Research ArticleAPPLIED SCIENCES AND ENGINEERING

Trabecular bone organoid model for studying the regulation of localized bone remodeling

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Science Advances  20 Jan 2021:
Vol. 7, no. 4, eabd6495
DOI: 10.1126/sciadv.abd6495
  • Fig. 1 Development and characterization of DBP.

    (A) A bovine femur was cut into blocks, (i) cleaned, and (ii) demineralized in HCl solution. (iii) Demineralization was limited by the diffusion limit. (B) We accelerated demineralization with a programmable cyclic pressure chamber. (C) Cross-sectional images of the bones processed in three different conditions and soaked in rhodamine dye show demineralization depth (n = 5). (D) Radiographs of processed and unprocessed bone blocks confirmed full demineralization (n = 3). (E) (i) Demineralized bone was sectioned into 20- to 100-μm slices to form DBP and (ii) cut into disks that fit multiwell plates. (F) (i) Cross sections of DBP with three thicknesses and corresponding (ii) optical transparency [percentage that of tissue culture plate (TCP)] and (iii) stiffness (n = 6). (G) DBP preserves the micro/nano collagen structure of bone. (i) Transverse-sectioned DBP has concentric lamellae and (ii) vertically sectioned DBP has parallel lamellae and (iii) densely aligned collagen fibril bundles [scanning electron micrographs (SEM)]. (H) Biochemical integrity of collagen is preserved versus heat-denatured control. (i) Fluorescent collagen hybridizing peptide stained images; (ii) multiphoton second harmonic generation images. (I) Removal of cellular materials by SDS was confirmed by nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining (n = 30) (a.u., arbitrary units; *P < 0.05, **P < 0.01). Photo credit: Yongkuk Park, University of Massachusetts Amherst.

  • Fig. 2 Osteoblasts rapidly mineralize DBP in a way that preserves the underlying lamellar structure.

    (A) (i) Osteoblasts (OBs) harvested from DsRed mice. (ii) Fluorescent micrograph of OBs migrating out from mouse bone chips. (B) Morphology of OBs grown on vertically sectioned DBP and TCP for 1 week: (i) immunofluorescent staining of actin filaments; (ii) circular histogram of cell alignment angles (n = 100). (C) Collagen deposition by OBs on DBP and TCP for 1 week: (i) multiphoton second harmonic generation (SHG) images; (ii) circular histogram of collagen fiber alignment angles (n = 100). (D) Mineral deposition by OBs on DBP and TCP: (i) alizarin red mineral stain on days 0 and 4; (ii) time-course measurement of mineral deposition for 16 days (n = 3). (E) (i) Confocal images of fluorescent calcein staining show mineral deposition pattern on DBP and TCP after 1-week culture. (ii) z-staked cross-sectional image. (F) Cross section of 100-μm-thick DBP stained with alizarin red after 3-week culture of OBs (n = 3). (G and H) Comparison of the mineral layer deposited by 3-week culture of OBs and chemical reaction in simulated body fluid without OBs (both subjected to thermal decomposition): (G) bright-field micrographs. (H) SEM and surface roughness quantified by optical profiler (n = 6) (*P < 0.05, **P < 0.01). Photo credit: Yongkuk Park, University of Massachusetts Amherst.

  • Fig. 3 OBs on DBP acquire the bone lining cell phenotype.

    (A) OBs on DBP migrated more slowly than those on TCP (n = 25). (B) OB on DBP proliferated less than those on TCP (n = 3). (C) OBs on DBP developed gap junction–mediated intercellular communication. (i) Immunofluorescent staining of connexin 43. (ii) Time-lapse monitoring of fluorescent Ca2+ flux in adjacent cells. (D) Bone surface healing assay: (i) Repair of mineral surface was monitored after scratch in 1-week culture of OBs on DBP; (ii) quantitative measurement of transiently increased OB migration during healing process (n = 3 to 5). (E) OBs cultured on DBP for 1-week regained proliferative activity when cultured on TCP. (i) Fluorescent images of mature OBs migrating out of DBP treated with collagenase and proliferating on TCP. (ii) Within 1 week, mature OBs released from DBP and grown on TCP regained mitogenic activity similar to that of OBs grown continuously on TCP (n = 10). (F) OB phenotypic switching assay: (i) experimental procedure and metabolic state changes in OBs. (ii) The cycle of switching from resting state on DBP to proliferative state on TCP was successfully repeated three times (n = 4) (*P < 0.05, **P < 0.01, ns, not significant).

  • Fig. 4 The bone remodeling cycle is recapitulated by coculture of bone lining cells and BMMs on DBP under chemical stimulation.

    (A) Experimental procedure to simulate a bone remodeling cycle. (B) Stimulation of bone lining cells on DBP with VD3 and PGE2 caused a temporary increase in RANKL/OPG secretion ratio (n = 3 to 5). (C) Representative fluorescent images show that activated OBs (green) induce BMMs (red) to differentiate into osteoclasts (OCs). (D) SEM confirmed functional mineral resorption by OCs. (E) Stimulated OBs migrated two times faster than their unstimulated counterparts on both DBP and TCP (n = 4). (F) OC migration in single culture and coculture on DBP and TCP (n = 10). (G) On DBP, OCs underwent both cell fission and cell fusion. (H) On TCP, OBs were readily pushed by large multinucleated OCs, whereas on DBP, the OBs stayed in place. (I) On TCP, OCs underwent cell fusion repeatedly until cells became giant and underwent apoptosis. After apoptosis, the large actin-ring structure of the OC remained and prevented immediate migration of neighboring OBs. (J) (i) Alkaline phosphatase (ALP) staining of OBs and (ii) quantitative comparison (n = 3 to 6). (K) (i) Tartrate-resistant acid phosphatase (TRAP) staining of multinucleated OCs and (ii) quantitative comparison (n = 10) (*P < 0.05, **P < 0.01, ns, not significant).

  • Fig. 5 The trabecular bone organoid model recapitulates coexisting active and resting bone surfaces.

    (A) Excessive bone remodeling results bone loss and changes in the trabecular bone morphology, which could compromise anatomical regulation of localized bone remodeling. (B) (i) DBP inserts were prepared by securing 100-μm-thick DBP between two acrylic O-rings. (ii) DBP inserts were suspended above DBP disks with ring-shaped spacers. OBs cultured on a DBP insert. (C) The trabecular bone organoid model consists of a DBP insert that has been activated by VD3 and PGE2 suspended over a DBP disk containing bone lining cells. This juxtaposition of active and resting surfaces with a shared microenvironment simulates in vivo–relevant gradients of stimulatory and suppressive molecules. (D) (i) OBs cultured on DBP inserts acquired the bone lining cell secretory profile. (ii) VD3 and PGE2 stimulation increased RANKL secretion in proportion to surface area of insert (n = 5). (E) Experimental design. (F and G) DBP with bone lining cells was cocultured with BMMs and (F) three insert sizes or (G) three spacer heights. After 6 days of coculture, TRAP+ multinucleated OCs and total area of ALP+ OBs were measured (n = 3) (*P < 0.05, **P < 0.01, ns, not significant). Photo credit: Yongkuk Park, University of Massachusetts Amherst.

  • Fig. 6 Quantitative spatial mapping of OBs and OCs captures paracrine- and cellular contact–mediated regulation of bone remodeling.

    (A) DBP disks in a well plate were scanned with fluorescent channels for TRAP, ALP, GFP, and DAPI. The resulting 218 multiplex images were stitched together to represent the entire surface of the DBP disk. (B) Illustration of quantitative spatial imaging analysis for creating heatmaps of counts of TRAP+ and multinucleated (>3) OCs (top) and areas of ALP+ and GFP+ OBs (bottom). (C) (i) The DBP surface was discretized into seven concentric zones. (ii) Plot of activity levels for each region in control. (D) Representative heatmaps of ALP+ OB activity and regional OB and OC activities with linear analysis to correlate localized bone remodeling cellular activity and paracrine signaling from coculture experiments (n = 3 to 5). (E) Representative images of TRAP and ALP in central zone 1 and peripheral zone 6. (F) (i) Representative multiplex images showing delineation of OCs and OBs. (ii) Quantitative comparison of ALP+ OBs that are and are not in direct contact with OCs (n = 30). (G) Proposed mechanism of localized trabecular bone remodeling regulation (*P < 0.05, **P < 0.01, ns, not significant).

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