Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture.

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

Characterization of articular chondrocytes isolated from 211 osteoarthritic patients.

We analyzed specific features of chondrocytes as cellular yield, cell doubling rates and the dependence between these parameters and patient-related data in a set of 211 osteoarthritic (OA) patients undergoing total joint replacement. For each patient the data available were joint type, age and gender. Knee samples chosen randomly among all biopsies were graded according to ICRS score. Patients' age ranged between 30 and 90 years with a mean age of 66 ± 9.7 years. Patients were divided into age classes and statistically significant differences in proliferation rate at passage 1 were found between chondrocytes derived from young and old donors, with the last ones characterized by a lower proliferation rate. A similar trend was observed for proliferation rate at passage 2. For all the samples, cellular yields ranged between 0.1 and 5.5 million cells/g of tissue. No significant correlation was observed between the level of cartilage degeneration (ICRS score) and cellular yield and proliferation rates. However, in samples with a high degree of cartilage degeneration (ICRS score 4) the cellular yield was lower compared to the other three groups (ICRS scores 1-3). In this study we performed a systematic characterization of basic parameters of chondrocytes originating from a wide group of OA patients. Considering the use of autologous chondrocytes in chondral treatments, the characterization of cell basic features may represent an important step to determine the quality of the cell source which is a major determinant in the outcome of cell-based therapies.

Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing.

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cell/extracellular matrix interaction. We applied femtosecond laser two-photon polymerization (2PP) to fabricate three-dimensional (3-D) microscaffolds, or "niches", using a hybrid organic-inorganic photoresist called SZ2080. The niches, of sizes fitting in a volume of 100×100×100 µm(3), were made by an external containment grid of horizontal parallel elements and by an internal 3-D lattice. We developed two niche heights, 20 and 80-100 µm, and four lattice pore dimensions (10, 20, 30 µm and graded). We used primary rat mesenchymal stem cells (MSCs) to study cell viability, migration and proliferation in the niches, up to 6 culture days. MSCs preferentially stayed on/in the structures once they ran into them through random migration from the surrounding flat surface, invaded those with a lattice pore dimension greater than 10 µm, and adhered to the internal lattice while the cell nuclei acquired a roundish morphology. In the niches, the highest MSC density was found in those areas where proliferation was observed, corresponding to the regions where the scaffold surface density available for cell adhesion was highest. The microgeometry inducing the highest cell density was 20 µm high with graded pores, in which cell invasion was favored in the central region of large porosity and cell adhesion was favored in the lateral regions of high scaffold surface density. Cell density in the niches, 17±6 cells/(100×100 µm(2)), did not significantly differ from that of the flat surface colonies. This implies that MSCs spontaneously homed and established colonies within the 3-D niches. This study brings to light the crucial role played by the niche 3-D geometry on MSC colonization in culture, with potential implications for the design of biomaterial scaffolds for synthetic niche engineering.

Two-photon laser polymerization: from fundamentals to biomedical application in tissue engineering and regenerative medicine.

Three-dimensional material microstructuring by femtosecond laser-induced two-photon polymerization is emerging as an important tool in biomedicine. During two-photon polymerization, a tightly focused femtosecond laser pulse induces a crosslinking photoreaction in the polymer confined within the focal volume. As a rapid-prototyping technique, two-photon polymerization enables the fabrication of truly arbitrary three-dimensional micro- and nano-structures directly from computer models, with a spatial resolution down to 100 nm. In this review, we discuss the fundamentals, experimental methods, and materials used for two-photon polymerization; in addition, we present some applications of this technology related to microfluidics and to biomaterial scaffolds for tissue engineering and regenerative medicine.

A miniaturized, optically accessible bioreactor for systematic 3D tissue engineering research.

Perfusion bioreactors are widely used in tissue engineering and pharmaceutical research to provide reliable models of tissue growth under controlled conditions. Destructive assays are not able to follow the evolution of the growing tissue on the same construct, so it is necessary to adopt non-destructive analysis. We have developed a miniaturized, optically accessible bioreactor for interstitial perfusion of 3D cell-seeded scaffolds. The scaffold adopted was optically transparent, with highly defined architecture. Computational fluid dynamics (CFD) analysis was useful to predict the flow behavior in the bioreactor scaffold chamber (that was laminar flow, Re = 0.179, with mean velocity equal to 100 microns/s). Moreover, experimental characterization of the bioreactor performance gave that the maximum allowable pressure was 0.06 MPa and allowable flow rate up to 25 ml/min. A method, to estimate quantitatively and non destructively the cell proliferation (from 15 to 43 thousand cells) and tissue growth (from 2% to 43%) during culture time, was introduced and validated. An end point viability test was performed to check the experimental set-up overall suitability for cell culture with successful results. Morphological analysis was performed at the end time point to show the complex tridimensional pattern of the biological tissue growth. Our system, characterized by controlled conditions in a wide range of allowable flow rate and pressure, permits to systematically study the influence of several parameters on engineered tissue growth, using viable staining and a standard fluorescence microscope.

Breakthroughs in computational modeling of cartilage regeneration in perfused bioreactors.

We report about two specific breakthroughs, relevant to the mathematical modeling and numerical simulation of tissue growth in the context of cartilage tissue engineering in vitro. The proposed models are intended to form the building blocks of a bottom-up multiscale analysis of tissue growth, the idea being that a full microscale analysis of the construct, a 3-D partial differential equation (PDE) problem with internal moving boundaries, is computationally unaffordable. We propose to couple a PDE microscale model of a single functional tissue subunit with the information computed at the macroscale by 2-D-0-D models of reduced computational cost. Preliminary results demonstrate the effectiveness of the proposed models in describing the interplay among interstitial perfusion flow, nutrient delivery, and consumption and tissue growth in realistic scaffold geometries.

Comparative chondrogenesis of human cells in a 3D integrated experimental-computational mechanobiology model.

We present an integrated experimental-computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2 weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075 mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m(2)/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8 mPa (0.018 dyn/cm(2)). In the non-perfused pellets, the lowest oxygen concentration was 0.003 mM (0.3% tension) and the maximal oxygen flux was 102.4 nmol/m(2)/s. A local correlation was observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results show up-regulation of hyaline matrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications.