucial mechanistic and preclinical data, more advanced and robust in vitro/ex vivo cartilage and IVD models, with appropriate extracellular matrix cues and allowing for cellular crosstalk, have seen an exponential growth in the last decade. Due to the complexity of articular microenvironments, adequate in vitro/ex vivo/in vivo models are essential to study the molecular mechanisms underlying articular diseases and develop new therapies for repair/regeneration.

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Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture

Cited by 60 publications

References 46 publications

Mechanical confinement regulates cartilage matrix formation by chondrocytes

Mechanical confinement regulates cartilage matrix formation by chondrocytes

In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

Articular Repair/Regeneration in Healthy and Inflammatory Conditions: From Advanced In Vitro to In Vivo Models

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