Jolanda R. Vetsch

Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature

Graphical abstract

Micro-Computed Tomography Based Computational Fluid Dynamics for the Determination of Shear Stresses in Scaffolds Within a Perfusion Bioreactor

Perfusion bioreactors are known to exert shear stresses on cultured cells, leading to cell differentiation and enhanced extracellular matrix deposition on scaffolds. The influence of the scaffold’s porous microstructure is investigated for a polycaprolactone (PCL) scaffold with a regular microarchitecture and a silk fibroin (SF) scaffold with an irregular network of interconnected pores. Their complex 3D geometries are imaged by micro-computed tomography and used in direct pore-level simulations of the entire scaffold–bioreactor system to numerically solve the governing mass and momentum conservation equations for fluid flow through porous media. The velocity field and wall shear stress distribution are determined for both scaffolds. The PCL scaffold exhibited an asymmetric distribution with peak and plateau, while the SF scaffold exhibited a homogenous distribution and conditioned the flow more efficiently than the PCL scaffold. The methodology guides the design and optimization of the scaffold geometry.

Effect of fetal bovine serum on mineralization in silk fibroin scaffolds

Fetal bovine serum (FBS) is a common media supplement used in tissue engineering (TE) cultures. The chemical composition of FBS is known to be highly variable between different brands, types or batches and can have a significant impact on cell function. This study investigated the influence of four different FBS types in osteogenic or control medium on mineralization of acellular and cell-seeded silk fibroin (SF) scaffolds. In bone TE, mineralized tissue is considered as the final product of a successful cell culture. Calcium assays and micro-computed tomography scans revealed spontaneous mineralization on SF scaffolds with certain FBS types, even without cells present. In contrast, cell-mediated mineralization was found under osteogenic conditions only. Fourier transform infrared spectroscopy analysis demonstrated a similar ion composition of the mineralization present in scaffolds, whether cell-mediated or spontaneous. These results were confirmed by scanning electron microscopy. This study shows clear evidence for the influence of FBS type on mineralization on SF scaffolds. The suitability of FBS medium supplementation in TE studies is highly questionable with regard to reproducibility of studies and comparability of obtained results. For future TE studies, alternatives to conventional FBS such as defined FBS or serum-free media should be considered, as suggested decades ago.

The evolution of simulation techniques for dynamic bone tissue engineering in bioreactors

Bone tissue engineering aims to overcome the drawbacks of current bone regeneration techniques in orthopaedics. Bioreactors are widely used in the field of bone tissue engineering, as they help support efficient nutrition of cultured cells with the possible combination of applying mechanical stimuli. Beneficial influencing parameters of in vitro cultures are difficult to find and are mostly determined by trial and error, which is associated with significant time and money spent. Mathematical simulations can support the finding of optimal parameters. Simulations have evolved over the last 20 years from simple analytical models to complex and detailed computational models. They allow researchers to simulate the mechanical as well as the biological environment experienced by cells seeded on scaffolds in a bioreactor. Based on the simulation results, it is possible to give recommendations about specific parameters for bone bioreactor cultures, such as scaffold geometries, scaffold mechanical properties, the level of applied mechanical loading or nutrient concentrations. This article reviews the evolution in simulating various aspects of dynamic bone culture in bioreactors and reveals future research directions. Copyright

Pressureless Mechanical Induction of Stem Cell Differentiation Is Dose and Frequency Dependent

Movement is a key characteristic of higher organisms. During mammalian embryogenesis fetal movements have been found critical to normal tissue development. On the single cell level, however, our current understanding of stem cell differentiation concentrates on inducing factors through cytokine mediated biochemical signaling. In this study, human mesenchymal stem cells and chondrogenesis were investigated as representative examples. We show that pressureless, soft mechanical stimulation precipitated by the cyclic deformation of soft, magnetic hydrogel scaffolds with an external magnetic field, can induce chondrogenesis in mesenchymal stem cells without any additional chondrogenesis transcription factors (TGF-β1 and dexamethasone). A systematic study on the role of movement frequency revealed a classical dose-response relationship for human mesenchymal stem cells differentiation towards cartilage using mere mechanical stimulation. This effect could even be synergistically amplified when exogenous chondrogenic factors and movement were combined.

Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.

Silk fibroin scaffolds with inverse opal structure for bone tissue engineering.

Abstract How scaffold porosity, pore diameter and geometry influence cellular behavior is‐although heavily researched ‐ merely understood, especially in 3D. This is mainly caused by a lack of suitable, reproducible scaffold fabrication methods, with processes such as gas foaming, lyophilization or particulate leaching still being the standard. Here we propose a method to generate highly porous silk fibroin scaffolds with monodisperse spherical pores, namely inverse opals, and study their effect on cell behavior. These silk fibroin inverse opal scaffolds were compared to salt‐leached silk fibroin scaffolds in terms of human mesenchymal stem cell response upon osteogenic differentiation signals. While cell number remained similar on both scaffold types, extracellular matrix mineralization nearly doubled on the newly developed scaffolds, suggesting a positive effect on cell differentiation. By using the very same material with comparable average pore diameters, this increase in mineral content can be attributed to either the differences in pore diameter distribution or the pore geometry. Although the exact mechanisms leading to enhanced mineralization in inverse opals are not yet fully understood, our results indicate that control over pore geometry alone can have a major impact on the bioactivity of a scaffold toward stem cell differentiation into bone tissue.

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

The influence of curvature on three-dimensional mineralized matrix formation under static and perfused conditions: an in vitro bioreactor model

Bone remodelling is the continuous turnover of bone by resorption and formation. It is controlled by interstitial fluid flow sensed by osteocytes. The refilling of bone resorption sites has been shown to be curvature driven. In vitro, curvature influences tissue growth and cytoskeletal arrangements under static and perfused conditions. Nevertheless, this has only been demonstrated for non-mineralized tissue in limited three-dimensional volumes. This study aims at investigating the influence of three different channel curvatures (S, −2.00 mm−1; M, −1.33 mm−1; L, −0.67 mm−1) on mineralized tissue formation in three dimensions under static and perfused conditions. The ingrowth of mineralized tissue into the channels was dependent on curvature and was higher under perfusion (M and S channels). L channels were not closed in any group compared with partially (static) or fully (perfused) closed M and S channels. Mineralized tissue morphology was cortical-like in static samples and trabecular-like in perfused samples. Our results suggest that the three-dimensional in vitro model presented is not only able to reveal effects of curvature on mineralized tissue formation, but may be used as an in vitro model for critical size defects in trabecular or cortical bone.

The choice of fetal bovine serum influences the degree of spontaneous mineralization on silk fibroin scaffolds in 3D cell cultures

Conclusions These results show that variations in FBS have a significant effect on spontaneous mineralization rates in acellular SF scaffolds. We need to learn more about the process of mineralization and how variations in media composition affect tissue calcification in 3D cell culture. Results & Discussion Though we expected calcium content to remain low in acellular scaffolds, by week 7 the calcium content in some acellular scaffolds was nearly half the amount of their cellularized counterparts. Calcium content varied significantly between FBS brands in acellular scaffolds, but not in cellularized scaffolds, and μCT analysis showed similar trends. PAA Gold and PAA Standard Quality both displayed relatively low levels of mineralization when compared to Biochrom’s FBS Superior or FBS Standard from Gibco.