Cartilage-bone interactions play a critical role in joint diseases and the osteochondral junction has been identified as a locus of osteoarthritis development. However, it is challenging to study osteochondral (OC) interaction in vitro, since cartilage and bone require very different environments. We developed a new medium-to-high throughput osteochondral microphysiological system bioreactor to culture biphasic native or engineered constructs and that can be used to study any musculoskeletal tissue interfaces. We developed engineered constructs from hMSCs on a porous polymeric matrix with a gradient in pore size to assess the supportive effect of the local topology on cartilaginous and osseous differentiation. Furthermore, we developed a triphasic, vascualized osteochondral constructs based on porous polycaprolactone and methacrylated gelatin scaffolds to study the specific effects of vasculature on cartilage and bone. We also cultured native OC tissues from postmenopausal women, exposing either cartilage or bone to sex hormones studying their protective effects. Finally, our bioreactor is being implemented for use on the International Space Station to study countermeasures against microgravity bone loss. Overall, our bioreactor maintains media separation for in vitro culture and engineering of OC tissues and constructs of progressively greater complexity, and it preserves the possibility of direct cartilage-bone crosstalk opening new opportunities to study interactions across the osteochondral junction.

After surgical tendon repair, the tendon-to-bone enthesis often doesn't regenerate, which leads to high numbers of rupture recurrences. To remedy this, tissue engineering techniques are being pursued to strengthen the interface and improve regeneration. In this study, we used biphasic 3D printed PLGA scaffolds with aligned pores at the tendon side and random pores at the bone side to mimic the native enthesis. We seeded these with mesenchymal stem cells and inserted them into dual-flow bioreactors, allowing us to employ tenogenic and chondrogenic differentiation medium in separate flows. MTS assay demonstrated metabolism in dual-flow bioreactors at levels similar to tissue culture plate and rotating bioreactors. After 7, 14 and 21 days, samples were collected and analyzed by histology, RT-PCR and GAG production. H&E staining confirmed a compact cell layer attached to fibers and between porous cavities of scaffolds that increased with time of culture. Interestingly, cultured constructs in dual-flow bioreactors biased towards a chondrogenic fate regardless of which flow they were exposed to, possibly due to high porosity of the scaffold allowing for fluid mixture. Sox9 was upregulated at all timepoints (up to 30× compared to control), and by day 21 Col2A1 was also highly upregulated. Additionally, GAG production in treated constructs (serum-free) was able to match constructs exposed to 10% FBS in controls, demonstrating the functional matrix forming capabilities of this system. Overall, we have validated this dual-flow system as a potential platform to form the enthesis, and future studies will further optimize parameters to achieve distinctly biphasic constructs.

We have explored indentation-type scanning force microscopy (IT SFM) that allows for a direct, quantitative inspection of cartilage morphology and biomechanical properties from the millimeter to the nanometer scale ex vivo, and ultimately, in situ (Stolz et al., 2004). Here we present three examples of using IT SFM where morphological and biomechanical changes could only be spotted at the sub-micrometer scale:

We employed IT SFM for quality control of engineered cartilage cultured under various conditions. These measurements harbor the prospect to optimize and yield engineered cartilage that exhibits long-term mechanical stability, functionality and biocompatibility for joint arthroplasty.

Accordingly, the stiffness of the aging cartilage increased concomitant with a decrease of its glycosaminoclycan (GAG) moiety. Frequently, aging articular cartilage takes a pathological turn called osteoarthritis (OA), which usually ends with a complete disappearance of the articular cartilage layer. Towards an early detection of OA in the human body, we inspected the morphological and biomechanical status of articular cartilage biopsies representing different grades of OA according to the ‘Outerbridge scale’. Most significantly, the early changes (grades 0 to 2) were only detectable at the nanometer scale, but not at the micrometer or millimeter scale. Based on such ex vivo indentation testing, we started to move from the bench to the patient, aiming to directly inspect the quality of human articular knee cartilage by an arthroscopic SFM (Imer et al., 2006). The arthroscopic SFM might just be the beginning of a new generation of nano tools designed for endoscopic or catheter-based interventions of other parts of the body. For such prophylactic interventions to eventually being tolerated by the patient, not only have these to be ambulant and minimally invasive, but they will require a change of paradigm vis-à-vis the patient, namely to undergo an invasive procedure without feeling sick – indeed a big challenge for nanomedicine and managed health care!