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Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro.

Kato S, Saito M, Funasaki H, Marumo K - Knee Surg Sports Traumatol Arthrosc (2013)

Bottom Line: A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression.In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation.This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan, soki@jikei.ac.jp.

ABSTRACT

Purpose: Differences in the tissue-specific collagen maturation process between tendon and ligament are still unknown. Collagen cross-link formation is crucial for the collagen maturation process. The aim of this study is to examine collagen maturation processes of anterior cruciate ligament (ACL), medial collateral ligament (MCL), and patellar tendon (PT) in vitro, in order to determine the optimal cell source for tissue engineering of ligament.

Methods: Cells derived from the ACL, MCL, and PT of New Zealand white rabbits were isolated. Each cell type was cultured for up to 4 weeks after reaching confluence. Cell-matrix layers were evaluated and compared for their morphology, collagen cross-links, and gene expression levels of lysine hydroxylase 1 and 2, lysyl oxidase (LOX), tenomodulin, collagen1A1 (Col1A1), and collagen3A1 (Col3A1).

Results: Transmission electron microscopy photomicrographs verified that collagen fibrils were secreted from all three types of fibroblasts. A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression. The gene expression of LOX, which regulates the total amount of enzymatic cross-linking, and the gene expression levels of Col1A1 and Col3A1 were higher in the ACL matrix than in the MCL and PT matrices.

Conclusion: ACL, MCL, and PT cells have distinct collagen maturation processes at the cellular level. In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation. This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

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Gene expression levels of PLOD2/PLOD1 (a), LOX (b), Col1A1 (c), Col3A1 (d), and tenomodulin (e) were determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The expression levels were relative to that of ACL-derived cells. The error bars represent the standard deviation of the mean in cells harvested in triplicate from five separate populations 3 weeks after reaching confluence in culture. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels for each cell type. The gene expression levels of PLOD2/PLOD1 (a) and LOX (b) were consistent with the results of the cross-linking patterns and the total amount of enzymatic cross-linking, respectively. The gene expression levels of Col1A1 (c) and Col3A1 (d) were higher in the ACL matrix than in the MCL and PT matrices. The gene expression level of tenomodulin (e) was significantly higher in the PT matrix than in the ACL and MCL matrices. [*significant difference compared to ACL, †significant difference compared to MCL and ‡significant difference compared to PT (p < 0.05)]
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Fig3: Gene expression levels of PLOD2/PLOD1 (a), LOX (b), Col1A1 (c), Col3A1 (d), and tenomodulin (e) were determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The expression levels were relative to that of ACL-derived cells. The error bars represent the standard deviation of the mean in cells harvested in triplicate from five separate populations 3 weeks after reaching confluence in culture. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels for each cell type. The gene expression levels of PLOD2/PLOD1 (a) and LOX (b) were consistent with the results of the cross-linking patterns and the total amount of enzymatic cross-linking, respectively. The gene expression levels of Col1A1 (c) and Col3A1 (d) were higher in the ACL matrix than in the MCL and PT matrices. The gene expression level of tenomodulin (e) was significantly higher in the PT matrix than in the ACL and MCL matrices. [*significant difference compared to ACL, †significant difference compared to MCL and ‡significant difference compared to PT (p < 0.05)]

Mentions: The PLOD2/PLOD1 ratio, which regulates distinct collagen cross-linking patterns, was significantly higher in the ACL and MCL matrices than in the PT matrix (n.s. and p < 0.05, respectively) (Fig. 3a). The gene expression of LOX and Col1A1 showed higher trend in the ACL matrix than in the MCL and PT matrices (Fig. 3b, c). The expression of the Col3A1 gene was significantly higher in the ACL matrix than in the MCL and PT matrices (n.s. and p < 0.05, respectively) (Fig. 3d). The expression of the tenomodulin gene was significantly higher in the PT matrix than in the ACL and MCL matrices (both p < 0.05) (Fig. 3e).Fig. 3


Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro.

Kato S, Saito M, Funasaki H, Marumo K - Knee Surg Sports Traumatol Arthrosc (2013)

Gene expression levels of PLOD2/PLOD1 (a), LOX (b), Col1A1 (c), Col3A1 (d), and tenomodulin (e) were determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The expression levels were relative to that of ACL-derived cells. The error bars represent the standard deviation of the mean in cells harvested in triplicate from five separate populations 3 weeks after reaching confluence in culture. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels for each cell type. The gene expression levels of PLOD2/PLOD1 (a) and LOX (b) were consistent with the results of the cross-linking patterns and the total amount of enzymatic cross-linking, respectively. The gene expression levels of Col1A1 (c) and Col3A1 (d) were higher in the ACL matrix than in the MCL and PT matrices. The gene expression level of tenomodulin (e) was significantly higher in the PT matrix than in the ACL and MCL matrices. [*significant difference compared to ACL, †significant difference compared to MCL and ‡significant difference compared to PT (p < 0.05)]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Gene expression levels of PLOD2/PLOD1 (a), LOX (b), Col1A1 (c), Col3A1 (d), and tenomodulin (e) were determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The expression levels were relative to that of ACL-derived cells. The error bars represent the standard deviation of the mean in cells harvested in triplicate from five separate populations 3 weeks after reaching confluence in culture. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels for each cell type. The gene expression levels of PLOD2/PLOD1 (a) and LOX (b) were consistent with the results of the cross-linking patterns and the total amount of enzymatic cross-linking, respectively. The gene expression levels of Col1A1 (c) and Col3A1 (d) were higher in the ACL matrix than in the MCL and PT matrices. The gene expression level of tenomodulin (e) was significantly higher in the PT matrix than in the ACL and MCL matrices. [*significant difference compared to ACL, †significant difference compared to MCL and ‡significant difference compared to PT (p < 0.05)]
Mentions: The PLOD2/PLOD1 ratio, which regulates distinct collagen cross-linking patterns, was significantly higher in the ACL and MCL matrices than in the PT matrix (n.s. and p < 0.05, respectively) (Fig. 3a). The gene expression of LOX and Col1A1 showed higher trend in the ACL matrix than in the MCL and PT matrices (Fig. 3b, c). The expression of the Col3A1 gene was significantly higher in the ACL matrix than in the MCL and PT matrices (n.s. and p < 0.05, respectively) (Fig. 3d). The expression of the tenomodulin gene was significantly higher in the PT matrix than in the ACL and MCL matrices (both p < 0.05) (Fig. 3e).Fig. 3

Bottom Line: A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression.In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation.This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan, soki@jikei.ac.jp.

ABSTRACT

Purpose: Differences in the tissue-specific collagen maturation process between tendon and ligament are still unknown. Collagen cross-link formation is crucial for the collagen maturation process. The aim of this study is to examine collagen maturation processes of anterior cruciate ligament (ACL), medial collateral ligament (MCL), and patellar tendon (PT) in vitro, in order to determine the optimal cell source for tissue engineering of ligament.

Methods: Cells derived from the ACL, MCL, and PT of New Zealand white rabbits were isolated. Each cell type was cultured for up to 4 weeks after reaching confluence. Cell-matrix layers were evaluated and compared for their morphology, collagen cross-links, and gene expression levels of lysine hydroxylase 1 and 2, lysyl oxidase (LOX), tenomodulin, collagen1A1 (Col1A1), and collagen3A1 (Col3A1).

Results: Transmission electron microscopy photomicrographs verified that collagen fibrils were secreted from all three types of fibroblasts. A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression. The gene expression of LOX, which regulates the total amount of enzymatic cross-linking, and the gene expression levels of Col1A1 and Col3A1 were higher in the ACL matrix than in the MCL and PT matrices.

Conclusion: ACL, MCL, and PT cells have distinct collagen maturation processes at the cellular level. In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation. This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

Show MeSH