Saartje Impens

Characterization and Optimization of Cell Seeding in Scaffolds by Factorial Design: Quality by Design Approach for Skeletal Tissue Engineering

Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.

Production and characterisation of porous calcium phosphate structures with controllable hydroxyapatite/β-tricalcium phosphate ratios

Abstract Because bone tissue engineering (TE) strategies encounter patient specificities, like site of implantation and different bone turnover rate, scaffolds with tunable characteristics are required. With this regard, custom made open porous calcium phosphate (CaP) structures with variable chemical and structural compositions were produced using an optimised gel casting technique. The phase composition was altered by changing the initial hydroxyapatite (HA) to β-tricalcium phosphate (β-TCP) powder ratio. After sintering, an HA/β-TCP ratio between 100 : 0 and 0 : 100 was predictably attained. Average pore sizes ranged from 182 to 466 m m and corresponded to porosity values of respectively 71 and 86%. Mechanical strength and dynamic E-modulus respectively decreased with decreasing HA content from 10·1 to 0·4 MPa and 9·46 to 0·27 GPa. It was concluded that this optimised gel casting method enables the production of CaP scaffolds with a custom made HA/β-TCP ratio with variable pore size, porosity and corresponding mechanical strength.

Numerical modeling of perfusion flow in irregular scaffolds

Direct perfusion of 3D tissue engineered constructs is known to enhance osteogenesis, which can be partly attributed to enhanced nutrient and waste transport. In addition flow mediated shear stresses are known to upregulate osteogenic differentiation and mineralization. A quantification of the hydrodynamic environment is therefore crucial to interpret and compare results of in vitro bioreactor experiments. In this study a 3D CFD model for the creeping perfusion flow inside two irregular bone scaffold structures is developed, simulating the velocity field including shear stress distribution. eCT imaging techniques were used to reconstruct the geometry of both a titanium and a hydroxyapatite scaffold, starting from 430 images with a resolution of 8 µm. The resulting CFD models are built with the 3D unstructured mesher TGrid and solved with the finite volume code Fluent (ANSYS, Inc.). With a flow rate of 0.04ml/min we obtained average wall shear stresses (WSS) of 1.46mPa for the hydroxyapatite scaffold compared to 1.95mPa for the Titanium scaffold. Influence of boundary conditions and scaffold micro architecture heterogeneity has been investigated. This methodology allows to get more insight in the complex concept of tissue engineering and will likely help to understand and eventually improve the fluidmechanical aspects.

In vitro dissolution behavior of custom made CaP scaffolds for bone tissue engineering

The degradation rate of custom made calcium phosphate scaffolds, designed for bone tissue engineering applications, influences the healing process of critical size bone defects. An optimal degradation rate exists at which the neo-formed bone replaces the CaP (calcium phosphate) scaffold [1]. Consequently investigating the complex degradation behavior (dissolution, reprecipitation, osteoclast activity) of custom made CaP structures gains interest. In this work different in vitro dissolution experiments were performed to study the degradation behavior of 4 by composition different calcium phosphates. Ideally these experiments should have a predictive power regarding the in vivo degradation behavior. In vitro dissolution tests still lack standardization. Therefore this study focuses on the influence of two dissolution constraints: (i) the material’s macrostructure (porous - dense), (ii) the regenerated fluid flow (bath shaking - perfusion). From 4 different CaP compositions porous structures and as a reference dense disks were produced, using the same starting powder and heat treatment. To compare the different dissolution tests, all data was normalized to the CaP surface area. Results show that besides the structural appearances of the CaP structures, also the design of the dissolution test influences the in vitro dissolution behavior. Moreover there is a need to take the morphology of the dissolved material into account. The CaP perfusion tests show dissolution dynamics that resemble the in vivo reality more closely than the shaking bath experiments.

Guided Bone Engineering: Healing of Large Bone Defects

Tissue engineering (TE) aims/seeks to achieve the substitution of organ transplantation by the creation of living, functional tissues. It has been suggested that biocompatible porous materials (scaffolds) and a controllable 3D environment are required to aid in the 3D cell organisation and their development into functional tissue. Our research envisions a TE-approach towards the repair of large, load bearing defects in long bones. In vitro standardised, systematic, quantitative screening of potential bone scaffolds is required to understand how scaffolds can affect cell behaviour. This screening will avoid a trial-and-error approach and thus limit the number of animal experiments. Such a screening should be based on the knowledge of mechanical, physical and (bio)chemical scaffold properties and their interaction with cell behaviour. In addition, the design and production of a clinically relevant scaffold requires control over its mechanical behaviour and a new approach for cell seeding in a 3D scaffold, as well as providing nutrition for the engrafted cells. The objective of this research is to gain substantial knowledge about guided bone regeneration and to develop quantitative methodologies that can lead to consistent and reproducible bone regeneration.