Molecular and phenotypic modulations of primary and immortalized canine chondrocytes in different culture systems

Farooq, Muhammad; Rachakonda, P. Sivaramakrishna; Manning, Kizzie; Palissa, Christiane; Sittinger, Michael; Ringe, Jochen; Schmidt, Michael F. G.

doi:10.1016/j.rvsc.2009.04.008

Cited by 10 publications

(8 citation statements)

References 33 publications

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“…Chondrocytes were isolated as previously described [30]. Briefly, cartilage was diced into 2–3 mm 2 slices and digested in spinner flasks using a cocktail of enzymes consisting of 1 U/ml Collagenase P (Roche Diagnostics, Mannheim, Germany), 330 U/ml Collagenase CLS II (Biochrom, Berlin, Germany) and 30 U/ml Hyaluronidase (Roche Diagnostics, Mannheim, Germany) at 37°C for 16–18 h. The resulting suspension was filtered through a 100 µm cell strainer and chondrocytes were collected by centrifugation at 400 g for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

et al. 2011

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ObjectiveTo elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions.MethodsMature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4.ResultscIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE2 in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent.ConclusionsRegulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.

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Section: Methodsmentioning

confidence: 99%

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

et al. 2011

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“…36 As noted earlier, chondrocytes cultured in 3D scaffolds retain their phenotype, so changes are a result of a biological response and not biased by dedifferentiation. 37,38 However, it is yet to be established whether a micromass culture has any dedifferentiating impact on primary cell populations used in bone tissue engineering. Some studies note a 3D morphology is critical to the retention of the chondrogenic phenotype.…”

Section: Indirect Coculturesmentioning

confidence: 99%

“…Some studies note a 3D morphology is critical to the retention of the chondrogenic phenotype. 38 For example, embryonic bodies derived from embryonic stem cells differentiate better in the chondrogenic medium when cultured in a 3D scaffold as compared to a monolayer culture when subjected to an identical induction. 25 Other studies come to a similar conclusion in their review of endothelial cell-osteoblast interactions.…”

Section: Indirect Coculturesmentioning

confidence: 99%

Coculture Strategies in Bone Tissue Engineering: The Impact of Culture Conditions on Pluripotent Stem Cell Populations

Janardhanan

Wang²,

Fisher³

2012

Tissue Engineering Part B: Reviews

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“…Despite its suitability for implantation, the nondegradable nature of the gel limits its application in tissue-engineering. Alginate, a naturally derived polysaccharide gel, has been successfully shown to support cell retention and the chondrocytic phenotype by maintaining cell shape through encapsulation (Guo et al, 1989;Häuselmann et al, 1994;Rai et al, 2009). On the other hand, its inferior biomechanical properties as well as concerns over its immunogenicity have raised biocompatibility issues (Kulseng et al, 1999).…”

Section: Natural Scaffold Materialsmentioning

confidence: 99%

Conditioning and Scaffolding of Chondrocytes: Smart Steps Towards Osteoarthritis Gene Therapy

Farooq¹,

Lang²,

Sieber³

et al. 2011

Gene Therapy Applications

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Molecular and phenotypic modulations of primary and immortalized canine chondrocytes in different culture systems

Cited by 10 publications

References 33 publications

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

Coculture Strategies in Bone Tissue Engineering: The Impact of Culture Conditions on Pluripotent Stem Cell Populations

Conditioning and Scaffolding of Chondrocytes: Smart Steps Towards Osteoarthritis Gene Therapy

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