The development and validation of a hydrostatic pressure bioreactor for applications in bone tissue engineering

Abstract

Current orthopaedics treatments of bone defects often involve the use of implanted fixatives and/or autograft procedures to restore function to the afflicted area following injury. Fixatives and implants are usually temporary solutions, since they are intrinsically prone to failure. In addition to this, replacing implants involve expensive and invasive procedures that cause great hardship to patients. Whilst autografts can provide an excellent outcome in healing of the initial injury site, donor site morbidity from the autologous bone graft can lead to complications such as infection, chronic pain and an abnormal walking gait. Bone tissue engineering is a field of science aiming to address these limitations by providing in vitro manufactured bone to replace autografts, and also limit the use of temporary fixatives. Hydrostatic force bioreactors are currently being developed within this field to attempt improve the outcome of the tissue engineered bone by mimicking the forces typically experienced by cells in the native bone niche. Based on this principle, it is hoped that such systems will aid the translation of research in bone tissue engineering from the lab to the clinic.

This research aims to investigate and validate the use of a hydrostatic force bioreactor for improving the outcome of in vitro manufactured bone using a clinically relative strategy employing human mesenchymal stem cells seeded in 3D scaffolds. The research first describes a validation process to determine the initial response of cells to hydrostatic pressure in monolayer cultures. The outcome of this study indicated that mechanical responsiveness in cells can vary according to cell phenotype and the integrity of the f-actin cytoskeleton. Next it was demonstrated that hydrostatic pressure can improve the outcome of in vitro bone formation by MG-63 human osteoblast like cells, validating the bioreactor as a potential preconditioning platform. Following this, a model of bone formation in hMSCs/collagen scaffolds was described, whereby a predictable rate of bone
formation was determined by adjusting cellular distribution and protein concentration in
collagen type-1 scaffolds.

Finally, an organotypic fracture repair model was established using explanted
embryonic chick femurs to test the hypothesis that hydrostatic preconditioning of
hMSC/collagen hydrogels can improve the outcome of fracture repair. The results of this
study showed that bioreactor stimulation could enhance the outcome of repair using a
combination of undifferentiated hMSC/collagen type-1 scaffolds, and global mechanical
signalling (stimulation of entire femur constructs). It was then shown that hydrostatic
preconditioning of hMSC seeded hydrogels prior to implantation did not increase the rate
of in vitro bone formation. Following implantation of the hydrogels into the fracture
repair model, it was demonstrated that highly mineralised preconditioned implants
actually inhibited the fracture repair process. In addition to this, it was shown that
preconditioned implants with a lower level of mineralisation allowed invasion and bone
formation by native cells from the host tissue. Collectively, the results implied that the
outcome of repair using this model relied on three main factors: the presence of global
hydrostatic stimulation; the lineage commitment of hMSCs in collagen scaffolds at the
time of implantation; and the permeability and cell invasion capacity of the implant.