Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the engineered cartilage grafts so far proposed have successfully shown to mimic the morphological and biochemical appearance of hyaline cartilage, they are generally mechanically inferior to the natural tissue¹. In this study a new bioreactor device was realized to test innovative scaffolds under physiological stimulation (i.e. perfusion fluid flow and dynamic compression), with the aim to produce a more functional engineered tissue construct for articular applications.

The computer-controlled bioreactor system has been properly designed to simultaneously provide static or dynamic compression and/or continuous perfusion to 3D engineered constructs, reproducing the physiological loads to which the articular cartilage is subjected. The specifically designed bioreactor comprises a chamber where the grafts are accommodated, a porous piston connected to a linear stepper motor (Dings, Model 34-2080-4-300), which controls its movement to provide mechanical stimulation and a peristaltic pump (Watson-Marlow, Model 323S), connected by joints and pipes to the culture chamber to ensure a continuous media perfusion. As piston for compression, a sintered stainless steel filter (43% of porosity) was adopted to allow the perfusion of the culture media during physical stimulation. The culture chamber is composed by a hollow cylinder (30 mm × 40,5 mm) and a base realized as a single object. They are made in polycarbonate for its characteristics of transparency and infrangibility and linked to a Nylon cover through four brass tie rods unscrewable from above. The chamber has been designed to accommodate simultaneously different constructs of any size and shape and stimulate them with perfusion and/or dynamic compression. A finites elements program was used to mimic the effects of perfusion and compression regime on the scaffolds cultured within the bioreactor chamber.

The bioreactor was properly designed and developed. Particular attention has been paid to the implementation of a simple, compact and economical system. It was then tested by using different polymeric porous scaffolds (PVA, collagen, Gelatin grafts, both porous and not) cultured with mesenchymal cells up to two weeks. The system has been validated in terms of sterility, experimental reproducibility and ease to use. The structural stability of grafts over time has been observed; moreover cells adhesion, proliferation and matrix production under different chemical-physical stimuli conditions is under investigation.

We have realized a novel bioreactor system representing an artificial articular niche, where a dynamic compression combined with fluid perfusion allows to functionally and mechanically validate tissue substitutes, besides investigating the response of engineered cartilaginous tissues to physical stimuli mimicking the natural cartilage micro-environment. Such bioreactor may be in fact adopted as a sort of articular simulator for promoting and standardizing the new tissue formation in vitro, preconditioning cell fate through the application of proper artificial stimuli. Moreover, they can be valid tools to investigate physiological processes and novel therapeutic approaches avoiding controversial animal models.

Hydrogels have been widely used for articular tissue engineering application, due to their controllable biodegradability and high water content mimicking the biological extracellular matrix. However, they often lack the mechanical support and signaling cues needed to properly guide cells. Graphene and its derivatives have recently emerged as promising materials due to their unique mechanical, physical, chemical proprieties [1]. Although not yet widely used for medical applications, preliminary works suggest that both structural and functional properties of polymeric substrates may be enhanced when combined with graphene oxide (GO) [2]. In this work, reinforced 3D GO/alginate (Alg) hydrogels have been realized and the opportunity of tuning hydrogels mechanical properties in relation to the required physiological needs has been investigated.

After preparing GO nanosheets (Sigma Aldrich) aqueous suspension (1 mg/ml) by ultrasonic treatment, alginate (Manugel GMB, FMC Biopolymer) composite solutions were produced (0, 0.5, 2 wt% GO/Alg). Moulds of agarose (1% w/v in CaCl 0,1M) were prepared to allocate GO/Alg solutions and chemically cross-link gels via diffusion (2 hr. at 37 °C).

GO/Alg hydrogels were characterized through optical/ AFM and FTIR analysis. Biocompatibility tests were performed embedding 3T3 fibroblasts (8 millions/ml) in the GO/Alg hydrogels; cell viability was evaluated at different time points up to two weeks with Dead/alive kit.

Gels mechanical proprieties were assessed via Dynamic Mechanical- Analysis (DMA) up to 28 days of culture (with and w/o cells) at different time points. All tests were performed in triplicate and statistical analyisis carried out (Mann–Whitney U test, n=9, p<0,005).

3D composite GO/alginate hydrogels were successfully realized (3 mm height, 5 mm diameter). Cell viability tests showed that the presence of GO does not decrease cell viability, confirming absence of toxicity, at least up to 2% wt GO/Alg. For all time points cell viability was statistically higher in presence of GO, while there was no significant difference between 0.5 wt% and 2 wt% GO/Alg. Hydrogels functionalized with GO exhibit an Elastic modulus about 3 fold higher than the Alg control at T0. After an initial decreasing of the Young Modulus for the all GO/Alg samples, possibly due to a partial degradation of alginate, a drastic recovery was observed up to 28 days of culture only for GO functionalized samples. The mechanical features improvement was neither mediated nor triggered by cells activity.

We successfully realized a natural-based 3D hydrogel nano-functionalized with graphene, where both mechanical and biological properties were successfully improved. The delayed stabilization of GO/Alg mechanical proprieties may be due either to a chemical interplay between GO and alginate matrix or to GO self-assembling processes over time. Future developments will be carried out to decouple the chemical and topological role of GO on the results observed up to now. Moreover, functional tests will be performed to evaluate the GO effects on in vitro cell differentiation for possible articular clinical applications.