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Bone Tissueengineering

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Michael B Keogh
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Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-beta(1)) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-beta(1) (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.
Michael B Keogh
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Introduction Flow perfusion bioreactors may be used to provide mechanostimulatory effects to cells and to improve cell distribution within a biomaterial. This study assessed the use of a flow perfusion bioreactor to improve cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold. Materials and Methods CG scaffolds were fabricated by a lyophilisation technique and cut to size (12mmØ) as previously reported 1 . 4x10 6 hFOB 1.19 pre-osteoblast cells were seeded onto each scaffold and pre-cultured under standard conditions for 6 days. Bioreactor groups were exposed to 3 x 1 hr bouts of steady flow (1ml/min) with each bout being followed by 7 hrs of no flow (to prevent cellular desensitization) 2 for one day. Constructs were then cultured under osteogenic conditions for a further 28 days. Cellular distribution, mineralization, gene and protein expression of a number of bone formation markers and mechanical properties of the constructs were analyzed using techniques such as Haematoxylin and Eosin and alizarin red staining, real-time PCR and compression testing. Results Both metabolic viability and cell number appeared similar between bioreactor and static culture groups. Histologically, cells in the constructs following bioreactor culture appeared in clusters which increased in distribution over time. In comparison, the static groups demonstrated a more uniform distribution, however, cells tended to aggregate on the periphery causing encapsulation. Osteogenesis was supported in both static & bioreactor groups. The early bone formation marker alkaline phosphatase gave a 3 fold increase in bioreactor groups at 21 days. The mid stage markers osteopontin and osteonectin showed similar trends with bioreactor groups providing higher expression levels earlier than the static groups. The late stage marker of bone formation, osteocalcin gave a 1.25 fold increase at 21 days. A 2 fold increase in alizarin red mineralisation was found in static groups at 28 days over bioreactor groups, which was due to the encapsulation effect. No difference was observed in mechanical strength between static or bioreactor groups. Fig.1. Early and late stage osteogenic gene expression; static versus bioreactor Discussion and Conclusions Flow perfusion bioreactors have been shown to stimulate osteoblasts by mechanoregulation 1 . We find that the bioreactor produced a more mature osteogenic state than static culture as well as discouraging peripheral encapsulation. This may be useful for in vitro applications as the presence of a capsule restricts nutrient diffusion and waste removal from a cell seeded construct. There was also no detrimental effect on the mechanical properties of the scaffold.
In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long-term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre-cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24-h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell-encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls.