Inflammation-associated ectopic mineralization.

Ectopic mineralization refers to the deposition of mineralized complexes in the extracellular matrix of soft tissues. Calcific aortic valve disease, vascular calcification, gallstones, kidney stones, and abnormal mineralization in arthritis are common examples of ectopic mineralization. They are debilitating diseases and exhibit excess mortality, disability, and morbidity, which impose on patients with limited social or financial resources. Recent recognition that inflammation plays an important role in ectopic mineralization has attracted the attention of scientists from different research fields. In the present review, we summarize the origin of inflammation in ectopic mineralization and different channels whereby inflammation drives the initiation and progression of ectopic mineralization. The current knowledge of inflammatory milieu in pathological mineralization is reviewed, including how immune cells, pro-inflammatory mediators, and osteogenic signaling pathways induce the osteogenic transition of connective tissue cells, providing nucleating sites and assembly of aberrant minerals. Advances in the understanding of the underlying mechanisms involved in inflammatory-mediated ectopic mineralization enable novel strategies to be developed that may lead to the resolution of these enervating conditions.

Requirement of Na+/H+ Exchanger NHE1 for Vasopressin-Induced Osteogenic Signaling and Calcification in Human Aortic Smooth Muscle Cells.

BACKGROUND/AIMS: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including upregulation of transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- and glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include upregulation of Na+/H+ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs. METHODS: Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using quantitative real-time polymerase chain reaction, protein abundance by Western blotting, ROS generation with 2',7'-dichlorofluorescein diacetate fluorescence, and ALP activity and calcium content by using colorimetric assays. RESULTS: Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 silencing. Vasopressin increased ROS accumulation, an effect significantly blocked by the NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 or cariporide. CONCLUSION: Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.

Argonaute (AGO) proteins play an essential role in mediating BMP9-induced osteogenic signaling in mesenchymal stem cells (MSCs).

As multipotent progenitor cells, mesenchymal stem cells (MSCs) can renew themselves and give rise to multiple lineages including osteoblastic, chondrogenic and adipogenic lineages. It's previously shown that BMP9 is the most potent BMP and induces osteogenic and adipogenic differentiation of MSCs. However, the molecular mechanism through which BMP9 regulates MSC differentiation remains poorly understood. Emerging evidence indicates that noncoding RNAs, especially microRNAs, may play important roles in regulating MSC differentiation and bone formation. As highly conserved RNA binding proteins, Argonaute (AGO) proteins are essential components of the multi-protein RNA-induced silencing complexes (RISCs), which are critical for small RNA biogenesis. Here, we investigate possible roles of AGO proteins in BMP9-induced lineage-specific differentiation of MSCs. We first found that BMP9 up-regulated the expression of Ago1, Ago2 and Ago3 in MSCs. By engineering multiplex siRNA vectors that express multiple siRNAs targeting individual Ago genes or all four Ago genes, we found that silencing individual Ago expression led to a decrease in BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs. Furthermore, we demonstrated that simultaneously silencing all four Ago genes significantly diminished BMP9-induced osteogenic and adipogenic differentiation of MSCs and matrix mineralization, and ectopic bone formation. Collectively, our findings strongly indicate that AGO proteins and associated small RNA biogenesis pathway play an essential role in mediating BMP9-induced osteogenic differentiation of MSCs.

Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene.

BACKGROUND: In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. RESULTS: In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). CONCLUSIONS: Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.

A functional autophagy pathway is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are capable of differentiating into bone, cartilage and adipose tissues. We identified BMP9 as the most potent osteoinductive BMP although detailed mechanism underlying BMP9-regulated osteogenesis of MSCs is indeterminate. Emerging evidence indicates that autophagy plays a critical role in regulating bone homeostasis. We investigated the possible role of autophagy in osteogenic differentiation induced by BMP9. We showed that BMP9 upregulated the expression of multiple autophagy-related genes in MSCs. Autophagy inhibitor chloroquine (CQ) inhibited the osteogenic activity induced by BMP9 in MSCs. While overexpression of ATG5 or ATG7 did not enhance osteogenic activity induced by BMP9, silencing Atg5 expression in MSCs effectively diminished BMP9 osteogenic signaling activity and blocked the expression of the osteogenic regulator Runx2 and the late marker osteopontin induced by BMP9. Stem cell implantation study revealed that silencing Atg5 in MSCs profoundly inhibited ectopic bone regeneration and bone matrix mineralization induced by BMP9. Collectively, our results strongly suggest a functional autophagy pathway may play an essential role in regulating osteogenic differentiation induced by BMP9 in MSCs. Thus, restoration of dysregulated autophagic activity in MSCs may be exploited to treat fracture healing, bone defects or osteoporosis.

A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology.

Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.

Stimulation of ORAI1 expression, store-operated Ca2+ entry, and osteogenic signaling by high glucose exposure of human aortic smooth muscle cells.

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 µM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs.

Reversal of phosphate-induced ORAI1 expression, store-operated Ca2+ entry and osteogenic signaling by MgCl2 in human aortic smooth muscle cells.

In chronic kidney disease, renal phosphate retention leads to hyperphosphatemia with subsequent vascular osteogenic signaling and calcification. Osteogenic signaling involves up-regulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme stimulating calcification by degrading the calcification inhibitor pyrophosphate. Stimulation of osteogenic signaling and calcification by phosphate donor ß-glycerophosphate in human aortic smooth muscle cells (HAoSMCs) is attenuated by MgCl2, an effect mimicked by Ca2+-sensing receptor agonist GdCl3. Most recent observations revealed that the effect of ß-glycerophosphate on osteogenic signaling requires ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE), which is stimulated by Ca2+-sensor STIM1. The present study explored whether ORAI1 and/or STIM1 expression and, thus, SOCE and osteogenic signaling in HAoSMCs are sensitive to MgCl2 and/or GdCl3. To this end, transcript levels were estimated using q-RT-PCR, protein abundance with western blotting, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1  µM). As a result, 24 h exposure to ß-glycerophosphate (2 mM) significantly enhanced transcript levels of ORAI1 and STIM1 as well as SOCE, effects significantly blunted or virtually abrogated by 1.5 mM MgCl2 and by 50  µM GdCl3. In conclusion, MgCl2 and GdCl3 are powerful inhibitors of ORAI1 and STIM1 expression and store-operated Ca2+-entry, effects affecting osteogenic signalling in vascular smooth muscle cells.

Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor ß-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, ß-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of ß-glycerophosphate on SOCE. Moreover, the ß-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

Signaling pathways involved in vascular smooth muscle cell calcification during hyperphosphatemia.

Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.

Role of PKB/SGK-dependent phosphorylation of GSK-3α/ß in vascular calcification during cholecalciferol overload in mice.

Medial vascular calcification is a highly regulated process involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells. Both, protein kinase B (PKB) and serum- and glucocorticoid-inducible kinase 1 (SGK1) are involved in the intracellular signaling of vascular calcification and both phosphorylate and inactivate glycogen synthase kinase 3 (GSK-3). The present study explored whether PKB/SGK-dependent phosphorylation of GSK-3α/ß is involved in vascular calcification. Experiments were performed in Gsk-3α/ß double knockin mice lacking functional PKB/SGK phosphorylation sites (gsk-3KI) and corresponding wild-type mice (gsk-3WT) following high-dosed cholecalciferol treatment as well as ex vivo in aortic ring explants from gsk-3KI and gsk-3WT mice treated without and with phosphate. In gsk-3WT mice, high-dosed cholecalciferol induced vascular calcification and aortic osteo-/chondrogenic signaling, shown by increased expression of osteogenic markers Msx2, Cbfa1 and tissue-nonspecific alkaline phosphatase (Alpl). All these effects were suppressed in aortic tissue from gsk-3KI mice. Cholecalciferol decreased aortic Gsk-3α/ß phosphorylation (Ser21/9) in gsk-3WT mice, while no phosphorylation was observed in gsk-3KI mice. Moreover, the mRNA expression of type III sodium-dependent phosphate transporter (Pit1) and plasminogen activator inhibitor 1 (Pai1) was increased following cholecalciferol treatment in aortic tissue of gsk-3WT mice, effects again blunted in gsk-3KI mice. In addition, phosphate treatment induced mineral deposition and osteogenic markers expression in aortic ring explants from gsk-3WT mice, effects reduced in aortic ring explants from gsk-3KI mice. In conclusion, vascular PKB/SGK-dependent phosphorylation of GSK-3α/ß contributes to the osteoinductive signaling leading to vascular calcification.

Therapeutic Interference With Vascular Calcification-Lessons From Klotho-Hypomorphic Mice and Beyond.

Medial vascular calcification, a major pathophysiological process associated with cardiovascular disease and mortality, involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). In chronic kidney disease (CKD), osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification is mainly driven by hyperphosphatemia, resulting from impaired elimination of phosphate by the diseased kidneys. Hyperphosphatemia with subsequent vascular calcification is a hallmark of klotho-hypomorphic mice, which are characterized by rapid development of multiple age-related disorders and early death. In those animals, hyperphosphatemia results from unrestrained formation of 1,25(OH)2D3 with subsequent retention of calcium and phosphate. Analysis of klotho-hypomorphic mice and mice with vitamin D3 overload uncovered several pathophysiological mechanisms participating in the orchestration of vascular calcification and several therapeutic opportunities to delay or even halt vascular calcification. The present brief review addresses the beneficial effects of bicarbonate, carbonic anhydrase inhibition, magnesium supplementation, mineralocorticoid receptor (MR) blockage, and ammonium salts. The case is made that bicarbonate is mainly effective by decreasing intestinal phosphate absorption, and that carbonic anhydrase inhibition leads to metabolic acidosis, which counteracts calcium-phosphate precipitation and VSMC transdifferentiation. Magnesium supplementation, MR blockage and ammonium salts are mainly effective by interference with osteo-/chondrogenic signaling in VSMCs. It should be pointed out that the, by far, most efficient substances are ammonium salts, which may virtually prevent vascular calcification. Future research will probably uncover further therapeutic options and, most importantly, reveal whether these observations in mice can be translated into treatment of patients suffering from vascular calcification, such as patients with CKD.

Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB.

Background The high cardiovascular morbidity and mortality of patients with CKD may result in large part from medial vascular calcification, a process promoted by hyperphosphatemia and involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Reduced serum zinc levels have frequently been observed in patients with CKD, but the functional relevance of this remains unclear.Methods We performed experiments in primary human aortic VSMCs; klotho-hypomorphic (kl/kl), subtotal nephrectomy, and cholecalciferol-overload mouse calcification models; and serum samples from patients with CKD.Results In cultured VSMCs, treatment with zinc sulfate (ZnSO4) blunted phosphate-induced calcification, osteo-/chondrogenic signaling, and NF-κB activation. ZnSO4 increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3, also known as A20), a suppressor of the NF-κB pathway, by zinc-sensing receptor ZnR/GPR39-dependent upregulation of TNFAIP3 gene expression. Silencing of TNFAIP3 in VSMCs blunted the anticalcific effects of ZnSO4 under high phosphate conditions. kl/kl mice showed reduced plasma zinc levels, and ZnSO4 supplementation strongly blunted vascular calcification and aortic osteoinduction and upregulated aortic Tnfaip3 expression. ZnSO4 ameliorated vascular calcification in mice with chronic renal failure and mice with cholecalciferol overload. In patients with CKD, serum zinc concentrations inversely correlated with serum calcification propensity. Finally, ZnSO4 ameliorated the osteoinductive effects of uremic serum in VSMCs.Conclusions Zinc supplementation ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation of VSMCs and vascular calcification through an active cellular mechanism resulting from GPR39-dependent induction of TNFAIP3 and subsequent suppression of the NF-κB pathway. Zinc supplementation may be a simple treatment to reduce the burden of vascular calcification in CKD.

Evidence for constitutive bone morphogenetic protein-2 secretion by M1 macrophages: Constitutive auto/paracrine osteogenic signaling by BMP-2 in M1 macrophages.

Mechanisms mediating vascular calcification recapitulate osteogenic processes encompassing bone formation and imply participation of bone related proteins such as bone morphogenetic protein-2 (BMP-2). Macrophages are amongst the cells that contribute to vascular ossification by releasing cytokines that induce an osteogenic program in vascular smooth muscle cells, and also by becoming themselves osteoclast-like cells. In inflammatory vascular disease, the macrophage population in the vascular wall is diverse, with the M1 or inflammatory, and the M2 or anti-inflammatory macrophage types being dominant. Yet, the osteogenic potential of M1 and M2 macrophages remains unknown. Prompted by recent studies from our laboratory showing that in macrophages the Transient Receptor Potential Canonical 3 (TRPC3) channel contributes to endoplasmic reticulum (ER) stress-induced apoptosis in M1, but not in M2 macrophages, and given the strong relationship between ER stress and vascular calcification, we wished to examine whether TRPC3 would play a role in the osteogenic signaling of polarized macrophages. The findings reported here indicate that a constitutive BMP-2-dependent signaling operates in M1 macrophages, which is not affected by deletion of Trpc3 and is not subject to regulation by ER stress. Our studies suggest operation of an auto/paracrine mechanism by which BMP-2 secreted by M1 macrophages maintains constitutive activation of a BMP-2 receptor/SMAD1/5 signaling axis.

Osteogenic signaling on silk-based matrices.

Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders.