Michael J. Jaasma

The effect of dehydrothermal treatment on the mechanical and structural properties of collagen-GAG scaffolds.

The mechanical properties of tissue engineering scaffolds are critical for preserving the structural integrity and functionality during both in vivo implantation and long-term performance. In addition, the mechanical and structural properties of the scaffold can direct cellular activity within a tissue-engineered construct. In this context, the aim of this study was to investigate the effects of dehydrothermal (DHT) treatment on the mechanical and structural properties of collagen-glycosaminoglycan (CG) scaffolds. Temperature (105-180 degrees C) and exposure period (24-120 h) of DHT treatment were varied to determine their effect on the mechanical properties, crosslinking density, and denaturation of CG scaffolds. As expected, increasing the temperature and duration of DHT treatment resulted in an increase in the mechanical properties. Compressive properties increased up to twofold, while tensile properties increased up to 3.8-fold. Crosslink density was found to increase with DHT temperature but not exposure period. Denaturation also increased with DHT temperature and exposure period, ranging from 25% to 60% denaturation. Crosslink density was found to be correlated with compressive modulus, whilst denaturation was found to correlate with tensile modulus. Taken together, these results indicate that DHT treatment is a viable technique for altering the mechanical properties of CG scaffolds. The enhanced mechanical properties of DHT-treated CG scaffolds improve their suitability for use both in vitro and in vivo. In addition, this work facilitates the investigation of the effects of mechanical properties and denaturation on cell activity in a 3D environment.

Biomechanical effects of intraspecimen variations in tissue modulus for trabecular bone

Although recent nanoindentation studies have revealed the existence of substantial variations in tissue modulus within single specimens of trabecular bone, little is known regarding the biomechanical effects of such intraspecimen variations. In this study, high-resolution finite element modeling was used to investigate these effects. With limited literature information on the spatial distribution of intraspecimen variations in tissue modulus, two plausible spatial distributions were evaluated. In addition, three specimens (human femoral neck, human vertebral body, and bovine proximal tibia) were studied to assess the role of trabecular architecture. Results indicated that for all specimen/distribution combinations, the apparent modulus of the whole specimen decreased nonlinearly with increasing coefficient of variation (COV) of tissue modulus within the specimen. Apparent modulus decreased by <4% when tissue modulus COV was increased from 0% to 20% but decreased by 7-24%, depending on the assumed spatial distribution, for an increase in tissue modulus COV from 20% to 50%. For compressive loading to the elastic limit, increasing tissue modulus COV from 20% to 50% caused up to a 28-fold increase in the amount of failed tissue, depending on assumed spatial distribution and trabecular architecture. We conclude that intraspecimen variations in tissue modulus, if large, may have appreciable effects on trabecular apparent modulus and tissue-level failure. Since the observed effects depended on the assumed spatial distribution of the tissue modulus variations, a description of such distributions, particularly as a function of age, disease, and drug treatment, may provide new insight into trabecular bone structure-function relationships.

Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.

Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds.

In tissue engineering, flow perfusion bioreactors can be used to enhance nutrient diffusion while mechanically stimulating cells to increase matrix production. The goal of this study was to design and validate a dynamic flow perfusion bioreactor for use with compliant scaffolds. Using a non-permanent staining technique, scaffold perfusion was verified for flow rates of 0.1-2.0 mL/min. Flow analysis revealed that steady, pulsatile and oscillatory flow profiles were effectively transferred from the pump to the scaffold. Compared to static culture, bioreactor culture of osteoblast-seeded collagen-GAG scaffolds led to a 27-34% decrease in cell number but stimulated an 800-1200% increase in the production of prostaglandin E(2), an early-stage bone formation marker. This validated flow perfusion bioreactor provides the basis for optimisation of bioreactor culture in tissue engineering applications.

A Comparative Study of Shear Stresses in Collagen-Glycosaminoglycan and Calcium Phosphate Scaffolds in Bone Tissue-Engineering Bioreactors

The increasing demand for bone grafts, combined with their limited availability and potential risks, has led to much new research in bone tissue engineering. Current strategies of bone tissue engineering commonly use cell-seeded scaffolds and flow perfusion bioreactors to stimulate the cells to produce bone tissue suitable for implantation into the patients body. The aim of this study was to quantify and compare the wall shear stresses in two bone tissue engineering scaffold types (collagen-glycosaminoglycan (CG) and calcium phosphate) exposed to fluid flow in a perfusion bioreactor. Based on micro-computed tomography images, three-dimensional numerical computational fluid dynamics (CFD) models of the two scaffold types were developed to calculate the wall shear stresses within the scaffolds. For a given flow rate (normalized according to the cross-sectional area of the scaffolds), shear stress was 2.8 times as high in the CG as in the calcium-phosphate scaffold. This is due to the differences in scaffold geometry, particularly the pore size (CG pore size approximately 96 microm, calcium phosphate pore size approximately 350 microm). The numerically obtained results were compared with those from an analytical method that researchers use widely experimentalists to determine perfusion flow rates in bioreactors. Our CFD simulations revealed that the cells in both scaffold types were exposed to a wide range of wall shear stresses throughout the scaffolds and that the analytical method predicted shear stresses 12% to 21% greater than those predicted using the CFD method. This study demonstrated that the wall shear stresses in calcium phosphate scaffolds (745.2 mPa) are approximately 40 times as high as in CG scaffolds (19.4 mPa) when flow rates are applied that have been experimentally used to stimulate the release of prostaglandin E(2). These findings indicate the importance of using accurate computational models to estimate shear stress and determine experimental conditions in perfusion bioreactors for tissue engineering.

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.

Mechanical loading by fluid shear is sufficient to alter the cytoskeletal composition of osteoblastic cells

Many structural modifications have been observed as a part of the cellular response to mechanical loading in a variety of cell types. Although changes in morphology and cytoskeletal rearrangement have been widely reported, few studies have investigated the change in cytoskeletal composition. Measuring how the amounts of specific structural proteins in the cytoskeleton change in response to mechanical loading will help to elucidate cellular mechanisms of functional adaptation to the applied forces. Therefore, the overall hypothesis of this study was that osteoblasts would respond to fluid shear stress by altering the amount of specific cross-linking proteins in the composition of the cytoskeleton. Mouse osteoblast cell line MC3T3-E1 and human fetal osteoblasts (hFOB) were exposed to 2 Pa of steady fluid shear for 2 h in a parallel plate flow chamber, and then the amount of actin, vimentin, alpha-actinin, filamin, and talin in the cytoskeleton was measured using Western blot analyses. After mechanical loading, there was no change in the amount of actin monomers in the cytoskeleton, but the cross-linking proteins alpha-actinin and filamin that cofractionated with the cytoskeleton increased by 29% (P<0.01) and 18% (P<0.02), respectively. Localization of the cross-linking proteins by fluorescent microscopy revealed that they were more widely distributed throughout the cell after exposure to fluid shear. The amount of vimentin in the cytoskeleton also increased by 15% (P<0.01). These results indicate that osteoblasts responded to mechanical loading by altering the cytoskeletal composition, which included an increase in specific proteins that would likely enhance the mechanical resistance of the cytoskeleton.

The effect of pore size, crosslinking and collagen content on mechanical properties of collagen-GAG scaffolds

Tissue-engineered bone provides an alternative source of bone grafts. Collagen-GAG scaffolds (porosity 99.5%) have shown great potential as a construct on which to culture bone cells [1], but the mechanical properties of the scaffold are currently not suitable for use as a bone graft. Therefore, the goal of this study was to investigate the effects of pore size, dehydrothermal (DHT) crosslinking and collagen content on the mechanical properties of collagen-GAG scaffolds. Scaffolds were produced via a freeze-drying process, using two compositions of collagen-GAG slurry (0.5% and 1% collagen). Pore size was altered by varying the final freezing temperature of the freeze-drying process (pore size range: 69-96~tm). DHT crosslinking was then carried out at a pressure of 0.05 bar. DHT duration and temperature were varied between 24 and 96 hours and 105°C and 120°C, respectively. Compression testing was carried out on dry samples to determine elastic modulus. Pore size did not have a significant effect on modulus (p > 0.05), which agrees with previous work [3]. For DHT crosslinking, prolonged exposure did not produce an increase in modulus at 105°C, but at 120°C there was s 50% increase in modulus after three days of crosslinking (p<0.05). Increasing the temperature from 105°C to 120°C produced a 2to 5-fold increase in modulus (p <0.001). Doubling the collagen content produced a 10-fold increase in modulus (p<0.001). Results show that the modulus can be substantially increased both by using DHT treatment and by increasing the collagen content in the scaffold. The combined effects of increasing DHT temperature and doubling the collagen content are being investigated to further increase scaffold modulus. Acknowledgements: Science Foundation Ireland, Integra Life Sciences.