• bioreactor
• cartilage
• hydrodynamic

The Regenerative medicine, or tissue engineering, is a multidisciplinary field involving engineering, biochemistry, biology, medicine and physics, with the purpose to repair, replace or regenerate tissue or organs. The advent of tissue engineering and regenerative medicine introduced a potential revolutionizing treatment for patients. In fact, tissue and organ failure is still one of the main and more expensive health problems, as yet solved only with organ transplantation or medical devices/implants. Both of those solutions have some problems: organ transplantation demands organs availability, but the number of donors is too low to answer this need. On the other hand, the medical devices or implants could be a good solution for few years, but they often present biocompatibility problems, as well as difficult acceptance from patients. Tissue engineering represents a massive and interesting market which promises radical changes in diagnostic as well as therapeutic applications. For diagnostic purposes, in vitro cell cultures have been developed as alternative methods in the pharmaceutical field to test drug efficacy and toxicity. For therapeutic purposes, the intention is to regenerate damaged tissues and organs through tissue engineering techniques by stimulating previously irreparable organs, so they can heal themselves. In addition, regenerative medicine promises to allow in vitro tissues and organs growth, implanting them if the body cannot be prompted to heal itself.
In this context, tissue engineering of cartilage is a growing and promising field to produce cartilage replacement as good as native tissue. Diseases of hyaline cartilage represent one of the major health problems, especially in industrialized countries with high life expectancy. The erosion of the articulating surfaces of joints, known as osteoarthritis, currently affects more than 200 million citizens worldwide, about 400 thousands people in the Italy, and more than 50% of the patients need or will need a surgical treatment.
Articular cartilage is a three dimensional avascular tissue, which covers the ends of all synovial joints. During normal daily function, articular cartilage can be repeatedly subjected to forces up to several time body weight, but it is able to provide articulating joints with a nearly frictionless motion. Despite its tremendously important function, articular cartilage has limited capacity for auto regeneration after degenerative and rheumatic diseases, like arthritis, as well as traumatic injuries. Cartilage problems are a huge and still unsolved medical issue, which therefore represents one of the most important tissue engineering targets requiring high quality products as fast as possible. For this reason, the possibility to recreate in vitro cartilage substitutes as a real alternative to total joint replacement represents an increasing and hopeful market, in which many research groups are still working.
At the moment, one of the main findings in in-vitro cartilage studies is the importance of the role of mechanical stimuli and dynamic loads for the chondrocytes growth and differentiation. Several studies using cartilage explants or chondrocytes seeded in 3D scaffolds have shown that mechanical compressive loads affect the cells metabolic activity and their matrix production. In order to simulate the in vivo environment, the use of bioreactors is becoming fundamental: bioreactors can provide the chemical and mechanical signals that optimize tissue development. Furthermore, bioreactors could be an important instrument to reduce the cost of clinical studies, used as in vitro predictors of in vivo performance. In this way, the use of bioreactors can reduce animal studies, helping the scientists to focus their attention in the right direction before starting pre-clinical studies, which are usually more expensive than preliminary research.
In the past few years, several systems for the application of different mechanical stimuli to chondrocytes have been developed. Most of these can generate biomechanical-like forces such as the direct compression, tensile and shear forces, or hydrostatic pressure, in order to stimulate the articular chondrocyctes to increase their matrix production. Generally, the most important requirements that a culture system has to satisfy are high reliability and usability, perfect sterility, easy control of all the important culture parameters and low cost.
During this work, we start from a critical benchmarking between already available systems to design and validate a new system able to apply an innovative type of stimulation on articular chondrocytes. Starting from the first prototype of this new bioreactor, the aim of this work was to optimize and test a new version of this system, in order to obtain an easy and ready to use bioreactor for long time experiments on chondrocytes cell culture.
The basic principle of this new system is the generation of a localized contact less overpressure on articular chondrocytes, using a simple vertical piston movement. When the piston moves down, a controlled hydrodynamic overpressure and a low shear stress is generated over the cell surface, stimulating cell matrix production. Starting from an early version of the SQPR (SQueeze Pressure) system, the geometry of the internal chamber was optimized performing FEM simulations and parametric analysis, in order to increase the area of the culture surface and allow the use of different constructs or tissues. The working zone of the system was identified as compromise between the overpressure and wall shear stress applied on the bottom of the SQPR chamber. For the parametric analysis, an analytic model of squeeze lubrication theory is used, considering the fluidodynamic inside a meatus between a fixed unconfined bottom plate and approaching planar piston. The meatus was also modeled using FEM simulation in Comsol® Multiphysics software, in order to analyze the fluidodynamics conditions in the real confined geometry.
Then, the whole chamber was designed and realized respecting mechanical stability and biocompatibility requirements, to improve the bioreactor usability and reduce the total size of the system. During this step, different materials were used, and several versions of the system were realized and tested, in order to satisfy all the sterilizability and usability requirements. Furthermore, the external frame was completely re-designed, in order to obtain a small, portable and easy to use system. Moreover, the affordance and usability problems were analyzed and solved developing a purpose-made UserGuide and a easy to read frontal label which helps the final user during all the experimental work, from the setting phase to the end of experiment.
Finally, the SQPR 2.1 was tested in a collaborating laboratory at the University of Sheffield, during a three months long training. The goal of this training period was to test the hypothesis that this system is able to perform long time contactless stimulation of chondrocytes, seeded in 3D constructs, promoting their differentiation and increasing their matrix production. For this reason, Bovine Adult Chondrocytes (BAC) were seeded in different constructs and subjected to a controlled, contactless overpressure, using the SQPR 2.1. An experimental protocol was also identified and validated, in order to test the culture conditions and evaluate the total glycosaminoglycan content, fundamental protein of the cartilage matrix directly correlated with the engineered cartilage strenght. Both the viability and the matrix production results were compared to the static cultures as positive controls. During this works, several constructs like coverslips, PGA scaffolds and Agarose gel are stimulated in 24h long experiments. All these tests showed that the hydrodynamic environment presents in the SQPR chamber stimulates the development of a better engineered tissue, allowing a high viability and at the same time the increase of the medium content of glycosaminoglycan. The high cell viability demonstrates that the fluid movement and the lateral holes allow the right oxygenation of the construct, and the right concentration of nutrients. Due to its small volume, this system allows a more sensitive analysis on cell metabolic products. During the experimental works, 48h long experiments using cell-seeded scaffolds were also performed, and a GAG production rate was identified. Comparing the stimulated constructs and the static controls, it is possible to note that after 24h the matrix production of the static control tends to decrease, indicating that these cultures gradually losed the ability to produce ECM. The curve for the stimulated scaffolds in the bioreactor does not shows this production rate variation, indicating that the contact-less pressure induces the constructs to maintain increased metabolic activity.
The SQPR 2.1 system also shows a high flexibility and usability, adapting to the different laboratory needs and protocols. The high flexibility is demonstrated because it is possible to stimulate different types of 3D constructs, as Agarose/cell gel or PGA scaffolds, just changing the brace of the bioreactor and the controller parameters. The simplicity to use has been tested during the experimental phase: the bioreactor is portable thanks to its lightness and small size, and the assembling procedure is fast and easy, particularly considering the limited working space in the laminar hood
Finally, we can conclude that this work has lead to the development of a patented and reliable product for the stimulation of cartilage tissue engineered constructs, using an easy to use controller. The stimulation can be autonomously maintained for long time with high stability, allowing a easy sample collection during the experiment. Thanks to the low price materials and the simple actuation, the SQPR 2.1 system is a competitive bioreactor system able to increase the GAG production, particularly in short experiment using fresh scaffolds, on the contrary the other systems that apply a direct compression on scaffolds 3-4 weeks post seeding.
One of the main limits observed is the impossibility to stimulate more than one 3D construct per time. For this reason, one of the future works should be oriented to the design of a new SQPR version able to stimulate several scaffolds in parallel. Furthermore, a perfusion system can be added, analyzing the GAG production rate in the time without stopping the experiment, assuring at the same time the right nutrient concentration.