Sclerostin Directly Stimulates Osteocyte Synthesis of Fibroblast Growth Factor-23.

Osteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo. In the current study, we sought to address this using a cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 h. Both rhPTH1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH1-34 and rhSCL was antagonised by pre-treatment with the NF-κß signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.

SaOS2 Osteosarcoma cells as an in vitro model for studying the transition of human osteoblasts to osteocytes.

The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST, DMP1, PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1. SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.

3D Bioprinting of Methylcellulose/Gelatin-Methacryloyl (MC/GelMA) Bioink with High Shape Integrity.

The advent of three-dimensional (3D) bioprinting offers a feasible approach to construct complex structures suitable for tissue regeneration, during which cell-laden materials are dispensed on a substrate according to a predesigned structure. However, the lack of ideal printable bioinks with high shape fidelity and improved biological stability remains a major challenge. In this study, methylcellulose/gelatin-methacryloyl (MC/GelMA) bioink with high shape integrity is presented, which takes advantage of the printability of MC and the permanent photo-cross-linking of GelMA under UV irradiation. Although MC demonstrates good printability at room temperature, the lack of cross-linking ability causes distortion and finally dissociation of printed MC in biological media within a few days. However, UV-cross-linked MC/GelMA bioink remains stable in biological media over a period of several months. The shape integrity of MC/GelMA was systematically characterized in terms of yield stress and complex modulus. Unlike pure MC ink, the MC/GelMA ink demonstrated self-supporting behavior once printed due to the higher complex modulus and yield stress induced by GelMA in the system. Shape integrity of MC/GelMA ink resulted in higher resolution and printability which are evaluated by the successful printing of various 1D, 2D, and 3D constructs. Moreover, human primary osteoblasts encapsulated within the MC/GelMA hydrogel show cell viability of >95%. Overall, this work introduces MC/GelMA bioink with high shape integrity and improved biological stability and highlights the importance of rheological properties and post-cross-linking for fabrication of physiologically scaled tissue implants.

Evidence for Gender-Specific Bone Loss Mechanisms in Periprosthetic Osteolysis.

Osteolysis adjacent to total hip replacement (THR) prostheses is a major cause of their eventual failure. Periprosthetic osteolysis is associated with the production of bioactive particles, produced by the wear of articulating prosthesis surfaces. Wear particles invade the periprosthetic tissue, inducing inflammation and bone resorption. Previous studies have shown that osteocytes, the most numerous cell type in mineralised bone, can respond to wear particles of multiple orthopaedic material types. Osteocytes play important roles in bone resorption, regulating bone resorption by osteoclasts and directly through osteocytic osteolysis, also known as perilacunar remodelling. In this study, we perform a histological analysis of bone biopsies obtained from cohorts of male and female patients undergoing either primary THR surgery or revision THR surgery for aseptic loosening. The osteocyte lacunae area (Ot.Lac.Ar) and percentage lacunar area/bone area (%Ot.Lac.Ar/B.Ar) were significantly larger overall in revision THR bone than bone from similar sites in primary THR. Analysis by patient gender showed that increased Ot.Lac.Ar, indicative of increased perilacunar remodelling, was restricted to female revision samples. No significant differences in osteoclast parameters were detectable between the cohorts. These findings suggest previously unrecognised gender-specific mechanisms of bone loss in orthopaedic wear particle-induced osteolysis in humans.

Osteocytes respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal alloys by up-regulation of resorptive and inflammatory pathways.

Periprosthetic osteolysis is a major cause of implant failure in total hip replacements. Aseptic loosening caused by osteolytic lesions is associated with the production of bioactive wear particles from the articulations of implants. Wear particles infiltrate the surrounding tissue of implants, promoting inflammation as well as bone resorption. Osteocytes have been shown to both regulate physiological osteoclastogenesis and directly remodel their perilacunar bone matrix by the process of osteocytic osteolysis. We hypothesise that osteocytes respond to wear debris of orthopaedic implant materials by adopting a pro-catabolic phenotype and thus contribute to periprosthetic osteolysis through the known pathways of bone loss. Osteocyte responses to particles derived from clinically relevant materials, ultra-high molecular weight polyethylene (UHMWPE), highly cross-linked polyethylene (XLPE) and metal alloys, Ti6Al4V and CoCrMo, were examined in vitro in human primary osteocyte-like cultures. Osteocyte-like cells exposed to both polyethylene and metal wear particle types showed upregulated expression of catabolic markers associated with osteocytic osteolysis, MMP13, carbonic anhydrase 2 (CA2) and cathepsin K (CTSK). In addition, pro-osteoclastogenesis markers RANKL and M-CSF were induced, as well as the expression of pro-inflammatory cytokines, IL-6 and TNFα, albeit with different kinetics. These findings suggest a previously unrecognised action of wear particles of multiple orthopaedic materials on osteocytes, and suggest a multifaceted role for osteocytes in periprosthetic osteolysis. STATEMENT OF SIGNIFICANCE: This study addresses periprosthetic osteolysis, a major clinical problem leading to aseptic loosening of orthopaedic implants. It is well accepted that wear particles of polyethylene and of other implant materials stimulate the activity of bone resorbing osteoclasts. Our recent work provided evidence that commercial particles of ultra-high molecular weight polyethylene (UHMWPE) stimulated osteocytes to adopt a bone catabolic state. In this study we demonstrate for the first time that particles derived from materials in clinical use, conventional UHMWPE, highly cross-linked polyethylene (XLPE), and Ti6Al4V and CoCrMo metal alloys, all stimulate human osteocyte activities of osteocyte-regulated osteoclastogenesis, osteocytic osteolysis, proinflammatory responses, osteocyte apoptosis, albeit to varying extents. This study provides further mechanistic insight into orthopaedic wear particle mediated bone disease in terms of the osteocyte, the most abundant and key controlling cell type in bone.

Evidence that osteocyte perilacunar remodelling contributes to polyethylene wear particle induced osteolysis.

Periprosthetic osteolysis (PO) leading to aseptic loosening, is the most common cause of failure of total hip replacement (THR) in the mid- to long-term. Polyethylene (PE) particulates from the wear of prosthesis liners are bioactive and are implicated in the initiation and or progression of osteolysis. Evidence exists that cells of the osteoblast/osteocyte lineage are affected by PE particles and contribute to the catabolic response by promoting osteoclastic bone resorption. In this study, we hypothesised that osteocytes contribute directly to PO by removing bone from their perilacunar matrix. Osteocyte responses to ultra-high molecular weight PE (UHMWPE) particles were examined in vitro in human primary osteocyte-like cultures, in vivo in the mouse calvarial osteolysis model, and in the acetabulum of patients undergoing revision total hip replacement (THR) surgery for PO. Osteocytes exposed to UHMWPE particles showed upregulated expression of catabolic markers, MMP-13, carbonic anhydrase 2 (CA2), cathepsin K (CTSK) and tartrate resistant acid phosphatase (TRAP), with no effect on cell viability, as assessed by Caspase 3 activity. Consistent with this catabolic activity causing perilacunar bone loss, histological analysis of calvarial sections from mice exposed to UHMWPE revealed a significant (p<0.001) increase in osteocyte lacunar area (Lac.Ar) compared to sham-operated animals. Furthermore, acetabular biopsies from patients with PO also showed significantly (p<0.001) increased osteocyte lacunar size in trabecular bone adjacent to PE particles, compared with osteocyte lacunar size in bone from primary THR patients. Together, these findings suggest a previously unrecognised action of UHMWPE wear particles on osteocytes, which directly results in a loss of osteocyte perilacunar bone. This action may exacerbate the indirect pro-osteoclastic action of UHMWPE-affected osteocytes, previously shown to contribute to aseptic loosening of orthopaedic implants. STATEMENT OF SIGNIFICANCE: This study addresses the clinical problem of periprosthetic osteolysis, bone loss in response to polyethylene wear particles derived from materials used in orthopaedic implants. Periprosthetic osteolysis has been thought to be due largely to wear particles stimulating the activity of bone resorbing osteoclasts. However, in this study we demonstrate for the first time that polyethylene particles stimulate another type of bone loss, mediated by the direct activity of bone mineral embedded osteocytes, termed osteocytic osteolysis or osteocyte perilacunar remodelling. This study provides new mechanistic insight into wear-particle mediated bone loss and represents a new paradigm for the way in which bone cells, namely osteocytes, the key controlling cell type in bone, react to biomaterials.

Regulation of FGF23 expression in IDG-SW3 osteocytes and human bone by pro-inflammatory stimuli.

Fibroblast growth factor-23 (FGF23), produced by osteocytes, is the key physiological regulator of phosphate homeostasis. Sepsis patients often experience transient hypophosphataemia, suggesting the regulation of FGF23 levels by pro-inflammatory factors. Here, we used the osteocyte-like cell line IDG-SW3 to investigate the effect of pro-inflammatory stimuli on FGF23 production. In differentiated IDG-SW3 cultures, basal Fgf23 mRNA was dose-dependently up-regulated by pro-inflammatory cytokines TNF, IL-1ß and TWEAK, and bacterial LPS. Similar effects were observed in human bone samples. TNF- and IL-1ß-induced Fgf23 expression was NF-κB-dependent. Conversely, mRNA encoding negative regulators of FGF23, Phex, Dmp1 and Enpp1, were suppressed by TNF, IL-1ß, TWEAK and LPS, independent of NF-κß signalling. Galnt3, the protein product of which protects intact FGF23 protein from furin/furin-like proprotein convertase cleavage, increased in response to these treatments. C-terminal FGF23 and intact FGF23 protein levels also increased, the latter only in the presence of Furin inhibitors, suggesting that enzymatic cleavage exerts critical control of active FGF23 secretion by osteocytes. Our results demonstrate in principle that pro-inflammatory stimuli are capable of increasing osteocyte secretion of FGF23, which may contribute to hypophosphataemia during sepsis and possibly other inflammatory conditions.

Analysis of vitamin D metabolism gene expression in human bone: evidence for autocrine control of bone remodelling.

The metabolism of 25-hydroxyvitamin D (25D) to active 1α,25-dihydroxyvitamin D (1,25D) by endogenous expression of 25D 1-α hydroxylase (CYP27B1) in bone cells appears to have functional effects in both osteoclasts and osteoblasts. To examine relationships between CYP27B1 expression in bone and its potential function in vivo, we examined the expression of vitamin D metabolism genes (CYP27B1, CYP24A1, VDR) in human trabecular bone samples and compared them by linear regression analysis with the expression of osteoclast (TRAP, CA2, CATK, NFATC1), osteoblast (TNAP, COL1A1, OCN, MEPE, BRIL), osteocyte (DMP1, SOST, PHEX, MEPE, FGF23)-related gene markers, genes associated with osteoblast/osteocyte control of osteoclastogenesis (RANKL, M-CSF, OPG, IL-8, TWEAK) and transcription factors (NFATC1, RUNX2, OSX, MSX2, HIF1A). This revealed multiple significant gene expression relationships between CYP27B1 and the transcription factors RUNX2, NFATC1, consistent with the coordinated expression of this gene by both osteoblast and osteoclast-lineage cells, and with MSX2 and the hypoxia-inducible transcription factor, HIF1A. CYP27B1 expression associated mainly with gene markers of bone resorption. VDR mRNA expression was also associated with resorption-related genes. Against expectations, CYP27B1 expression did not associate with bone expressed genes known to be 1,25D responsive, such as OCN, RANKL and DMP1. The major implication of these relationships in gene expression is that endogenous 1,25D synthesis and the response to 1,25D in human trabecular bone is linked with coordinated functions in both the osteoclastic and osteoblastic compartments towards the control of bone remodelling. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Calcium induces pro-anabolic effects on human primary osteoblasts associated with acquisition of mature osteocyte markers.

Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.

Sclerostin regulates release of bone mineral by osteocytes by induction of carbonic anhydrase 2.

The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.