(628b) In Vitro Bone Model Capture Molecular Regulation of Bone Remodeling

Bone is a dynamic tissue that undergoes repeated remodeling for the maintenance of integrity throughout the lifetime, which includes bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs). This process occurs under the regulation of local growth factors and cytokines. These are stimulatory and suppressive molecules, such as the receptor activator of nuclear factor B ligand (RANKL) and its soluble decoy receptor, osteoprotegerin (OPG). RANKL induces OC differentiation by binding to its receptor, RANK. Whereas, OPG competitively binds to RANKL and inhibits the RANK-RANKL, signaling for OC differentiation. This molecular crosstalk between OB and OC is tightly regulated to localize bone remodeling activity and prevent unnecessary bone formation and resorption. Imbalanced bone remodeling can cause osteoporosis, decreased bone marrow hematopoietic activity, or increased risk of bone metastasis. To develop effective treatments for these conditions, it is imperative to understand the regulation of localized bone remodeling in bone tissue. However, one of the main challenges is that it is difficult to investigate the details of cellular and molecular processes in vivo, since bone tissue is anatomically inaccessible. In an effort to overcome such limitations, bone cells have been studied in vitro by using commercially available culture plates, which allows greater experimental control and access. However, these studies have not been able to reproduce the unique phenotype of bone cells and bone remodeling. A lot of effort has gone into developing in vitro bone tissue models, but no other material system has proven reliable for bone cell cultures.

Here, we report on a new model of in vitro bone tissue using a demineralized bone slice that retains the intrinsic complexity of the extracellular matrix of bones with semi-optical transparency as well as controllable thickness and area (1). To mimic the unmineralized osteoid using demineralized bovine compact bone tissue, we devised a sequential method to rapidly demineralize the bone matrix. After dissolving connective tissue and fat in methanol and chloroform, the clean bone blocks (4- to 5-cm) in 1.2 N hydrochloric acid were placed in a hydrostatic pressure chamber at 4-bar to accelerate the demineralization process. Furthermore, hydrostatic pressure with a 10-s on/off interval highly increases the demineralization depth. We confirmed that the bone blocks fully demineralized after 5 days, whereas demineralization progressed little over the next 4 weeks without the pressure chamber. Then, a demineralized bone block was cryosectioned and tailored into a circular shape to fit in multi-well plates, called demineralized bone paper (DBP) (Fig. 1a). We found that DBP is semi-transparent enough to confirm the functions of bone cells using real-time imaging and enzyme immunoassay. In addition, the fibrillar collagen structure of bone extracellular matrix (ECM) was well-preserved. Widely used tissue culture plates (TCP) and DBP are similarly standardized, bioactive and have optical transparency. However, TCP does not contain bone ECM. Thus, our engineered material could be a good alternative to TCP as a reliable platform for in vitro bone cell culture.

We further characterized the mineralization of mouse OB on DBP and TCP. Alizarin red staining made apparent that OBs fully demineralized the DBP surface after 4 days of culture and kept depositing minerals for more than 2 weeks (Fig. 1b). Alternatively, OBs on TCP deposited few mineral nodules in the same time period, indicating that DBP served as a template for rapid mineralization of OBs in osteoid bone in vivo (2). Interestingly, OBs acquire the bone lining cell phenotype on DBP, which is a quiescent OB. Seeded OBs showed decreased migration and proliferation over time. After 2 weeks of culture on DBP, OBs showed two times lower the migration rate than that of OBs on TCP. Immunofluorescent staining with the mitogenic marker Ki67+ after a 1-week culture showed four times down-regulated proliferation on DBP but three times up-regulated Ki67 expression on TCP (Fig. 1c). Not only was the migration and proliferation of bone lining cell phenotypes of OBs on DBP, there was also a higher OPG secretion and lower RANKL secretion than OBs on TCP. Interestingly, we confirmed that the bone lining cells on DBP are capable of regaining OB activity when they have been reseeded on TCP in the phenotypic switching assay, physically disrupted in the bone surface healing assay, or chemically stimulated with Vitamin D3 (VD3) and Prostaglandin E2 (PGE2). When the bone lining cells on DBP were isolated from mineralized ECM or returned to the damaged area, their migration and proliferation increased, indicating that they retained the ability to revert to active OBs (Fig. 1d). Likewise, under chemical stimulation, the bone lining cells switched from a suppressive secretory profile (high OPG and low RANKL) to a stimulatory secretory profile (low OPG and high RANKL). This result suggests that bone lining cells use paracrine signaling to actively regulate the extent and duration of localized bone remodeling. These results are consistent with recent evidence that bone lining cells are a major source of OBs in vivo (3). Bone lining cells cover non-remodeling bone surfaces and are directly involved in the biochemical regulation of bone remodeling, but little is known about them because there are no definitive surface markers to specifically identify them. Our experiments captured phenotypic differences and the transition from bone lining cells to OBs. To the best of our knowledge, this is the first demonstration of the controlled and reversible activation of bone lining cells under physiologically relevant chemical stimulation. This makes it possible to study the initiation and termination of the bone remodeling cycle, which is difficult to achieve with existing models.

By taking advantage of DBP, we developed the trabecular organoid model to investigate the impact of spatiotemporal profiles of regulatory molecules on OC differentiation and OB activity. In healthy trabecular bone, remodeling activity is locally regulated by surrounding resting surfaces. The active and resting surfaces secrete different profiles of stimulatory and suppressive molecules with a unique spatiotemporal pattern. Unbalanced remodeling could cause excessive bone resorption, decreasing bone thickness and increasing cavity diameter. We hypothesized that localized bone remodeling is maintained by not only integrated metabolic but also morphological regulation. To test this hypothesis, coexisting resting and active bone surfaces were simulated in a well plate (Fig. 1e). A resting bone surface was reproduced by culturing DBP disks with resting-state bone lining cells. Then, active OBs were cultured on the DBP insert, which was made by fastening DBP between two concentric O-rings. The insert was separately activated with VD3 and PGE2 since it was transferable to different well plates. Furthermore, we added ring-shaped spacers between the DBP disk and the DBP insert to mimic trabecular bone cavities. Bone marrow mononuclear cells (BMMs) were also co-cultured to investigate the impact of regulatory molecules on OC differentiation (Fig. 1f). Molecular and morphological changes of trabecular bone spaces were simulated by using 6-, 10-, and 14-mm-diameter inserts to represent different-sized areas of the active bone surface, and 0.5-, 1.5-, and 4.5-mm spacers to simulate different-sized trabecular bone spaces. After being co-cultured for 1 week, the effects of spatiotemporal profiles of regulatory molecules on BMMs and bone lining cells was elucidated based on the quantitative multiplex immunostaining. This targeted tartrate-resistant acid phosphatase (TRAP) to monitor OC emergence and alkaline phosphatase (ALP) to measure OB activation. The larger areas of activated DBP inserts increased the OC differentiation of BMMs and activation of bone lining cells.

On the other hand, the size of the gap between the bone disk and insert showed a weak effect, possibly because the tested dimensions were too large to capture the diffusion gradient of regulatory molecules and paracrine signaling. In addition, quantitative image analysis revealed the functional coupling of OB and OC via direct cell-to-cell contact. ALP expression in OBs that make contact with mature OCs highly decreased, indicating that mature OCs activate resting OBs via direct contact. Collectively, the trabecular bone organoid model demonstrated the effect of spatiotemporal profiles of regulatory molecules and OB-OC coupling via direct contact for localized bone remodeling.

Finally, we created a tissue-inspired trabecular bone model in a well plate by co-layering stimulated and resting bone tissue surfaces together, reproducing cellular connection and paracrine signaling between OB and OC. The molecular and cellular crosstalk of these cells was closely investigated using the high fidelity and analytical power of the trabecular bone organoid model. Moreover, in future work, we will create the humanized bone organoid model by replacing bovine bone and mouse cells with human bone and cells. The humanized model could improve preclinical studies and drug screening for osteoporosis. We envision that the established model will facilitate a better understanding of the numerous aspects of bone remodeling biology with high experimental control and access.

References

1. Park et al., Trabecular bone organoid model for studying the regulation of localized bone remodeling, Sci. Adv. 7:eabd6495 (2021)

2. Boonrungsiman et al., The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc. Natl. Acad. Sci. U.S.A. 109, 14170–14175 (2012)

3. Matic et al., Quiescent bone lining cells are a major source of osteoblasts during adulthood. Stem Cells 34, 2930–2942 (2016)