Engineering approaches to manipulate osteoclast behavior for bone regeneration

Abstract

Extensive research has delved into the multifaceted roles of osteoclasts beyond their traditional function in bone resorption in recent years, uncovering their significant influence on bone formation. This shift in understanding has spurred investigations into engineering strategies aimed at leveraging osteoclasts to not only inhibit bone resorption but also facilitate bone regeneration. This review seeks to comprehensively examine the mechanisms by which osteoclasts impact bone metabolism. Additionally, it explores various engineering methodologies, including the modification of bioactive material properties, localized drug delivery, and the introduction of exogenous cells, assessing their potential and mechanisms in aiding bone repair by targeting osteoclasts. Finally, the review proposes current limitations and future routes for manipulating osteoclasts through biological and material cues to facilitate bone repair.

Keywords: Biomaterials; Bone coupling; Bone regeneration; Drug delivery; Osteoclasts.

Graphical abstract

Fig. 1

A scheme of osteoclast differentiation and the interaction between osteoclasts and other bone resident cells in the bone microenvironment. Mature (activated) osteoclasts develop from the fusion of multiple individual peripheral blood-borne mononuclear cells, and are characterized by cytomembrane, multiple nuclei and ruffled border responsible for the degradation of bone matrix. Osteoclasts, pre-osteoblasts, osteoblasts, and osteocytes have reciprocal interactions by different cytokines to maintain bone homeostasis. Plus signs and minus signs indicate positive and negative regulators, respectively.

Fig. 2

Osteoclast signaling pathways. M-CSF and RANKL signaling are two predominant pathways involved in osteoclast differentiation. The interaction between M-CSF and cFms results in recruitment of PI3K and Grb2, which plays an important role in maintenance of the survival of osteoclast. The RANKL-RANK interaction activates several signaling cascades, including MAPKs, NF-κB, and AKT pathways, to induce osteoclast differentiation, activation and proliferation via the TRAF adaptor proteins (mainly TRAF6). Besides, RANKL signaling can evoke Ca²⁺ oscillations via activation of PLCγ, which induces the release of Ca²⁺ from intracellular Ca²⁺ store sites such as the mitochondria and endoplasmic reticulum, to prompt calcineurin-dependent dephosphorylation and activation of NFATc1, allowing the differentiation of osteoclasts. Ca²⁺ entry through TRP2/4 (Ca²⁺-permeable channels) occurs simultaneously with intracellular Ca²⁺ release, also contributing to the Ca²⁺ oscillations and affecting osteoclast differentiation. Wnt5a can also activate calcium signaling via calcineurin and PKC signals by binding to a receptor complex including Ror 1/2 and a frizzled receptor. As a co-stimulatory way of RANK signaling, Ig-like receptors (OSCAR and TREM2) associate with transmembrane adapter proteins (FcRγ and DAP12), which contain ITA motifs, the phosphorylation of the motifs leads to the activation of PLCγ, Ca²⁺, β-catenin, and ERK signaling, which are critical for osteoclast cell proliferation and cytoskeleton rearrangement. DAP12 can solely work with α_vβ₃ integrin to regulate the osteoclast cytoskeleton and actin ring formation through activation of PLCγ, VAV and ERK.

Fig. 3

Osteoclast behavior modulated by ions. (A) The effects of Mg²⁺ and PO4³⁻ on osteoclast differentiation of RAW264.7 cells. (Reprinted with permission from Ref. [52], copyright 2017 Elsevier). (A1) Representative images showing Actin-stained cells (top) and TRAP-stained cells (bottom). (A2) The number of multinucleate cells with more than two nuclei. (A3-5) The expression of osteoclast-specific genes NFATc1, TRAP, and CTSK on mRNA level. WH: whitlockite. (B). The effect of silicic acid on osteoclast differentiation of RAW264.7 cells. (Reprinted with permission from Ref. [57], copyright 2015 Elsevier). (B1) TEM images of calcified (CCS), silicified (SCS), and their biphasic (BCS) mineralized collagen scaffolds. (B2) Cumulative release profiles of silicic acid and Ca²⁺ from the collagen scaffolds. (B3) SCS-conditioned MSCs showed up-regulation of OPG expression and down-regulation of RANKL expression; (B4) Effects of scaffold-conditioned MSCs on osteoclastogenesis and osteoclast function of RAW264.7 cells examined by TRAP staining and resorption pit assay.

Fig. 4

Osteoclast behavior modulated by topographical feature on material surfaces. (A1) RAW264.7 derived osteoclasts and (A2) primary mouse osteoclasts were fewer but exhibited bigger F-actin ring-like structures on smooth surface of titanium disk than those on rougher surfaces of titanium disks with low roughness (TiLR), medium roughness (TiMR), and high roughness (TiHR). (Reprinted with permission from Ref. [64], copyright 2018 The American Chemical Society). (B) Osteoclastic resorption and F-actin organization of rabbit osteoclast precursors after cultured on dentin slices, smoother HAP1, and micro-rough HAP3 surfaces. (Reprinted with permission from Ref. [65], copyright 2013 Elsevier). (B1) Resorption pits indicated by arrowheads and osteoclasts indicated by asterisks. (B2) F-actin (red) staining and DAPI (blue) staining. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Osteoclast behavior manipulated by exogenous cells. (A) TRAP positive cells increased in BCP implants with hMSCs. (Reprinted with permission from Ref. [70], copyright 2014 Elsevier). (B) PSCs mixed with autograft bone increased osteoblast to osteoclast ratio and promoted bone formation. (Reprinted with permission from Ref. [71], copyright 2020 Oxford University Press). (B1) Graft preparation for posterolateral spine fusion, bone graft harvest area (left), preparation of graft with bone morselizer (middle), and the surgical area (right). (B2) Viability of PKH pre-labeled PSC (appearing red) when seeded on bone graft at 1 and 2 h (left), and the kinetics of PSC adhesion to bone graft (middle and right). (B3) ALP staining showed increased osteoblastic activity while TRAP staining showed no change in osteoclasts formation among spine fusion segments. (B4) Culture of mice BMMs with PSC conditioned medium reduced TRAP-positive cells in vitro. (C) Endothelial cells inhibited osteoclast formation and activity. (Reprinted with permission from Ref. [79], copyright 2018 Karger Publishers). (C1) TRAP staining and the numbers of TRAP-positive multinucleated cells showed that ECs suppressed the differentiation of BMMs into osteoclasts in vitro. (C2) TRAP staining and the numbers of TRAP-positive multinucleated cells showed EC suppressed the differentiation of BMMs into osteoclasts by delivering TGF-β1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Osteoclast behavior modulated by mechanical stimuli. (A) Effect of material stiffness on osteoclast activity. (Reprinted with permission from Ref. [81], copyright 2021 John Wiley and Sons). (A1) PDMS substrates with different stiffness by changing the mass ratio of the curing agent to the liquid oligomeric base. (A2) Stiffer PDMS substrate upregulated the expression of NFATc1, CTSK, and DCST1 of osteoclasts. (A3) The number of mature TRAP-positive osteoclasts were decreased with the reduction in substrate stiffness (A4) Compared with decalcified bone slices (soft), untreated bone slices (stiff) could significantly promote the formation of TRAP-positive osteoclasts and resorption lacunae. (B) Effect of fluid shear stress (FSS) on osteoclast activity. (Reprinted with permission from Ref. [82], copyright 2020 International Scientific Information). FSS could reverse the formation of RANKL-induced (B1) TRAP-positive cells, (B2) bone resorption area, and (B3) expression of NFATc1, CTSK, TRAP, and MMP9 in protein level of RAW264.7 cells. (C) Effect of compressive force on osteoclast activity. Exposure to compressive force resulted in (C1) more TRAP‐positive cells and (C2) promotion of the expression of Tks5 and F‐actin and the cell fusion (indicated by dash line) of RAW264.7 cells. (Reprinted with permission from Ref. [83], copyright 2018 John Wiley and Sons). (C3) RAW264.7 cells were cultured on slips and reversed them onto the collagen gel layer to receive compressive force, the optimal compressive force to promote osteoclast formation was approximately 300 mg/7 slips. (Reprinted with permission from Ref. [84], copyright 2015 SPANDIDOS PUBLICATIONS).

Fig. 7

Osteoclast behavior modulated by bisphosphonate. (A) ZOL-loaded scaffolds facilitated bone regeneration through inhibition of osteoclastogenesis. (Reprinted with permission from Ref. [112], copyright 2020 IOP PUBLISHING LTD). (A1) Scheme of the fabrication of ZOL-loaded gelatin NPs integrated porous titanium scaffold. (A2) Morphology of osteoclasts attached on porous titanium scaffolds loaded with different concentrations of ZOL. (A3) The porous titanium scaffolds with high concentration of ZOL (50 μmol/L) inhibited the resorption pits formed by osteoclasts. (A4) micro-CT scans showed high concentration of ZOL-loaded scaffolds induced more bone reconstruction of the femoral condyle defection of OVX rabbits. (B) Col-GO-Aln sponges inhibited osteoclastogenesis; (Reprinted with permission from Ref. [116], copyright 2020 Elsevier). (B1) Scheme of the fabrication procedure and morphology of Col-GO-Aln sponges. Aln released from Col-GO-Aln sponges inhibited osteoclasts formation (B2) in vitro and (B3, B4) in vivo.