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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

RT-PCR products for the respective markers as indicated in each row obtained from 2D and 3D cultures grown in classical medium (CM) or newly defined medium (NDM). GAPDH indicates the loading control.
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fig9-2325967114539122: RT-PCR products for the respective markers as indicated in each row obtained from 2D and 3D cultures grown in classical medium (CM) or newly defined medium (NDM). GAPDH indicates the loading control.

Mentions: A comparative study of native AC and cells obtained from 2D/3D AC cultures was done by RT-PCR. Equal amounts of total RNA (0.8 ng) was used for estimating GAPDH (loading control), Col II, Agg, Col I, and CD44 expression levels. Eight independent experiments were performed, for which a representative experiment is shown in Figure 9. High levels of expression of Col II and Agg genetic markers were observed in NDM-grown 2D and 3D cultures, similar to that of the native AC, compared with that of CM-grown 2D and 3D cultures. Expression of Col I was seen at higher levels in samples grown in CM, whereas samples grown in NDM showed minimal expression, thus maintaining the LIII property. Expression of CD44 is almost negligible throughout, indicating that the native HC-collected 2D and 3D cultures are unaffected and are completely healthy.


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)

RT-PCR products for the respective markers as indicated in each row obtained from 2D and 3D cultures grown in classical medium (CM) or newly defined medium (NDM). GAPDH indicates the loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig9-2325967114539122: RT-PCR products for the respective markers as indicated in each row obtained from 2D and 3D cultures grown in classical medium (CM) or newly defined medium (NDM). GAPDH indicates the loading control.
Mentions: A comparative study of native AC and cells obtained from 2D/3D AC cultures was done by RT-PCR. Equal amounts of total RNA (0.8 ng) was used for estimating GAPDH (loading control), Col II, Agg, Col I, and CD44 expression levels. Eight independent experiments were performed, for which a representative experiment is shown in Figure 9. High levels of expression of Col II and Agg genetic markers were observed in NDM-grown 2D and 3D cultures, similar to that of the native AC, compared with that of CM-grown 2D and 3D cultures. Expression of Col I was seen at higher levels in samples grown in CM, whereas samples grown in NDM showed minimal expression, thus maintaining the LIII property. Expression of CD44 is almost negligible throughout, indicating that the native HC-collected 2D and 3D cultures are unaffected and are completely healthy.

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