Dataset Information

Optimization of TGF-?1-transduced chondrocytes for cartilage regeneration in a 3D printed knee joint model.

ABSTRACT: A cell therapy product of transforming growth factor (TGF)-?1-transduced chondrocytes has been commercialized to treat osteoarthritis of the knee via intra-articular injection. The need for arthroscopic application of the cells to simultaneously treat intra-articular pathologies of knee osteoarthritis is increasingly urgent. The purpose of this study was to optimize TGF-?1-transduced chondrocytes for arthroscopic application. The optimal composition of chondrocytes and thrombin was initially determined by measuring the consolidation time of a diverse ratio of chondrocytes and thrombin mixed with 1 ml of fibrinogen. The consolidation time of the diverse ratio of fibrinogen and atelocollagen mixed with the determined optimal ratio of chondrocytes and thrombin was evaluated. The mixture of the determined optimal ratio of TGF-?1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin was applied to the cartilage defect of the 3D printed knee joint model arthroscopically. The status of the mixture in the defect was then evaluated. Chondrogenic activities of TGF-?1-transduced chondrocytes mixed with atelocollagen were evaluated. The determined ratio of TGF-?1-transduced chondrocytes to thrombin was 8:2 and that of fibrin to atelocollagen was also 8:2. Excellent maintenance of conformation of the mixture of TGF-?1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin in the cartilage defect of the 3D printed knee joint model was observed arthroscopically. Increased chondrogenic activities were observed in the group of TGF-?1-transduced chondrocytes mixed with atelocollagen. TGF-?1-transduced chondrocytes can be applied arthroscopically to treat cartilage defects of the knee at an optimized mixing ratio of atelocollagen, fibrinogen, and thrombin.

SUBMITTER: Ahn J

PROVIDER: S-EPMC6532938 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Optimization of TGF-β1-transduced chondrocytes for cartilage regeneration in a 3D printed knee joint model.

Ahn Jiyong J Kim Seon Ae SA Kim Ki Won KW Oh Joon Hyuck JH Kim Seok Jung SJ

PloS one 20190523 5

A cell therapy product of transforming growth factor (TGF)-β1-transduced chondrocytes has been commercialized to treat osteoarthritis of the knee via intra-articular injection. The need for arthroscopic application of the cells to simultaneously treat intra-articular pathologies of knee osteoarthritis is increasingly urgent. The purpose of this study was to optimize TGF-β1-transduced chondrocytes for arthroscopic application. The optimal composition of chondrocytes and thrombin was initially det ...[more]

PMID: 31120999

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Project description:BackgroundOsteoarthritis (OA) is a common chronic degenerative joint disease. Due to the limited understanding of its complex pathological mechanism, there is currently no effective treatment that can alleviate or even reverse cartilage damage associated with OA. With improvement in public databases, researchers have successfully identified the key factors involved in the occurrence and development of OA through bioinformatics analysis. The aim of this study was to screen for the differentially expressed genes (DEGs) between the normal and OA cartilage through bioinformatics, and validate the function of the TGF-β1/Smad2/3 pathway-related neuron regeneration related protein (NREP) in the articular cartilage.MethodsThe DEGs between the cartilage tissues of OA patients and healthy controls were screened by bioinformatics, and functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression levels of the DEG in human and murine OA cartilage was verified by reverse transcription-quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry (IHC). RT-qPCR, Western-blotting, Cell Counting Kit-8(CCK8) and EdU assays were used to evaluate the effects of knocking down NREP in normal chondrocytes, and the molecular mechanisms were investigated by RT-qPCR, Western blotting and IHC.ResultsIn this study, we identified NREP as a DEG in OA through bioinformatics analysis, and found that NREP was downregulated in the damaged articular cartilage of OA patients and mouse model with surgically-induced OA. In addition, knockdown of NREP in normal chondrocytes reduced their proliferative capacity, which is the pathological basis of OA. At the molecular level, knock-down of NREP inactivated the TGF-β1/Smad2/3 pathway, resulting in the downregulation of the anabolic markers Col2a1 and Sox9, and an increase in the expression of the catabolic markers MMP3 and MMP13.ConclusionNREP plays a key role in the progression of OA by regulating the TGF-β1/Smad2/3 pathway in chondrocytes, and warrants further study as a potential therapeutic target.

Project description:UnlabelledArticular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6-13 cm(2)) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies.SignificanceInjury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to contribute to tissue renewal and homeostasis, the derivation, biological function, and application potential of stem/progenitor cells found in adult human articular cartilage are incompletely understood. This study reports the derivation of a population of cartilage stem/progenitor cells from fully differentiated chondrocytes under specific culture conditions, which have the potential to reassume their chondrocytic phenotype for efficient cartilage regeneration. These findings support the possibility of using in vitro amplified chondrocyte-derived progenitor cells for joint cartilage repair.

Project description:BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Dataset Information

Optimization of TGF-?1-transduced chondrocytes for cartilage regeneration in a 3D printed knee joint model.

Publications

Optimization of TGF-β1-transduced chondrocytes for cartilage regeneration in a 3D printed knee joint model.

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