Using bone explants to study bone remodeling

Results of in vitro experiments are often not representative of what is observed in vivo, potentially because the complex interplay between different bone cell types and their arrangement in the original extracellular matrix (ECM) is missing for the in vitro case.

Explants

For her PhD research, Esther Cramer studied bone remodeling and related factors that influence the bone remodeling process in bone explant cultures. In addition, Cramer investigated if bone explants could be used as a defect model to test new bone substitutes.

Explant cultures, also known as ex vivo cultures, include tissue which is explanted from living bone and maintained in vitro. These cultures preserve tissue specific cells in their native ECM, thereby providing a unique environment to study remodeling because a close representation of the natural bone environment is realized.

Explant cultures can provide insights on bridging the gap between in vitro and in vivo experimentation as well as addressing key ethical considerations to reduce, refine, and replace animal studies (the 3Rs), where such tissues are obtained from human tissue or left-over material from a slaughterhouse.

Challenge

A major challenge in the analysis of remodeling ex vivo is to recognize newly formed and resorbed tissue and to discriminate it from bone that already existed at the start of culture experiment.

Cramer and her collaborators developed a culture chamber that allowed longitudinal monitoring of bone formation and resorption using µCT scanning. With this technique, bone formation and resorption could be uncoupled, which is important in testing the effects of new therapies in an ex vivo model of tissue.  

Thus far, studies involving bone explants showed a clear focus on achieving bone formation and neglected osteoclast activity. Preservation of active osteoclastogenesis and resorption in explant cultures is challenging because of osteoclasts’ short lifespan and lack of vascularization ex vivo, limiting the source of osteoclast precursors.

To address this issue, Cramer added peripheral blood mononuclear cells (PBMCs) as a source of osteoclast precursors to the explants. It was demonstrated that these precursor cells were able to differentiate towards osteoclasts and showed early signs of enhanced resorption compared to explants without PBMCs. The addition of PBMCs provides a way of including osteoclast activity in ex vivo bone cultures, which is essential for investigating bone remodeling processes.

Turning to biomaterials

For the second part of her PhD thesis, Cramer demonstrated the applicability of ex vivo cultures as bone defect model by implanting different biomaterials.

A novel supramolecular ureido-pyrimidinone (UPy) hydrogel was functionalized with alendronate (ALN), a bisphosphonate used in the treatment of osteoporosis because it induces osteoclast apoptosis.

Initial in vitro experiments revealed an inhibiting effect of UPy-ALN hydrogels on osteoclast activity, while simultaneously facilitating osteogenic activity and matrix formation. Utilizing the ex vivo model, it was demonstrated that UPy-ALN cross-linked rapidly in a bony environment, preserved its structural integrity in culture, and could induce migration of cells from the surrounding bone.

Key contribution

Cramer’s research contributes significantly to the development of ex vivo models for the study of bone remodeling. The ex vivo bone defect model could be used to perform studies with novel bone substitutes to investigate parameters which are difficult to study in simple in vitro experiments.

By providing research methods that could improve explant bone cultures and analysis, Cramer’s work provides a foundation for future research, thereby contributing to the advance of ex vivo bone cultures into standardized models and endorsing translation from novel therapy design into clinical practice.

Title of PhD thesis: Ex vivo bone cultures for evaluation of remodeling and bone substitutes. Supervisors: Sandra Hofmann and Keita Ito.