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

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.

Figure 1. 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).

Figure 2. 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).

Figure 3. 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).

Figure 4. FESEM images of untreated and HHP-treated cartilage.

High pressure treatment did not affect the ECM structure of hyaline cartilage (upper panel: bar = 20 μm; lower panel: bar = 5 μm).

Figure 5. 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).

Figure 6. High hydrostatic pressure device HDR-100 for devitalisation of cartilage tissue.

Samples were placed in the hermetically sealed pressure chamber and treated for 10 min at 480 MPa.