Fergal J. O’Brien

Microcrack accumulation at different intervals during fatigue testing of compact bone

Fatigue damage in bone occurs in the form of microcracks. This microdamage contributes to the formation of stress fractures and acts as a stimulus for bone remodelling. A technique has been developed, which allows microcrack growth to be monitored during the course of a fatigue test by the application of a series of fluorescent chelating agents. Specimens were taken from bovine tibiae and fatigue tested in cyclic compression at a stress range of 80MPa. The specimens were stained before testing with alizarin and up to three other chelating agents were applied during testing to label microcracks formed at different times. Microcracks initiated in interstitial bone in the early part of a specimens life. Further accumulation of microcracks is then suppressed until the period late in the specimens life. Microcracks were found to be longer in the longitudinal than in the transverse direction. Only a small proportion of cracks are actively propagating; these are longer than non-propagating cracks. These results support the concept of a microstructural barrier effect existing in bone, whereby cracks initiate easily but slow down or stop at barriers such as cement lines.

Mesenchymal stem cell fate is regulated by the composition and mechanical properties of Collagen-Glycosaminoglycan scaffolds

In stem cell biology, focus has recently turned to the influence of the intrinsic properties of the extracellular matrix (ECM), such as structural, composition and elasticity, on stem cell differentiation. Utilising collagen-glycosaminoglycan (CG) scaffolds as an analogue of the ECM, this study set out to determine the effect of scaffold stiffness and composition on naive mesenchymal stem cell (MSC) differentiation in the absence of differentiation supplements. Dehydrothermal (DHT) and 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) crosslinking treatments were used to produce three homogeneous CG scaffolds with the same composition but different stiffness values: 0.5, 1 and 1.5 kPa. In addition, the effect of scaffold composition on MSC differentiation was investigated by utilising two glycosaminoglycan (GAG) types: chondroitin sulphate (CS) and hyaluronic acid (HyA). Results demonstrated that scaffolds with the lowest stiffness (0.5 kPa) facilitated a significant up-regulation in SOX9 expression indicating that MSCs are directed towards a chondrogenic lineage in more compliant scaffolds. In contrast, the greatest level of RUNX2 expression was found in the stiffest scaffolds (1.5 kPa) indicating that MSCs are directed towards an osteogenic lineage in stiffer scaffolds. Furthermore, results demonstrated that the level of up-regulation of SOX9 was higher within the CHyA scaffolds in comparison to the CCS scaffolds indicating that hyaluronic acid further influences chondrogenic differentiation. In contrast, enhanced RUNX2 expression was observed in the CCS scaffolds in comparison to the CHyA scaffolds suggesting an osteogenic influence of chondroitin sulphate on MSC differentiation. In summary, this study demonstrates that, even in the absence of differentiation supplements, scaffold stiffness can direct the fate of MSCs, an effect that is further enhanced by the GAG type used within the CG scaffolds. These results have significant implications for the therapeutic uses of stem cells and enhance our understanding of the physical effects of the in vivo microenvironment on stem cell behaviour.

The effects of collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering.

In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 degrees C for 24 h and [2] 150 degrees C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 degrees C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 degrees C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering.

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.

An improved labelling technique for monitoring microcrack growth in compact bone.

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells.

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-beta3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5+/-0.21%w/w vs. 0.94+/-0.03%w/w) and annulus (1.09+/-0.09%w/w vs. 0.59+/-0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.

Addition of hyaluronic acid improves cellular infiltration and promotes early-stage chondrogenesis in a collagen-based scaffold for cartilage tissue engineering.

The response of mesenchymal stem cells (MSCs) to a matrix largely depends on the composition as well as the extrinsic mechanical and morphological properties of the substrate to which they adhere to. Collagen-glycosaminoglycan (CG) scaffolds have been extensively used in a range of tissue engineering applications with great success. This is due in part to the presence of the glycosaminoglycans (GAGs) in complementing the biofunctionality of collagen. In this context, the overall goal of this study was to investigate the effect of two GAG types: chondroitin sulphate (CS) and hyaluronic acid (HyA) on the mechanical and morphological characteristics of collagen-based scaffolds and subsequently on the differentiation of rat MSCs in vitro. Morphological characterisation revealed that the incorporation of HyA resulted in a significant reduction in scaffold mean pore size (93.9 μm) relative to collagen-CS (CCS) scaffolds (136.2 μm). In addition, the collagen-HyA (CHyA) scaffolds exhibited greater levels of MSC infiltration in comparison to the CCS scaffolds. Moreover, these CHyA scaffolds showed significant acceleration of early stage gene expression of SOX-9 (approximately 60-fold higher, p<0.01) and collagen type II (approximately 35-fold higher, p<0.01) as well as cartilage matrix production (7-fold higher sGAG content) in comparison to CCS scaffolds by day 14. Combining their ability to stimulate MSC migration and chondrogenesis in vitro, these CHyA scaffolds show great potential as appropriate matrices for promoting cartilage tissue repair.

Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen–GAG scaffold

Anchorage-dependent cells respond to the mechanical and physical properties of biomaterials. One such cue is the mechanical stiffness of a material. We compared the osteogenic potential of collagen-glycosaminoglycan (CG) scaffolds with varying stiffness for up to 6 weeks in culture. The mechanical stiffness of CG scaffolds were varied by cross-linking by physical (dehydrothermal (DHT)) and chemical (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) and glutaraldehyde (GLUT)) methods. The results showed that all CG substrates allowed cellular attachment, infiltration and osteogenic differentiation. CG scaffolds treated with EDAC and GLUT were mechanically stiffer, retained their original scaffold structure and resisted cellular contraction. Consequently, they facilitated a 2-fold greater cell number, probably due to the pore architecture being maintained, allowing improved diffusion of nutrients. On the other hand, the less stiff substrates cross-linked with DHT allowed increased cell-mediated scaffold contraction, contracting by 70% following 6 weeks (P < 0.01) of culture. This reduction in scaffold area resulted in cells reaching the centre of the scaffold quicker up to 4 weeks; however, at 6 weeks all scaffolds showed similar levels of cellular infiltration, with higher cell numbers found on the stiffer EDAC- and GLUT-treated scaffolds. Analysis of osteogenesis showed that scaffolds cross-linked with DHT expressed higher levels of the late stage bone formation markers osteopontin and osteocalcin (P < 0.01) and increased levels of mineralisation. In conclusion, the more compliant CG scaffolds allowed cell-mediated contraction and supported a greater level of osteogenic maturation of MC3T3 cells, while the stiffer, non-contractible scaffolds resulted in lower levels of cell maturation, but higher cell numbers on the scaffold. Therefore, we found scaffold stiffness had different effects on differentiation and cell number whereby the increased cell-mediated contraction facilitated by the less stiff scaffolds positively modulated osteoblast differentiation while reducing cell numbers.

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen–glycosaminoglycan scaffold in vivo

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen-glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model.

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patients body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a microCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffolds pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.