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Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue

View Article: PubMed Central - PubMed

ABSTRACT

The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480 MPa over 10 minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage-specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair.

No MeSH data available.


Cultivation of chondrocytes and MSCs onto devitalised cartilage.Cells were cultured for 21 days onto devitalised cartilage in cell culture medium supplemented with ascorbic acid. (A) Chondrogenic differentiation was examined using gene expression analyses of Col I (collagen type I), RUNX (Runt-related transcription factor 2), SOX9 (Sry-related HMG box 9), COMP (cartilage oligomeric matrix protein) and ACAN (aggrecan) after 21 days of cultivation. Data are normalised to the respective controls (AD-MSCs, BM-MSCs and chondrocytes, respectively) cultured in fibrin glue on TCP. All three cell types show significantly increased expression rates of cartilage-specific genes compared to control (mean ± SD, n = 3, *p < 0.05). (B) Histological and immunohistological staining of cells (chondrocytes and MSCs) cultured onto devitalised cartilage to investigated chondrogenic differentiation. H&E staining were performed to get an overview of cultured cells. Heidenhain’s AZAN trichrome staining of cell/devitalised cartilage constructs indicated neocartilage-like ECM synthesised by AD-MSCs, BM-MSCs and chondrocytes. Collagen is stained blue and cell nuclei red. Immunohistochemical staining of collagen type II and aggrecan indicated a differentiation into chondrogenic lineage. Arrows display the crossover from cartilage tissue to cell layer. HHP-treated cartilage tissue where no cells were seeded on shows no sign of viable cells. This indicates that no vital cells could grow out of the cartilage. At the right site controls of antibody staining are provided (low magnification: bar = 100 μm; high magnification: 50 μm).
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f5: Cultivation of chondrocytes and MSCs onto devitalised cartilage.Cells were cultured for 21 days onto devitalised cartilage in cell culture medium supplemented with ascorbic acid. (A) Chondrogenic differentiation was examined using gene expression analyses of Col I (collagen type I), RUNX (Runt-related transcription factor 2), SOX9 (Sry-related HMG box 9), COMP (cartilage oligomeric matrix protein) and ACAN (aggrecan) after 21 days of cultivation. Data are normalised to the respective controls (AD-MSCs, BM-MSCs and chondrocytes, respectively) cultured in fibrin glue on TCP. All three cell types show significantly increased expression rates of cartilage-specific genes compared to control (mean ± SD, n = 3, *p < 0.05). (B) Histological and immunohistological staining of cells (chondrocytes and MSCs) cultured onto devitalised cartilage to investigated chondrogenic differentiation. H&E staining were performed to get an overview of cultured cells. Heidenhain’s AZAN trichrome staining of cell/devitalised cartilage constructs indicated neocartilage-like ECM synthesised by AD-MSCs, BM-MSCs and chondrocytes. Collagen is stained blue and cell nuclei red. Immunohistochemical staining of collagen type II and aggrecan indicated a differentiation into chondrogenic lineage. Arrows display the crossover from cartilage tissue to cell layer. HHP-treated cartilage tissue where no cells were seeded on shows no sign of viable cells. This indicates that no vital cells could grow out of the cartilage. At the right site controls of antibody staining are provided (low magnification: bar = 100 μm; high magnification: 50 μm).

Mentions: For application as a biomaterial, the influence of devitalised cartilage on the differentiation of MSCs has to be considered as well as the potential to redifferentiate chondrocytes which dedifferentiated while expansion in monolayer (Fig. 5A). Gene expression analyses demonstrated that SOX9, an essential transcription factor of chondrogenic differentiation, is upregulated in chondrocytes and AD-MSCs but not for BM-MSCs. However, both AD-MSCs and BM-MSCs cultured on devitalised cartilage showed significantly increased expression of the chondrogenic markers COMP (p = 0.037 (AD-MSC); p = 0.037 (BM-MSCs)) and aggrecan (p = 0.037 (AD-MSC); p = 0.046 (BM-MSCs)) compared to respective controls cultured in fibrin glue on TCP. Using BM-MSCs the collagen type I expression as an indicator of unwanted fibrocartilage formation was not increased. In chondrocytes, COMP expression was significantly increased (p = 0.037), pointing to initial redifferentiation of the cells. Collagen type I expression was also significantly increased in chondrocytes (p = 0.046) and even in AD-MSCs (p = 0.037). The expression of transcription factor RUNX-2 as a hypertrophy marker was not increased. Additionally, the devitalised cartilage without seeded cells were analysed as control. Thereby, Ct-values were similar to the negative control (DEPC-water) indicating an absence of gene expression.


Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue
Cultivation of chondrocytes and MSCs onto devitalised cartilage.Cells were cultured for 21 days onto devitalised cartilage in cell culture medium supplemented with ascorbic acid. (A) Chondrogenic differentiation was examined using gene expression analyses of Col I (collagen type I), RUNX (Runt-related transcription factor 2), SOX9 (Sry-related HMG box 9), COMP (cartilage oligomeric matrix protein) and ACAN (aggrecan) after 21 days of cultivation. Data are normalised to the respective controls (AD-MSCs, BM-MSCs and chondrocytes, respectively) cultured in fibrin glue on TCP. All three cell types show significantly increased expression rates of cartilage-specific genes compared to control (mean ± SD, n = 3, *p < 0.05). (B) Histological and immunohistological staining of cells (chondrocytes and MSCs) cultured onto devitalised cartilage to investigated chondrogenic differentiation. H&E staining were performed to get an overview of cultured cells. Heidenhain’s AZAN trichrome staining of cell/devitalised cartilage constructs indicated neocartilage-like ECM synthesised by AD-MSCs, BM-MSCs and chondrocytes. Collagen is stained blue and cell nuclei red. Immunohistochemical staining of collagen type II and aggrecan indicated a differentiation into chondrogenic lineage. Arrows display the crossover from cartilage tissue to cell layer. HHP-treated cartilage tissue where no cells were seeded on shows no sign of viable cells. This indicates that no vital cells could grow out of the cartilage. At the right site controls of antibody staining are provided (low magnification: bar = 100 μm; high magnification: 50 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037397&req=5

f5: Cultivation of chondrocytes and MSCs onto devitalised cartilage.Cells were cultured for 21 days onto devitalised cartilage in cell culture medium supplemented with ascorbic acid. (A) Chondrogenic differentiation was examined using gene expression analyses of Col I (collagen type I), RUNX (Runt-related transcription factor 2), SOX9 (Sry-related HMG box 9), COMP (cartilage oligomeric matrix protein) and ACAN (aggrecan) after 21 days of cultivation. Data are normalised to the respective controls (AD-MSCs, BM-MSCs and chondrocytes, respectively) cultured in fibrin glue on TCP. All three cell types show significantly increased expression rates of cartilage-specific genes compared to control (mean ± SD, n = 3, *p < 0.05). (B) Histological and immunohistological staining of cells (chondrocytes and MSCs) cultured onto devitalised cartilage to investigated chondrogenic differentiation. H&E staining were performed to get an overview of cultured cells. Heidenhain’s AZAN trichrome staining of cell/devitalised cartilage constructs indicated neocartilage-like ECM synthesised by AD-MSCs, BM-MSCs and chondrocytes. Collagen is stained blue and cell nuclei red. Immunohistochemical staining of collagen type II and aggrecan indicated a differentiation into chondrogenic lineage. Arrows display the crossover from cartilage tissue to cell layer. HHP-treated cartilage tissue where no cells were seeded on shows no sign of viable cells. This indicates that no vital cells could grow out of the cartilage. At the right site controls of antibody staining are provided (low magnification: bar = 100 μm; high magnification: 50 μm).
Mentions: For application as a biomaterial, the influence of devitalised cartilage on the differentiation of MSCs has to be considered as well as the potential to redifferentiate chondrocytes which dedifferentiated while expansion in monolayer (Fig. 5A). Gene expression analyses demonstrated that SOX9, an essential transcription factor of chondrogenic differentiation, is upregulated in chondrocytes and AD-MSCs but not for BM-MSCs. However, both AD-MSCs and BM-MSCs cultured on devitalised cartilage showed significantly increased expression of the chondrogenic markers COMP (p = 0.037 (AD-MSC); p = 0.037 (BM-MSCs)) and aggrecan (p = 0.037 (AD-MSC); p = 0.046 (BM-MSCs)) compared to respective controls cultured in fibrin glue on TCP. Using BM-MSCs the collagen type I expression as an indicator of unwanted fibrocartilage formation was not increased. In chondrocytes, COMP expression was significantly increased (p = 0.037), pointing to initial redifferentiation of the cells. Collagen type I expression was also significantly increased in chondrocytes (p = 0.046) and even in AD-MSCs (p = 0.037). The expression of transcription factor RUNX-2 as a hypertrophy marker was not increased. Additionally, the devitalised cartilage without seeded cells were analysed as control. Thereby, Ct-values were similar to the negative control (DEPC-water) indicating an absence of gene expression.

View Article: PubMed Central - PubMed

ABSTRACT

The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480&thinsp;MPa over 10&thinsp;minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage-specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair.

No MeSH data available.