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Articular cartilage defects are a global challenge, causing substantial disability. Repairing large defects is problematic, often exceeding cartilage's self-healing capacity and damaging bone structures. To tackle this problem, a scaffold-mediated therapeutic ion delivery system is developed. These scaffolds are constructed from poly(ε-caprolactone) and strontium (Sr)-doped bioactive nanoglasses (SrBGn), creating a unique hierarchical structure featuring macropores from 3D printing, micropores, and nanotopologies due to SrBGn integration. The SrBGn-embedded scaffolds (SrBGn-µCh) release Sr, silicon (Si), and calcium (Ca) ions, which improve chondrocyte activation, adhesion, proliferation, and maturation-related gene expression. This multiple ion delivery significantly affects metabolic activity and maturation of chondrocytes. Importantly, Sr ions may play a role in chondrocyte regulation through the Notch signaling pathway. Notably, the scaffold's structure and topological cues expedite the recruitment, adhesion, spreading, and proliferation of chondrocytes and bone marrow-derived mesenchymal stem cells. Si and Ca ions accelerate osteogenic differentiation and blood vessel formation, while Sr ions enhance the polarization of M2 macrophages. The findings show that SrBGn-µCh scaffolds accelerate osteochondral defect repair by delivering multiple ions and providing structural/topological cues, ultimately supporting host cell functions and defect healing. This scaffold holds great promise for osteochondral repair applications., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1 ) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin ( Ctsk )-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk + PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk + PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Exercise can prevent osteoporosis and improve immune function, but the mechanism remains unclear. Here, we show that exercise promotes reticulocalbin-2 secretion from the bone marrow macrophages to initiate bone marrow fat lipolysis. Given the crucial role of lipolysis in exercise-stimulated osteogenesis and lymphopoiesis, these findings suggest that reticulocalbin-2 is a pivotal regulator of a local adipose-osteogenic/immune axis. Mechanistically, reticulocalbin-2 binds to a functional receptor complex, which is composed of neuronilin-2 and integrin beta-1, to activate a cAMP-PKA signaling pathway that mobilizes bone marrow fat via lipolysis to fuel the differentiation and function of mesenchymal and hematopoietic stem cells. Notably, the administration of recombinant reticulocalbin-2 in tail-suspended and old mice remarkably decreases bone marrow fat accumulation and promotes osteogenesis and lymphopoiesis. These findings identify reticulocalbin-2 as a novel mechanosensitive lipolytic factor in maintaining energy homeostasis in bone resident cells, and it provides a promising target for skeletal and immune health., Competing Interests: Declaration of interests X-H.L. and H.P. disclose that they are inventors of a submitted patent application by Xiangya Hospital of Central South University covering recombinant RCN2., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A specific bone capillary subtype, namely type H vessels, with high expression of CD31 and endomucin, was shown to couple angiogenesis and osteogenesis recently. The number of type H vessels in bone tissue declines with age, and the underlying mechanism for this reduction is unclear. Here, we report that microRNA-188-3p (miR-188-3p) involves this process. miRNA-188-3p expression is upregulated in skeletal endothelium and negatively regulates the formation of type H vessels during ageing. Mice with depletion of miR-188 showed an alleviated age-related decline in type H vessels. In contrast, endothelial-specific overexpression of miR-188-3p reduced the number of type H vessels, leading to decreased bone mass and delayed bone regeneration. Mechanistically, we found that miR-188 inhibits type H vessel formation by directly targeting integrin β3 in endothelial cells. Our findings indicate that miR-188-3p is a key regulator of type H vessel formation and may be a potential therapeutic target for preventing bone loss and accelerating bone regeneration., (© 2022. The Author(s).)

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising alternative source of mesenchymal stem cells (MSCs) that are enormously attractive for clinical use. This study was designed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or osteogenic media (OMD) on bone regeneration of hUC-MSCs seeded on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in a rabbit model. The characteristics of stem cells were analyzed by plastic adherence, cell phenotype, and multilineage differentiation potential. Cell proliferation was examined using cell counting kit-8 assay. Osteogenic differentiation was evaluated by quantitative Ca 2+ concentration, PO 4 3- concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralized matrix formation. Bone regeneration was investigated in jaw bone defect repair in rabbit by microcomputed tomography, fluorescent labeling, and hematoxylin and eosin staining. Except for initial stress response, OMD and OMD + rhBMP-7 inhibited the proliferation of hUC-MSCs seeded on nHAC/PLA; rhBMP-7 inhibited cell proliferation in the nonlogarithmic phase and attenuated the inhibitory effect of OMD on cell proliferation. The inhibitory effects of OMD, rhBMP-7, and OMD + rhBMP-7 on cell proliferation were ranked as OMD > OMD + rhBMP-7 > rhBMP-7. OMD, rhBMP-7, and OMD + rhBMP-7 promoted Ca 2+ concentration, PO 4 3- concentration, ALP activity, OCN secretion, and mineralized matrix formation of hUC-MSCs seeded on nHAC/PLA. The promoting effects of OMD, rhBMP-7, and OMD+rhBMP-7 on Ca 2+ concentration, PO 4 3- concentration, ALP activity, OCN secretion, and mineralized matrix formation were ranked as rhBMP-7 > OMD > OMD + rhBMP-7, OMD > OMD + rhBMP-7 > rhBMP-7, OMD > rhBMP-7 > OMD + rhBMP-7, rhBMP-7 > OMD + rhBMP-7 > OMD, and OMD > rhBMP-7 > OMD + rhBMP-7, respectively. In rabbit jaw bone defect repair, OMD, rhBMP-7, and OMD + rhBMP-7 enhanced bone regeneration of hUC-MSCs seeded on nHAC/PLA, but the largest bone mineral apposition rate and bone formation were presented in cultures with rhBMP-7. These findings suggested that the combined use of rhBMP-7 and OMD may have no ideal synergistic effect on bone regeneration of hUC-MSCs seeded on nHAC/PLA in rabbit jaw bone defect.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The fabrication of bioactive coatings on metallic implants to enhance osseointegration has become a topic of general interest in orthopedics and dentistry. Hydroxyapatite (HA) coating has been shown to induce bone formation and promote bone-implant integration. Unfortunately, poor mechanical performance has hindered this from becoming a favorable coating material. The majority of present studies have focused in incorporating different elements into HA coatings to improve mechanical properties. In recent years, tantalum (Ta) has received increasing attention due to its excellent biocompatibility and corrosion resistance. The aim of on the present study was to investigate the fabrication and biological performance of Ta-incorporated HA coatings., Methods: Ta-incorporated HA coatings were fabricated using the plasma spray technique on a titanium substrate, and the surface characteristics and mechanical properties were examined. In addition, the effects of Ta-incorporated HA coatings on the biological behavior of mesenchymal stem cells (BMSCs) were investigated., Results: Ta-incorporated HA coatings with microporous structure had higher roughness and wettability. In addition, the bonding strength of Ta/HA coatings with the substrate was substantially superior to HA coatings. Furthermore, Ta-incorporated HA coatings not only facilitated initial cell adhesion and faster proliferation, but also promoted the osteogenic differentiation of BMSCs., Conclusion: These results indicate that the incorporation of Ta could improve mechanical performance and increase the osteogenic activity of HA coatings. The Ta-incorporated HA coating fabricated by plasma spraying is expected to be a promising bio-coating material for metallic implants.

The present study aims to investigate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteogenesis of periodontal ligament (PDL) cells. The expression vector of rhBMP-2 (pcDNA3.1-rhBMP-2) was established. PDL cells were obtained through the enzymatic digestion and tissue explant methods and verified by immunohistochemistry. Cells were classified into experimental (cells transfected with pcDNA3.1/rhBMP-2-EGFP), blank (cells with no transfection) and control group (cells transfected with empty plasmid). rhBMP-2 expression was assessed via Western blotting analysis. The mineralization ability, alkaline phosphatase (ALP) activity and level of related osteogenic biomarkers were detected to evaluate the osteogenic characteristics of PDL cells. The rhBMP-2 expression vector (pcDNA3.1-rhBMP-2) was successfully established. Primary PDL cells displayed a star or long, spindle shape. The cultured cells were long, spindle-shaped, had a plump cell body and homogeneous cytoplasm and the ellipse nucleus contained two or three nucleoli. Cells displayed a radial, sheaf-like or eddy-like arrangement after adherence growth. Immunohistochemical staining confirmed that cells originated from mesenchymal opposed to epithelium. The experimental group exhibited an enhanced mineralization ability, higher ALP activity and increased expression of rhBMP-2 and osteogenic biomarkers (Runx2, collagen type I and osteocalcin) than the blank and control group. The present study demonstrated that rhBMP-2 transfection enhances the osteogenesis of PDL cells and provides a possibility for the application of rhBMP-2 expression products in dental disease treatment., (© 2017 The Author(s).)

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β superfamily. Recent studies confirmed that GDF11 plays an important role in regulating the regeneration of brain, skeletal muscle, and heart during aging; however, its role in bone metabolism remains unclear. Thus, the aim of this study was to determine the effects of GDF11 on bone metabolism, including bone formation and bone resorption, both in vitro and in vivo. Our results showed that GDF11 inhibited osteoblastic differentiation of bone marrow mesenchymal stem cells in vitro. Mechanistically, GDF11 repressed Runx2 expression by inducing SMAD2/3 phosphorylation during osteoblast differentiation. Moreover, intraperitoneal injection of GDF11 inhibited bone formation and accelerated age-related bone loss in mice. Our results also showed that GDF11 had no effect on osteoclast differentiation or bone resorption both in vitro and in vivo. These results provide a further rationale for the therapeutic targeting of GDF11 for the treatment of age-related osteoporosis.

Periodontal ligament stem cells (PDLSCs) are tissue-specific mesenchymal stem cells (MSCs), having an important role in regenerative therapy for teeth loss. Interleukin-7 (IL-7) is a key cytokine produced by stromal cells including MSCs, and exhibits specific roles for B and T cell development and osteoblasts differentiation of multiple myeloma. However, the effect of IL-7 on osteogenic differentiation of PDLSCs remains unclear. Therefore, in the present study we determined whether IL-7 affects the proliferation and osteogenic differentiation of PDLSCs in vitro and explored the associated signaling pathways for IL-7-mediated cell differentiation. The results demonstrated that the isolated human PDLSCs possessed MSCs features, highly expressing CD90, CD44, CD105, CD29 and CD73, and almost did not expressed CD34, CD45, CD11b, CD14 and CD117. IL-7 could not significantly affect the proliferation of PDLSCs, but it decreased their osteogenic differentiation and inhibited alkaline phosphatase (ALP) activity. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting exhibited that the expression levels of Runx-2, SP7 and osteocalcin (OCN) were significantly reduced by IL-7. Further studies indicated that IL-7 did not significantly change JNK, ERK1/2 and p38 protein production, but markedly suppressed their phosphorylation levels. These data suggest that IL-7 inhibits the osteogenic differentiation of PDLSCs probably via inactivation of mitogen-activated protein kinase (MAPK) signaling pathway.

Deficiency in vascular endothelial growth factor (VEGF) or bone morphogenetic proteins (BMPs) results in fracture non-unions. Therefore, it is indispensable to comprehend the combined effect of VEGF and BMPs on the osteogenic differentiation of osteoprogenitor mesenchymal stem cells (MSCs) that are either naturally occurring at the fracture repair site or exogenously added to enhance the bone repair. We found that the combination of VEGF and BMP-6 enhanced COL1A2 expression, which correlated with upregulated expression of osterix, Dlx5, and Msx2 in human adipose-derived stem cells (hADSCs). Cross-talk between VEGF and BMP-6 pathways upregulated activation of p38 mitogen-activated kinase (p38 MAPK) and inhibited activation of protein kinase B (PKB, also known as Akt), whereas phosphorylation of "mothers against decapentaplegic" homologs 1/5/8 (Smads 1/5/8) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2) was not affected. Consistent with these findings, p38 inhibitor SB203580, or siRNA knockdown of osterix, abrogated crosstalk between the VEGF and BMP-6 pathways and significantly reduced the observed upregulation of COL1A2. Nuclear translocation of the phosphorylated form of osterix was also inhibited by SB203580. Although crosstalk between the VEGF-BMP-6 pathways did not show an effect on the extent of mineralization, inhibition of any one of the three components that were upregulated through the cross-talk, i.e., osterix, Dlx5, and p38 activation, led to a complete inhibition of mineralization. Inhibition of PKB/Akt activation, which is attenuated through the cross-talk, significantly enhanced ALP gene expression. These observations imply that crosstalk between the VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of MSCs., (© 2015 Wiley Periodicals, Inc.)

Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-dependent reduction in osteogenesis that is accompanied by an increased propensity toward adipocyte differentiation. This switch increases adipocyte numbers and decreases the number of osteoblasts, contributing to age-related bone loss. Here, we found that the level of microRNA-188 (miR-188) is markedly higher in BMSCs from aged compared with young mice and humans. Compared with control mice, animals lacking miR-188 showed a substantial reduction of age-associated bone loss and fat accumulation in bone marrow. Conversely, mice with transgenic overexpression of miR-188 in osterix+ osteoprogenitors had greater age-associated bone loss and fat accumulation in bone marrow relative to WT mice. Moreover, using an aptamer delivery system, we found that BMSC-specific overexpression of miR-188 in mice reduced bone formation and increased bone marrow fat accumulation. We identified histone deacetylase 9 (HDAC9) and RPTOR-independent companion of MTOR complex 2 (RICTOR) as the direct targets of miR-188. Notably, BMSC-specific inhibition of miR-188 by intra-bone marrow injection of aptamer-antagomiR-188 increased bone formation and decreased bone marrow fat accumulation in aged mice. Together, our results indicate that miR-188 is a key regulator of the age-related switch between osteogenesis and adipogenesis of BMSCs and may represent a potential therapeutic target for age-related bone loss.

Antioxidants were implicated as potential reagents to enhance osteogenesis, and nano-fullerenes have been demonstrated to have a great antioxidative capacity by both in vitro and in vivo experiments. In this study, we assessed the impact of a polyhydroxylated fullerene, fullerol, on the osteogenic differentiation of human adipose-derived stem cells (ADSCs). Fullerol was not toxic against human ADSCs at concentrations up to 10 μM. At a concentration of 1 μM, fullerol reduced cellular reactive oxygen species after a 5-day incubation either in the presence or in the absence of osteogenic media. Pretreatment of fullerol for 7 days increased the osteogenic potential of human ADSCs. Furthermore, when incubated together with osteogenic medium, fullerol promoted osteogenic differentiation in a dose-dependent manner. Finally, fullerol proved to promote expression of FoxO1, a major functional isoform of forkhead box O transcription factors that defend against reactive oxygen species in bone. Although further clarification of related mechanisms is required, the findings may help further development of a novel approach for bone repair, using combined treatment of nano-fullerol with ADSCs.

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

20-Hydroxyecdysone, an ecdysteroid hormone, can induce osteogenic differentiation in mesenchymal stem cells. Periodontal ligament stem cells (PDLS cells) have mesenchymal-stem-cell-like qualities and are considered as one of the candidates of future clinical application in periodontitis treatment. However, there are no studies describing the effect of 20-Hydroxyecdysone on PDLS cells. In this paper, we report a detailed study on the effect of 20-Hydroxyecdysone on PDLS cell proliferation in vitro. PDLS cells were developed from human PDL cells and were treated with 20-Hydroxyecdysone to understand different aspects of its effects. 20-Hydroxyecdysone promoted PDLS cell proliferation; significantly increased the gene expression levels of runt-related transcription factor 2, alkaline phosphatase (ALP), type I collagen, and osteocalcin. Moreover, 20-Hydroxyecdysone enhanced bone formation by PDLS cells and significantly increased bone morphogenetic protein-2 (BMP-2) mRNA and protein expression. However, 20-Hydroxyecdysonemediated increase in ALP activity was blocked with a BMP-2-specific neutralizing antibody or with the antagonist noggin; and20-Hydroxyecdysone mediated induction of BMP-2 expression and increase of ALP activity were abolished by the extracellular regulated protein kinase (ERK) MAPK pathway inhibitor PD98059. 20-Hydroxyecdysone also increased the phosphorylation of ERK. These findings provide evidence to state that 20-Hydroxyecdysone stimulates cell proliferation and induces osteogenic differentiation through the induction of BMP-2 expression in PDLS cells. It also shows that the ERK pathway is involved in 20-Hydroxyecdysone induced BMP-2 expression and osteogenic differentiation. These results are suggesting its potential as a drug for periodontal regenerative therapy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were found to suppress proliferation and induce cell death in cultured osteoblasts, and steroids were found to decrease the osteogenesis potential of mesenchymal stem cells. In this study, we further tested the effects of anti-inflammatory drugs (AIDs) on the functions of bone marrow mesenchymal stem cells (BMSCs). The BMSCs from mice (D1-cells) and humans (hBMSCs) were treated with dexamethasone (10(-7) to 10(-6) M), cyclooxygenase-2 (COX-2) selective NSAIDs (10(-6) to 10(-5) M) and non-selective NSAIDs (10(-5) to 10(-4) M). Drug effects on proliferation, cell cycle kinetics, cytotoxicity and mRNA and protein expressions of cell cycle regulators were tested. The osteogenesis potential of D1-cells were evaluated by testing mRNA expressions of type Ialpha collagen and osteocalcin 2-8 days after treatments, and testing mineralization 1-3 weeks after treatments. The results showed that all the tested drugs suppressed proliferation and arrested cell cycle of D1-cells, but no significant cytotoxic effects was found. Prostaglandin E1, E2 and F2alpha couldn't rescue the effects of AIDs on proliferation. The p27kip1 expression was up-regulated by indomethacin, celecoxib and dexamethasone in both D1-cells and hBMSCs. Higher concentrations of indomethacin and dexamethasone also up-regulated p21Cip1/Waf1 expression in hBMSCs, and so did celecoxib on D1-cells. Expressions of cyclin E1 and E2 were down-regulated by these AIDs in D-cells, while only cyclin E2 was down-regulated by dexamethasone in hBMSCs. All the tested NSAIDs revealed no obvious detrimental effects on osteogenic differentiation of D1-cells. These results suggest that the proliferation suppression of AIDs on BMSCs may act via affecting expressions of cell cycle regulators, but not prostaglandin-related mechanisms.