(M-501) Flexion Promotes in vitro hMSCs Chondrogenesis and Hypertrophy in a Customized Bioreactor

Introduction:: At least 2 million people are born annually worldwide with a skeletal birth defect. Greater than 60% of these cases are caused by a combination of genetic susceptibility and environmental exposures. Compared to the current in vivo models, which suffer from poor translation of the findings to humans, in vitro 3D tissue culture models of joint formation can be more efficient in investigating these defects. Flexion is thought to play a critical role in developing joints in vivo. We developed a bioreactor in which scaffold-free chondrogenic tissue can be subjected to dynamic flexion. In our study, we used this system to show that dynamic flexion can promote chemically-induced chondrogenesis and hypertrophy in hMSCs-based aggregates. Safranin-O and H&E histology staining showed that a bubble/void feature was developed in the region under higher stress of aggregates. Immunohistochemistry (IHC) results show that Col II (chondrogenesis) levels were decreased, Col X and VEGF (hypertrophy) levels were increased when subjected to flexion.


Materials and Methods:: We developed a perfusion bioreactor that can subject the chondrogenic/hypertrophic tissue formed from human mesenchymal stem cells (hMSCs) to mechanical flexion and chemical stimulation. A flexible tissue holder containing five (same seeding density, 1.25x10⁶ cells/channel) linear channels were integrated into the perfusion module, which allowed the perfusion of the media and the induction of chondrogenesis and hypertrophy through TGF-β1 and triiodothyronine (T3), respectively. The tissue holder used microscale anchors and a custom-designed membrane to minimize relative tissue movement and maximize flexion exposure. A pneumatically-controlled external actuator applied flexion onto the tissue indirectly. Chondrogenesis-inducing media (CIM) and hypertrophy-inducing media (HIM) were used to induce chondrogenesis or hypertrophy, respectively. hMSCs were cultured in the channels and allowed to form aggregates. In the chondrogenesis group, CIM was perfused for all 4 weeks of culture; in the hypertrophy group, CIM was perfused in the first two weeks and HIM for the remaining 2 weeks. For both groups, flexion was introduced on day 8 and continued for the remainder of the culture. Flexion of different strengths (deflection angles: 0^o, 7^o, or 15^o) and durations (1 or 6 hr/day) at a frequency of 1 Hz was induced. Both CIM and HIM were perfused at a flow rate equivalent to 0.4 ml/10⁶cells/day. The accumulated medium leaving the chamber was collected every two days and analyzed for amino acid uptake/secretion, glucose uptake, and lactate production. Tissue samples were harvested for histology and IHC staining on Col 1, Col 2, Col 10, and VEGF markers.


Results, Conclusions, and Discussions:: Tissue culture experiments showed that chondrogenesis and hypertrophy could be chemically induced with CIM and HIM, respectively. In comparison to HIM-treated control group and CIM-treated flexion group, flexion induced morphological change (void space and bubble structure) at the middle-top region in HIM-treated group, confirmed by Safranin-O and H&E staining and longer duration (6hr/day) increased the size of void space. This indicates that the morphological change is induced by the combination of flexion and T3 treatment. A solid mechanics simulation study shows that the region of tissue with morphological change undergoes higher stress than the end regions. We hypothesize that the morphological change was similar to a developmental phase during early stage of joint formation, called interzone emergence where a single cartilage is gradually separated into two parts where eventually the joint will form. The argument was proved by GAG analysis and IHC on Col 2, Col 10 and VEGF markers. During interzone emergence and formation, GAG content within this area drastically increased, chondrocytes became further hypertrophic, and secreted decreased deposition of Col 2 and increased the amount of Col 10. GAG content normalized to DNA content significantly increased in the middle area in HIM-treated group than HIM-treated controls and CIM-treated flexion group. This area also showed increased staining for Col 10 and VEGF, and weaker staining for Col 2. VEGF presence targets future capillary invasion and angiogenesis of the hypertrophic cartilage matrix. In conclusion, the cartilage tissue formed in the reactor can be subject to flexion using an external actuator in a minimally- invasive way without affecting the sterile environment of the tissue. The cooperative effect of flexion and T3 caused the morphological change at the higher-stress middle-top region. Our further analysis indicates that it is similar to interzone emergence and formation.




Acknowledgements (Optional): : This research was funded in part by a grant from the National Institutes of Health (P41-EB021911

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