Effect of High Hydrostatic Pressure on Human Trabecular Bone Regarding Cell Death and Matrix Integrity

Abstract

The reconstruction of critical size bone defects is still clinically challenging. Even though the transplantation of autologous bone is used as gold standard, this therapy is accompanied by donor site morbidities as well as tissue limitations. The alternatively used allografts, which are devitalized due to thermal, chemical or physical processing, often lose their matrix integrity and have diminished biomechanical properties. High Hydrostatic Pressure (HHP) may represent a gentle alternative to already existing methods since HHP treated human osteoblasts undergo cell death and HHP treated bone cylinders maintain their mechanical properties. The aim of this study was to determine the biological effects caused by HHP treatment regarding protein/matrix integrity and type of cell death in trabecular bone cylinders. Therefore, different pressure protocols (250 and 300 MPa for 10, 20 and 30 min) and end point analysis such as quantification of DNA-fragmentation, gene expression, SDS-PAGE, FESEM analysis and histological staining were performed. While both protein and matrix integrity was preserved, molecular biological methods showed an apoptotic differentiation of cell death for lower pressures and shorter applications (250 MPa for 10 and 20 min) and necrotic differentiation for higher pressures and longer applications (300 MPa for 30 min). This study serves as a basis for further investigation as it shows that HHP successfully devitalizes trabecular bone cylinders.

BMP-2 Long-Term Stimulation of Human Pre-Osteoblasts Induces Osteogenic Differentiation and Promotes Transdifferentiation and Bone Remodeling Processes

“…( Figure 4 b). Additionally, the analysis of FAS , FADD , and CASP8 , genes involved in apoptosis [ 30 ] did not show any significant influence of BMP-2 exposure on these processes ( Figure 4 c).…”

Systematic enhancement of microbial decontamination efficiency in bone graft processing by means of high hydrostatic pressure using Escherichia coli as a model organism

“…[30][31][32][33] This study aims to prove HHP as an all-encompassing and simplified method in tissue preparation, providing both decontamination and, as shown before, devitalization of bone. 35,47 Considering the few other studies on the major hurdles, as suggested by the varying success rates. 18,37,41 From a biomechanical point of view, an application of up to 600 MPa would be acceptable.…”

“…29,35 Since apoptotic devitalization is desired in terms of immunological host cell reactions, the selection of pressure levels below 300 MPa is preferable. 20,47 This led to the attempt to optimize the effectiveness of HHP treatment using moderate pressure levels slightly below and slightly above the designated pressure level. Our present study shows that HHP reduced cfu of E. coli despite only using moderate pressure levels of 250 and 350 MPa.…”

Osteogenic differentiation of mesenchymal stem cells cultured on allogenic trabecular bone grafts treated with high hydrostatic pressure

“…For HHP it was already shown that pressures up to 600 MPa can successfully disinfect bone contaminated with bone specific pathogens 14,45 . However, those pressure levels lead to necrosis of cells, which in turn results in the release of damage associated molecular patterns resulting in the activation of the immune system of the host 20,46 . Therefore, it would be optimal, to develop a process protocol to which cells react primarily apoptotic and a decontaminating effect can be achieved.…”

“…Following an incubation of 5 min, samples were treated as described before and according to manufacturer's manual. 20 RNA was then resuspended in Tris-EDTA-buffer (TE-buffer, pH 7.5) and RNA con- In addition to flow cytometry analysis the supernatants of MSC cultivation were also used for an enzyme linked immunosorbant assay (ELISA) that detects the type I C-terminal collagen propeptide (CICP), which is a by-product of collagen synthesis. Collagen I is secreted as procollagen, which is extracellularly cut at the carboxy-terminal and amino-terminal end into collagen I.…”