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Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

Nanduri V, Tattikota SM, T AR, Sriramagiri VR, Kantipudi S, Pande G - Orthop J Sports Med (2014)

Bottom Line: Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties.The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research-Centre for Cellular and Molecular Biology, Hyderabad, India.

ABSTRACT

Background: Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC).

Purpose: To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC.

Study design: Descriptive laboratory study.

Methods: Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.

Results: Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC.

Conclusion: This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage tissue with properties similar to the deep layer of HC in vitro.

Clinical relevance: The HC tissue obtained by the method described can be used to develop an implantable product for the replacement of damaged or malformed AC, especially in younger patients where the lesions are caused by trauma or mechanical stress.

No MeSH data available.


Related in: MedlinePlus

Two-dimensional cultures of chondrocytes grown in either classical medium (CM) or newly defined medium (NDM), at 10× magnification. (A) Passage 0 (P0) cells on day 19; (B) passage 1 (P1) on day 10. Increased ECM formation is seen on P1 cultures grown in NDM compared with CM.
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fig4-2325967114539122: Two-dimensional cultures of chondrocytes grown in either classical medium (CM) or newly defined medium (NDM), at 10× magnification. (A) Passage 0 (P0) cells on day 19; (B) passage 1 (P1) on day 10. Increased ECM formation is seen on P1 cultures grown in NDM compared with CM.

Mentions: Primary chondrocytes from normal LIII of AC were maintained either in CM or NDM for a total period of 21 (P0) or 14 (P1) days. The phase contrast microscopy images of these cells are shown in Figure 4. We clearly noticed increased ECM formation in P1 chondrocytes grown in NDM as compared with CM. To confirm this observation, immunofluorescence of Col I, Col II, Agg, and CD44 was performed, and the results from a representative experiment are shown in Figure 5 (similar observations were seen in 8 different experiments). Col II and Agg were highly expressed, whereas Col I and CD44 were maintained at low levels in P1 chondrocytes grown in NDM as compared with the same grown in CM. Thus, our results reproducibly show that P1 chondrocytes grown in NDM exhibit matrix properties similar to that of LIII in native AC.


Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

Nanduri V, Tattikota SM, T AR, Sriramagiri VR, Kantipudi S, Pande G - Orthop J Sports Med (2014)

Two-dimensional cultures of chondrocytes grown in either classical medium (CM) or newly defined medium (NDM), at 10× magnification. (A) Passage 0 (P0) cells on day 19; (B) passage 1 (P1) on day 10. Increased ECM formation is seen on P1 cultures grown in NDM compared with CM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4555603&req=5

fig4-2325967114539122: Two-dimensional cultures of chondrocytes grown in either classical medium (CM) or newly defined medium (NDM), at 10× magnification. (A) Passage 0 (P0) cells on day 19; (B) passage 1 (P1) on day 10. Increased ECM formation is seen on P1 cultures grown in NDM compared with CM.
Mentions: Primary chondrocytes from normal LIII of AC were maintained either in CM or NDM for a total period of 21 (P0) or 14 (P1) days. The phase contrast microscopy images of these cells are shown in Figure 4. We clearly noticed increased ECM formation in P1 chondrocytes grown in NDM as compared with CM. To confirm this observation, immunofluorescence of Col I, Col II, Agg, and CD44 was performed, and the results from a representative experiment are shown in Figure 5 (similar observations were seen in 8 different experiments). Col II and Agg were highly expressed, whereas Col I and CD44 were maintained at low levels in P1 chondrocytes grown in NDM as compared with the same grown in CM. Thus, our results reproducibly show that P1 chondrocytes grown in NDM exhibit matrix properties similar to that of LIII in native AC.

Bottom Line: Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties.The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research-Centre for Cellular and Molecular Biology, Hyderabad, India.

ABSTRACT

Background: Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC).

Purpose: To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC.

Study design: Descriptive laboratory study.

Methods: Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.

Results: Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC.

Conclusion: This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage tissue with properties similar to the deep layer of HC in vitro.

Clinical relevance: The HC tissue obtained by the method described can be used to develop an implantable product for the replacement of damaged or malformed AC, especially in younger patients where the lesions are caused by trauma or mechanical stress.

No MeSH data available.


Related in: MedlinePlus