- Participants will acquire knowledge on how patients may react to therapies based on cell, tissue, and material implantation.
19.3.1 - Cell Therapy for Cartilage Repair-Modelling - Increasing Complexity of Cartilage Models
Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are taken into account when performing in vivo studies, in vivo studies are still largely performed under static conditions.
We and others have shown that mechanical load can greatly influence chondroprogenitor and chondrocyte phenotype, generally beneficially, with factors such as TGF and nitric oxide being implicated. Taken together there is an increased need to implement mechanical stimulation during in vitro studies and to further understand the relative role of the specific load applied. This requires an increasing complexity of in vitro culture models, with the ultimate aim to recreate the articulating joint as accurately as possible. Building on previous studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs (1, 2), we have been further increasing the complexity of our in vitro/ ex vivo models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition (3). In addition, combining osteochondral defect models where viable cartilage and bone tissue can provide soluble cross talk, and a potential cell source, with an implanted therapeutic provides a multicellular environment more representative of the in vivo situation.
We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. While being able to apply multiple stimuli, selecting the stimuli for the vast number possible is a major challenge. Utilization of a design of experiment (DoE) approach can allow for a rapid screening of multiple factors, and their interactions, allowing for a smaller number to be selected for longer term studies. Design of higher throughput, multiwell multiaxial load bioreactors further increases the possibility to test multiple conditions or materials simultaneously opening more opportunities for in vitro screening. In vitro models have the major advantage that human cells from skeletally mature adults can be used, thus providing more clinically relevant data.
Improved understanding of the underlying mechanism of mechanically induced chondrogenesis can also assist with designing better protocols and streamlining biomaterial testing. Latent TGFβ can be mechanically activated and investigating the ability of cell free materials to activate TGFβ under mechanical load provides an insight to their performance under kinematic loading conditions (4).
The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable development pathways.
1. Gardner OFW, Fahy N, Alini M, Stoddart MJ. Joint mimicking mechanical load activates TGFbeta1 in fibrin-poly(ester-urethane) scaffolds seeded with mesenchymal stem cells. Journal of tissue engineering and regenerative medicine. 2017;11(9):2663-6.
2. Li Z, Kupcsik L, Yao SJ, Alini M, Stoddart MJ. Mechanical Load Modulates Chondrogenesis of Human Mesenchymal Stem Cells through the TGF-beta Pathway. J Cell Mol Med. 2010;14(6A):1338-46.
3. Monaco G, El Haj AJ, Alini M, Stoddart MJ. Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells. Eur Cell Mater. 2021;41:616-32.
4. Behrendt P, Ladner Y, Stoddart MJ, Lippross S, Alini M, Eglin D, et al. Articular Joint-Simulating Mechanical Load Activates Endogenous TGF-beta in a Highly Cellularized Bioadhesive Hydrogel for Cartilage Repair. Am J Sports Med. 2020;48(1):210-21.
This work was funded by the Swiss National Science Foundation (31003A_179438) and the AO Foundation.
19.3.3 - Targeted Joint Therapeutics: Mechano-Activated Microcapsule Drug Delivery
19.3.4 - Interaction of Cellular Grafts with Osteoarthritic Joint Compartments
Current therapies of the degenerative joint disease osteoarthritis (OA) are limited to alleviate the main symptoms of pain and stiffness until at the end stage of the disease a joint replacement by a prosthesis becomes inevitable. Such an irreversible surgical intervention is particularly problematic for younger patients due to the limited durability of a prosthesis and increased risk of revision surgeries. We previously showed that Tissue Engineered Cartilage generated with autologous Nasal chondrocytes (N-TEC) is suitable for the repair of focal articular cartilage defects (Mumme et al., Lancet 2016). We then evaluated the compatibility of N-TEC in the more challenging OA environment where chronical inflammation is present (Acevedo et al., Sci Transl Med, 2021). Specifically, we investigated the in vitro ability of N-TEC to sustain prolonged exposure to soluble factors simulating an OA environment and to regulate the inflammatory profile of cells typically present in an OA joint. We then explored in different small and large animal models in vivo N-TEC engraftment with OA tissues. Safety of autologous N-TEC was finally addressed in two individual patients with diagnosed advanced knee OA.
Material and Methods: N-TEC were generated with expanded human nasal chondrocytes (NC) and exposed in vitro to an inflammatory cytokine cocktail (IL1b, IL6 and TNFa) or conditioned medium from OA-synoviocytes. Additionally, inflammatory factors secreted by synoviocytes and chondrocytes isolated from OA joints were quantified upon culture with N-TEC conditioned medium.
N-TEC generated with GFP-labelled human NC were combined with a bone-like tissue engineered with osteoblasts or osteochondral tissue explants from patients with OA and subcutaneously implanted in mice for 8 weeks.
OA was induced in an orthotopic sheep model by generation of full thickness cartilage defects in the femoral condyles. Two months after OA induction, N-TEC generated with GFP-labelled autologous sheep NC (sN-TEC) were implanted into the degenerated cartilage defects for up to 12 months and the repair tissue quality assessed immuno-/histochemically. In addition, inflammatory factors were quantified in the synovial fluids harvested from joints of healthy (before OA induction), OA (at the time of treatment) and treated sheep (at the time of explantation).
Safety of autologous N-TEC transplantation was tested in 2 patients with radiological signs of medial compartment OA (Kellgren and Lawrence grades 3 and 4, age 34 and 36) who were otherwise considered for unicondylar knee arthroplasty. N-TEC implantation was combined with corrective high tibial osteotomy to reduce abnormal mechanical loading due to varus malalignment.
Results: We demonstrated that N-TEC were able to maintain cartilaginous properties in different inflammatory in vitro models simulating conditions of an osteoarthritic joint. Importantly, factors secreted by N-TEC significantly reduced the secretion of several specific inflammatory cytokines by OA-synoviocytes, including IL6 and TNFα.
We showed that these effects were at least partially mediated by WNT (wingless/integrated) signaling, a pathway that is chronically upregulated in OA. Interestingly our transcriptomic analyses revealed that this pathway instead is repressed in NC, while the WNT inhibitor sFRP1 (secreted frizzled-related protein-1) is highly expressed. We confirmed sFRP1 to also be secreted by NC and observed that its chemical and genetic inhibition reduced the capacity of N-TEC to maintain cartilaginous properties at OA-like conditions. These observations suggested repression of WNT signaling as a possible mechanism enabling NC to resist / reduce inflammation.
OA bone/osteoblasts are known to release factors that can lead to phenotypic changes and degradation of cartilage tissue. When N-TEC were combined with OA bone tissues in different ectopic in vivo models, we could nevertheless demonstrate cell survival, N-TEC integration and maintenance of the cartilaginous properties. Successful engraftment of N-TEC was also observed in a weight bearing orthotopic sheep OA model. Identification of GFP-positive NC in the explant tissues confirmed sustained survival of the implanted cells over the study duration. Observed high levels of the inflammatory cytokines Il1b, IL8 and TNFα in the synovial fluids from OA joints decreased after treatment with N-TEC to cytokine levels comparable to healthy joints.
No adverse reactions occurred in our first treated patients. In a patient's self-reported Knee Injury and Osteoarthritis Outcome Score (KOOS) both patients stated improvements in all the categories, including reduced pain, improved joint function and life quality until 14 months after the treatment. KOOS scores generally further increased with time until our last follow-up time points at 24 or 30 months.
Conclusion: Our findings demonstrated the compatibility of N-TEC with an OA environment, as it not only resisted, but also seemed to positively modulate the chronically inflamed joint environment. Our pre-clinical results indicated that implanted NC directly contribute to cartilage repair and engraftment in OA cartilage defects. To verify the regenerative capacity of N-TEC in patients, a suitably powered phase II clinical trial in a larger cohort of patients with OA is now required.
Mumme M, Barbero A, Miot S, Wixmerten A, Feliciano S, Wolf F, Asnaghi MA, Baumhoer D, Bieri O, Kretzschmar M, Pagenstert G, Haug M, Schaefer DJ, Martin I, Jakob M. Nasal chondrocytes-based engineered autologous cartilage tissue for the repair of articular cartilage defect: an observational first-in-human trial. Lancet 2016 Oct 22;388(10055):1985-1994. doi: 10.1016/S0140-6736(16)31658-0.
Acevedo L, Mumme M, Manferdini C, Darwiche S, Khalil A, Hilpert M, Buchner DA, Lisignoli G, Occhetta P, von Rechenberg B, Haug M, Schäfer D, Jakob M, Caplan A, Martin I, Barbero A, Pelttari K. Engineered nasal cartilage for the repair of osteoarthritic knee cartilage defects. Science Translational Medicine 2021 Sept;13(609). doi:10.1126/scitranslmed.aaz4499.
This project was supported by the Swiss National Science Foundation (310030_149614, PMPDP3_151396 and as part of the NCCR Molecular Systems Engineering SNSF 51NF40-141825), by Colombian Department of Science, Technology and Innovation (Colciencias), Burckhardt-Bürgin Foundation, Forschungsfond of the University of Basel, Swisslife and Deutsche Arthroseforschung.