In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools. Moreover, when equipped with automation, their use allows reducing user intervention and dependence, increasing reproducibility and the overall quality of the culture process. In this study, an automated uni-/bi-directional perfusion bioreactor combinable with pulsed electromagnetic field (PEMF) stimulation for culturing 3D bone tissue models is proposed. A user-friendly control unit automates the perfusion, minimizing the user dependency. Computational fluid dynamics simulations supported the culture chamber design and allowed the estimation of the shear stress values within the construct. Electromagnetic field simulations demonstrated that, in case of combination with a PEMF stimulator, the construct can be exposed to uniform magnetic fields. Preliminary biological tests on 3D bone tissue models showed that perfusion promotes the release of the early differentiation marker alkaline phosphatase. The histological analysis confirmed that perfusion favors cells to deposit more extracellular matrix (ECM) with respect to the static culture and revealed that bi-directional perfusion better promotes ECM deposition across the construct with respect to uni-directional perfusion. Lastly, the Real-time PCR results of 3D bone tissue models cultured under bi-directional perfusion without and with PEMF stimulation revealed that the only perfusion induced a ~ 40-fold up-regulation of the expression of the osteogenic gene collagen type I with respect to the static control, while a ~ 80-fold up-regulation was measured when perfusion was combined with PEMF stimulation, indicating a positive synergic pro-osteogenic effect of combined physical stimulations.

Anterior cruciate ligament (ACL) deficiency can result in serious degenerative stifle injuries. Although tibial plateau leveling osteotomy (TPLO) is a common method for the surgical treatment of ACL deficiency, alternative osteotomies, such as a leveling osteotomy based on the center of rotation of angulation (CBLO) are described in the literature. However, whether a CBLO could represent a viable alternative to a TPLO remains to be established. The aim of this study is to compare TPLO and CBLO effectiveness in treating ACL rupture. First, a computational multibody model of a physiological stifle was created using three-dimensional surfaces of a medium-sized canine femur, tibia, fibula and patella. Articular contacts were modeled by means of a formulation describing the contact force as function of the interpenetration between surfaces. Moreover, ligaments were represented by vector forces connecting origin and insertion points. The lengths of the ligaments at rest were optimized simulating the drawer test. The ACL-deficient model was obtained by deactivating the ACL related forces in the optimized physiological one. Then, TPLO and CBLO treatments were virtually performed on the pathological stifle. Finally, the drawer test and a weight-bearing squat movement were performed to compare the treatments effectiveness in terms of tibial anteroposterior translation, patellar ligament force, intra-articular compressive force and quadriceps force. Results from drawer test simulations showed that ACL-deficiency causes an increase of the anterior tibial translation by up to 5.2 mm, while no remarkable differences between CBLO and TPLO were recorded. Overall, squat simulations have demonstrated that both treatments lead to an increase of all considered forces compared to the physiological model. Specifically, CBLO and TPLO produce an increase in compressive forces of 54% and 37%, respectively, at 90° flexion. However, TPLO produces higher compressive forces (up to 16%) with respect to CBLO for wider flexion angles ranging from 135° to 117°. Conversely, TPLO generates lower forces in patellar ligament and quadriceps muscle, compared to CBLO. In light of the higher intra-articular compressive force over the physiological walking range of flexion, which was observed to result from TPLO in the current study, the use of this technique should be carefully considered.

Three-dimensional (3D) printing represents a key technology for rapid prototyping, allowing easy, rapid, and low-cost fabrication. In this work, 3D printing was applied for the in-house production of customized components of a mechanical stretching bioreactor with potential application for cardiac tissue engineering and mechanobiology studies. The culture chamber housing and the motor housing were developed as functional permanent parts, aimed at fixing the culture chamber position and at guaranteeing motor watertightness, respectively. Innovative sample holder prototypes were specifically designed and 3D-printed for holding thin and soft biological samples during cyclic stretch culture. The manufactured components were tested in-house and in a cell biology laboratory. Moreover, tensile tests and finite element analysis were performed to investigate the gripping performance of the sample holder prototypes. All the components showed suitable performances in terms of design, ease of use, and functionality. Based on 3D printing, the bioreactor optimization was completely performed in-house, from design to fabrication, enabling customization freedom, strict design-to-prototype timing, and cost and time effective testing, finally boosting the bioreactor development process.

V.

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