Researchers from Charité – Universitätsmedizin Berlin plan to use 3D in vitro models in their quest for new treatments against rheumatic disease, osteoarthrosis and bone diseases. 3D models also have the potential to reduce the need for animal research in the future.
Musculoskeletal research without any non-animal-based methods? In just a few years’ time, this could be reality. The feasibility of this prospect is currently being demonstrated by the research group led by Prof. Dr. Frank Buttgereit of Charité’s Department of Rheumatology and Clinical Immunology. The Buttgereit Lab’s four new 3D in vitro models – all made using human cells – are intended for use in the development of new treatment strategies against musculoskeletal disorders such as rheumatoid arthritis, osteoarthritis, osteoporosis and abnormalities in fracture healing. Support for these endeavors is being provided by the Federal Ministry of Education and Research (BMBF), the foundations ‘Elsbeth Bonhoff Stiftung’ and ‘Wolfgang Schulze Stiftung’, and industry sources. The outlook is promising.
“We expect our models to be more robust and meaningful for patients. This is because findings from animal research often have limited generalizability,” explains Dr. Annemarie Lang, a postdoctoral researcher in the Buttgereit Lab. She adds: “By using human models, we also hope to reduce the need for animal research. It is still common for animals to be used in research, mainly because there is a lack of suitable alternatives.” However, Dr. Lang is also keen to point out that animal research remains integral to developing new insights and testing new treatments. “It is therefore unlikely we will be able to dispense with them entirely.”
From hip bone to human experimental model
The raw material for these potential new alternatives to animal research is harvested from tissue which has been removed from patients undergoing hip surgery. Once harvested, it is used as a source for the cells used in human models. The first step is to isolate mesenchymal stromal cells from the bone marrow. These multipotent cells can differentiate into a variety of different tissues, including bone and cartilage. To make the models, clusters of cells are centrifuged and exposed to biomechanical stress. Once the samples have been condensed in this manner, they are used to create 3D structures. This process produces small, reproducible constructs with diameters ranging from millimeters to centimeters. Depending on the model required, different differentiation factors will then be used to produce constructs resembling either cartilage or bone. The final 3D model is constructed from the individual components. Where necessary, these may need to be combined with other immune cells, enzymes and/or other components. “The aim is to produce as complex a 3D model of a specific disease as possible,” emphasizes Dr. Lang.
Mathematical models to simulate osteoarthrosis
The osteoarthrosis model, for instance, was ‘built’ from in-house-produced in vitro articular cartilage and mimics specific aspects of osteoarthrosis, one of the most common joint disorders. Osteoarthrosis does not – as was previously thought – develop exclusively due to natural wear and tear. It also develops as a result of inflammatory processes on the molecular level. Certain endogenous messengers which stimulate the immune system, such as cytokines, therefore play a crucial role in progressive joint destruction. This is why the researchers joined forces with mathematicians from the Zuse Institute Berlin to develop a mathematical model which simulates what happens when the cartilage produced by the researchers in-house is exposed to cytokines. What happens is osteoarthrosis. “We now want to use this real-life osteoarthrosis model to test the first potential treatments,” says veterinary surgeon Dr. Lang.
In December 2019, Dr. Annemarie Lang and Dr. Reinhald Ehrig (Charité and Zuse Institute) received the Berlin Research Award for Alternatives to Animal Research for their ‘virtual joint’. It was the third time a Charité researcher had been honored with the award.
Bone fractures and osteoporosis cause problems in patients with rheumatic disease
Another model that is almost ready for practical application is the researchers’ bone model, which simulates bone fractures and bone healing. The long-term objective here is to use drugs to correct abnormal bone healing. “A plethora of reactions take place at the fracture site within the first hours after injury. For instance, a fracture hematoma will develop, immune cells invade the fracture site, and the healing response is initiated,” explains Moritz Pfeiffenberger, a doctoral student who forms part of the Buttgereit Lab. “This complex process is what we want to reconstruct, because this initial phase is particularly sensitive to negative influences which can impair the healing process.”
The researchers’ new insights may be of particular benefit to patients with rheumatic disease. This is because fracture healing is often impaired due to a dysfunctional immune system. Many people with rheumatic disease also have glucocorticoid-induced osteoporosis. This, in turn, increases their risk of fractures, and creates the vicious circle which the researchers are hoping to break. They will develop another new model specifically for the purpose of developing treatments for glucocorticoid-induced osteoporosis.
Everything a joint needs to live
“Our models are highly complex systems which we hope will help us to better understand cellular mechanisms and enable us to get a first idea of the potential efficacy of therapeutic strategies,” says doctoral student Alexandra Damerau, who was awarded the 2018 Lush-Prize for a model which provides an alternative to animal testing. The Berlin-based biotechnologist played a major role in the development of the group’s human arthritis model. She hopes the model will help reduce the need for animal research and deliver improved, human-based research results.
For this model, Damerau and her colleagues developed an entire joint, which consists of bone, cartilage, synovial fluid and synovial membrane. The researchers then add immune cells and inflammatory cytokines to the 3D model in order to adequately simulate the early stages of the autoimmune disease rheumatoid arthritis.
The joint model contains all the relevant tissue components and cell types. This enables interactions between cells, be that via cell contacts or via signaling molecules and metabolites, and closely mimics the in vivo situation in the living organism, explains Head of Laboratory Dr. Timo Gaber. Mid-2020 will see the researchers start to use the miniature joints to test potential drug-based treatments.
“Research to improve treatments and reduce the need for animal research”
Will alternative models render animal research unnecessary? Not quite, according to the researchers. However, human models can act as a filter, meaning that animal testing will only ever be necessary for truly promising candidate drugs. In other words, substances which fail at this initial hurdle will never move on to animal testing.
“It is our express aim to show that our models are capable of achieving better results, and that they could potentially replace animal testing,” emphasizes Prof. Dr. Frank Buttgereit.
As the researchers publish the protocols used to make their models, other institutions are able to copy their 3D models and use them for their own research purposes. This has a potentiating effect. Rheumatic disease researcher Prof. Dr. Frank Buttgereit reports: “There has already been enormous interest from within the wider research community, which suggests there are people all over the world who will want to copy our non-animal-based models.”
(Text: Beatrice Hamberger)
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