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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

(A) Anterior view of the knee exposing the diarthordial joint. (B) The 4 layers of the hyaline cartilage. Layer I (LI), or the superficial layer, consists of round-shaped cells, thin collagen fibrils, and low PG content; layer II (LII), or the intermediate layer, has moderate cell number and thick and thin collagen fibrils; layer III (LIII), or the deep layer, shows sparse cell population with elongated chondrocytes, thick and long collagen fibers, and a high proteoglycan content; layer IV (LIV), or the calcified layer, contains rich concentration or calcium salts, low proteoglycan composition, and less protein synthesis.
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fig1-2325967114539122: (A) Anterior view of the knee exposing the diarthordial joint. (B) The 4 layers of the hyaline cartilage. Layer I (LI), or the superficial layer, consists of round-shaped cells, thin collagen fibrils, and low PG content; layer II (LII), or the intermediate layer, has moderate cell number and thick and thin collagen fibrils; layer III (LIII), or the deep layer, shows sparse cell population with elongated chondrocytes, thick and long collagen fibers, and a high proteoglycan content; layer IV (LIV), or the calcified layer, contains rich concentration or calcium salts, low proteoglycan composition, and less protein synthesis.

Mentions: Articular cartilage (AC) is a form of hyaline cartilage (HC) primarily composed of extracellular matrix (ECM), rich in collagen II and proteolglycans (PG)—especially aggrecan. It has a very limited regenerative capacity due to lack of blood vessels and low mitotic activity in the small number of cells that it contains (only 5% of the total AC tissue). AC, which is located in the diarthrodial joint, is a high mechanical stress–bearing tissue and therefore, structurally it represents the uniqueness of HC organization.8,9,24,26 It is composed of 4 different layers (Figure 1) that differ in their protein content, proteoglycan composition, thickness of the collagen network, and cellular content.3,9,16,26,30 Among all the layers, the deep layer (layer III [LIII]) of the native AC is the most stress resistant because it comprises thick collagen fibrils, high PG content, and decreased water concentration.16,26,30 This layered structural organization of AC gets disturbed to variable degrees because of excessive mechanical stress caused by sports-related trauma or other day-to-day activities, resulting in osteoarthritis.5,6,11,15,34,38 The International Cartilage Repair Society has prescribed specific “grades of degradation” of AC, according to which when more than 50% thickness of LIII is degraded, AC tissue cannot be repaired without surgical intervention.7,23 For management of these conditions, various cell-based techniques such as autologous chondrocyte implantation, matrix-assisted chondrocyte implantation, autologous mesenchymal stem cell transplantation, and microfracture are used to ameliorate the pain and arrest the degradation of the tissue.2,3,15,17,37 New strategies are being developed for making scaffold-based tissue-engineered materials using synthetic and natural materials,1 biomolecules, growth factors, ECM,10 and genetically modified cells21 that would help in repairing the degraded AC.13,23,25 Based on the evidence,10 we selected the collagen gel–based Culture Matrix system (Invitrogen) for growing the 3-dimensional (3D) cultures of AC. The main consideration for choosing this system was that the collagen is a natural biomaterial highly biocompatible and absorbable in the tissue.


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)

(A) Anterior view of the knee exposing the diarthordial joint. (B) The 4 layers of the hyaline cartilage. Layer I (LI), or the superficial layer, consists of round-shaped cells, thin collagen fibrils, and low PG content; layer II (LII), or the intermediate layer, has moderate cell number and thick and thin collagen fibrils; layer III (LIII), or the deep layer, shows sparse cell population with elongated chondrocytes, thick and long collagen fibers, and a high proteoglycan content; layer IV (LIV), or the calcified layer, contains rich concentration or calcium salts, low proteoglycan composition, and less protein synthesis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig1-2325967114539122: (A) Anterior view of the knee exposing the diarthordial joint. (B) The 4 layers of the hyaline cartilage. Layer I (LI), or the superficial layer, consists of round-shaped cells, thin collagen fibrils, and low PG content; layer II (LII), or the intermediate layer, has moderate cell number and thick and thin collagen fibrils; layer III (LIII), or the deep layer, shows sparse cell population with elongated chondrocytes, thick and long collagen fibers, and a high proteoglycan content; layer IV (LIV), or the calcified layer, contains rich concentration or calcium salts, low proteoglycan composition, and less protein synthesis.
Mentions: Articular cartilage (AC) is a form of hyaline cartilage (HC) primarily composed of extracellular matrix (ECM), rich in collagen II and proteolglycans (PG)—especially aggrecan. It has a very limited regenerative capacity due to lack of blood vessels and low mitotic activity in the small number of cells that it contains (only 5% of the total AC tissue). AC, which is located in the diarthrodial joint, is a high mechanical stress–bearing tissue and therefore, structurally it represents the uniqueness of HC organization.8,9,24,26 It is composed of 4 different layers (Figure 1) that differ in their protein content, proteoglycan composition, thickness of the collagen network, and cellular content.3,9,16,26,30 Among all the layers, the deep layer (layer III [LIII]) of the native AC is the most stress resistant because it comprises thick collagen fibrils, high PG content, and decreased water concentration.16,26,30 This layered structural organization of AC gets disturbed to variable degrees because of excessive mechanical stress caused by sports-related trauma or other day-to-day activities, resulting in osteoarthritis.5,6,11,15,34,38 The International Cartilage Repair Society has prescribed specific “grades of degradation” of AC, according to which when more than 50% thickness of LIII is degraded, AC tissue cannot be repaired without surgical intervention.7,23 For management of these conditions, various cell-based techniques such as autologous chondrocyte implantation, matrix-assisted chondrocyte implantation, autologous mesenchymal stem cell transplantation, and microfracture are used to ameliorate the pain and arrest the degradation of the tissue.2,3,15,17,37 New strategies are being developed for making scaffold-based tissue-engineered materials using synthetic and natural materials,1 biomolecules, growth factors, ECM,10 and genetically modified cells21 that would help in repairing the degraded AC.13,23,25 Based on the evidence,10 we selected the collagen gel–based Culture Matrix system (Invitrogen) for growing the 3-dimensional (3D) cultures of AC. The main consideration for choosing this system was that the collagen is a natural biomaterial highly biocompatible and absorbable in the tissue.

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