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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

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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.

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Ratio of DNA content related to sample weight of HHP-treated cartilage and untreated controls.After 21 days of cultivation, the ratio was significantly decreased for HHP-treated cartilage compared to untreated controls. After HHP treatment, no sign of proliferation and viable cells could be detected within cartilage matrix (n = 5, mean ± SD, *p < 0.05).
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f2: Ratio of DNA content related to sample weight of HHP-treated cartilage and untreated controls.After 21 days of cultivation, the ratio was significantly decreased for HHP-treated cartilage compared to untreated controls. After HHP treatment, no sign of proliferation and viable cells could be detected within cartilage matrix (n = 5, mean ± SD, *p < 0.05).

Mentions: HHP-induced cell inactivation is accompanied by the degradation of DNA. The total DNA amount was quantified and normalised to the respective sample weight (Fig. 2). Immediately after HHP treatment, control and treated samples presented similar ratios of DNA/sample weight. Vital cartilage (untreated controls) showed a high DNA/sample weight ratio, which was constant up to 21 days of cultivation, whereas the ratio of HHP-treated cartilage was significantly decreased after 21 days (p = 0.029) compared to the control.


Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue
Ratio of DNA content related to sample weight of HHP-treated cartilage and untreated controls.After 21 days of cultivation, the ratio was significantly decreased for HHP-treated cartilage compared to untreated controls. After HHP treatment, no sign of proliferation and viable cells could be detected within cartilage matrix (n = 5, mean ± SD, *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Ratio of DNA content related to sample weight of HHP-treated cartilage and untreated controls.After 21 days of cultivation, the ratio was significantly decreased for HHP-treated cartilage compared to untreated controls. After HHP treatment, no sign of proliferation and viable cells could be detected within cartilage matrix (n = 5, mean ± SD, *p < 0.05).
Mentions: HHP-induced cell inactivation is accompanied by the degradation of DNA. The total DNA amount was quantified and normalised to the respective sample weight (Fig. 2). Immediately after HHP treatment, control and treated samples presented similar ratios of DNA/sample weight. Vital cartilage (untreated controls) showed a high DNA/sample weight ratio, which was constant up to 21 days of cultivation, whereas the ratio of HHP-treated cartilage was significantly decreased after 21 days (p = 0.029) compared to the control.

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.


Related in: MedlinePlus