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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.

No MeSH data available.


Investigation of cell viability after HHP treatment.(A) After tissue preparation, HHP treatment and subsequent digestion, trypan blue exclusion test indicated no viable chondrocytes in HHP-treated cartilage in contrast to untreated controls (n = 5, mean ± SD, *p < 0.05). (B,C) Live/dead staining of the cartilage matrix was analysed by confocal laser scanning microscopy and showed green fluorescent, viable cells in untreated controls compared to HHP-treated samples which displayed only red fluorescent, dead cells (white arrow).
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f1: Investigation of cell viability after HHP treatment.(A) After tissue preparation, HHP treatment and subsequent digestion, trypan blue exclusion test indicated no viable chondrocytes in HHP-treated cartilage in contrast to untreated controls (n = 5, mean ± SD, *p < 0.05). (B,C) Live/dead staining of the cartilage matrix was analysed by confocal laser scanning microscopy and showed green fluorescent, viable cells in untreated controls compared to HHP-treated samples which displayed only red fluorescent, dead cells (white arrow).

Mentions: After enzymatic digestion of HHP-treated and untreated cartilage tissue, the trypan blue exclusion test indicated cell viability of 75% ± 25% for untreated controls (Fig. 1A). However, pressure-loaded samples contained no viable cells (cell viability: 0%). Afterwards, digested cartilage samples were cultured for seven days in complete cell culture medium. Untreated controls contained adherent and viable cells which were determined by live/dead staining. Whereas untreated controls showed a high number of viable (green fluorescent) cells after cultivation, for HHP-treated cartilage no living cells were detected (data not shown).


Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue
Investigation of cell viability after HHP treatment.(A) After tissue preparation, HHP treatment and subsequent digestion, trypan blue exclusion test indicated no viable chondrocytes in HHP-treated cartilage in contrast to untreated controls (n = 5, mean ± SD, *p < 0.05). (B,C) Live/dead staining of the cartilage matrix was analysed by confocal laser scanning microscopy and showed green fluorescent, viable cells in untreated controls compared to HHP-treated samples which displayed only red fluorescent, dead cells (white arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Investigation of cell viability after HHP treatment.(A) After tissue preparation, HHP treatment and subsequent digestion, trypan blue exclusion test indicated no viable chondrocytes in HHP-treated cartilage in contrast to untreated controls (n = 5, mean ± SD, *p < 0.05). (B,C) Live/dead staining of the cartilage matrix was analysed by confocal laser scanning microscopy and showed green fluorescent, viable cells in untreated controls compared to HHP-treated samples which displayed only red fluorescent, dead cells (white arrow).
Mentions: After enzymatic digestion of HHP-treated and untreated cartilage tissue, the trypan blue exclusion test indicated cell viability of 75% ± 25% for untreated controls (Fig. 1A). However, pressure-loaded samples contained no viable cells (cell viability: 0%). Afterwards, digested cartilage samples were cultured for seven days in complete cell culture medium. Untreated controls contained adherent and viable cells which were determined by live/dead staining. Whereas untreated controls showed a high number of viable (green fluorescent) cells after cultivation, for HHP-treated cartilage no living cells were detected (data not shown).

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.