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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.


Histological and immunohistochemical staining of HHP-treated cartilage and untreated controls.Stainings were performed 14 days after HHP treatment. The first panel shows staining with haematoxylin and eosin (H&E). Using H&E cell nuclei were stained blue and cell cytoplasm as well as extracellular proteins were stained bright red. Second panel displays the cartilage-specific Heidenhain’s AZAN trichrome staining whereby collagen is stained blue and cell nuclei red. The panels below demonstrated the immunohistological stainings with collagen type II and aggrecan antibodies fluorescing in green. Counterstaining was performed with Hoechst 33342 visualising cell nuclei in blue. Alterations in ECM staining between HHP-treated and untreated cartilage could not be detected (low magnification: bar = 100 μm; high magnification: 50 μm).
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f3: Histological and immunohistochemical staining of HHP-treated cartilage and untreated controls.Stainings were performed 14 days after HHP treatment. The first panel shows staining with haematoxylin and eosin (H&E). Using H&E cell nuclei were stained blue and cell cytoplasm as well as extracellular proteins were stained bright red. Second panel displays the cartilage-specific Heidenhain’s AZAN trichrome staining whereby collagen is stained blue and cell nuclei red. The panels below demonstrated the immunohistological stainings with collagen type II and aggrecan antibodies fluorescing in green. Counterstaining was performed with Hoechst 33342 visualising cell nuclei in blue. Alterations in ECM staining between HHP-treated and untreated cartilage could not be detected (low magnification: bar = 100 μm; high magnification: 50 μm).

Mentions: Alterations in the structure of cartilage extracellular matrix (ECM) were investigated using histological and immunohistochemical staining as well as FESEM analyses. For an overview of cell morphology and ECM haematoxylin and eosin (H&E) as well as Heidenhain’s AZAN trichrome staining were performed (Fig. 3). The histological stainings exhibited intact cartilage tissue in both pressure-loaded and control samples without any lesions or destructions. The staining and dyability of extracellular proteins remained unchanged. Immunohistochemical staining of the prominent ECM components were conducted to observe shifts in protein conformation. Figure 3 shows that collagen type II and aggrecan could be detected even after HHP treatment. However, the cell nuclei stained using Hoechst 33342 shined brighter in untreated controls compared to cell nuclei of HHP-treated tissue indicative for a successful devitalisation.


Devitalisation of human cartilage by high hydrostatic pressure treatment: Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue
Histological and immunohistochemical staining of HHP-treated cartilage and untreated controls.Stainings were performed 14 days after HHP treatment. The first panel shows staining with haematoxylin and eosin (H&E). Using H&E cell nuclei were stained blue and cell cytoplasm as well as extracellular proteins were stained bright red. Second panel displays the cartilage-specific Heidenhain’s AZAN trichrome staining whereby collagen is stained blue and cell nuclei red. The panels below demonstrated the immunohistological stainings with collagen type II and aggrecan antibodies fluorescing in green. Counterstaining was performed with Hoechst 33342 visualising cell nuclei in blue. Alterations in ECM staining between HHP-treated and untreated cartilage could not be detected (low magnification: bar = 100 μm; high magnification: 50 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Histological and immunohistochemical staining of HHP-treated cartilage and untreated controls.Stainings were performed 14 days after HHP treatment. The first panel shows staining with haematoxylin and eosin (H&E). Using H&E cell nuclei were stained blue and cell cytoplasm as well as extracellular proteins were stained bright red. Second panel displays the cartilage-specific Heidenhain’s AZAN trichrome staining whereby collagen is stained blue and cell nuclei red. The panels below demonstrated the immunohistological stainings with collagen type II and aggrecan antibodies fluorescing in green. Counterstaining was performed with Hoechst 33342 visualising cell nuclei in blue. Alterations in ECM staining between HHP-treated and untreated cartilage could not be detected (low magnification: bar = 100 μm; high magnification: 50 μm).
Mentions: Alterations in the structure of cartilage extracellular matrix (ECM) were investigated using histological and immunohistochemical staining as well as FESEM analyses. For an overview of cell morphology and ECM haematoxylin and eosin (H&E) as well as Heidenhain’s AZAN trichrome staining were performed (Fig. 3). The histological stainings exhibited intact cartilage tissue in both pressure-loaded and control samples without any lesions or destructions. The staining and dyability of extracellular proteins remained unchanged. Immunohistochemical staining of the prominent ECM components were conducted to observe shifts in protein conformation. Figure 3 shows that collagen type II and aggrecan could be detected even after HHP treatment. However, the cell nuclei stained using Hoechst 33342 shined brighter in untreated controls compared to cell nuclei of HHP-treated tissue indicative for a successful devitalisation.

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.