Differentiation Of Pre-osteoblastic Cells Research Articles

Fabrication of poly(lactic acid)/Ti composite scaffolds with enhanced mechanical properties and biocompatibility via fused filament fabrication (FFF)–based 3D printing

Ideal bone substitutes should ensure good integration with bone tissue and are therefore required to exhibit good mechanical stability and biocompatibility. Consequently, the high elastic modulus (similar to that of bone), thermoplasticity, and biocompatibility of poly(lactic acid) (PLA) make it well suited for the fabrication of such substitutes by fused filament fabrication (FFF)-based 3D printing. However, the demands of present-day applications require the mechanical and biological properties of PLA to be further improved. Herein, we fabricated PLA/Ti composite scaffolds by FFF-based 3D printing and used thermogravimetric analysis to confirm the homogenous dispersion of Ti particles in the PLA matrix at loadings of 5–20 vol%. Notably, the thermal stability of these composites and the crystallization temperature/crystallinity degree of PLA therein decreased with increasing Ti content, while the corresponding glass transition temperature and melting temperature concomitantly increased. The compressive and tensile strengths of PLA/Ti composites increased with Ti increasing loading until it reached 10 vol% and were within the range of real bone values, while the impact strengths of the above composites significantly exceeded that of pure PLA. The incorporation of Ti resulted in enhanced in vitro biocompatibility, promoting the initial attachment, proliferation, and differentiation of pre-osteoblast cells, which allowed us to conclude that the prepared PLA/Ti composite scaffolds with enhanced mechanical and biological properties are promising candidates for bone tissue engineering applications.

Injectable Catalyst-Free Poly(Propylene Fumarate) System Cross-Linked by Strain Promoted Alkyne-Azide Cycloaddition Click Chemistry for Spine Defect Filling.

A new PPF-BCN/hyPCL32-N3 injectable system that can be cross-linked by catalyst-free, strain promoted alkyne-azide cycloaddition (SPAAC) click chemistry was developed for tissue engineering applications. The system consisted of two components: PPF-BCN, poly(propylene fumarate) (PPF) functionalized with (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH), and hyPCL32-N3, a hyper-branched 32-arm poly(ε-caprolactone) (PCL) dendrimer functionalized with azide as the cross-linker core. Fast SPAAC click reaction allowed the desired gelation of the system without using any toxic initiator or catalyst. Compared to the conventional injectable formulation, e.g., poly(methyl methacrylate) (PMMA), our PPF-BCN/hyPCL32-N3 (abbreviated as PFCL-Click) injectable system showed enhanced biocompatibility and low heat generation during cross-linking. After reaction, the cross-linked PFCL-Click scaffolds supported excellent proliferation and differentiation of preosteoblast cells on the surface. The PFCL-Click system can be successfully injected into vertebral bodies of rabbit spine and can be monitored by X-ray imaging after incorporating zirconium dioxide (ZrO2) powder. With these unique advantages, this injectable system has promising potential for bone defect repair and other tissue engineering and regenerative medicine applications.

Epigenomic Regulation of SATB2 Links Maternal Obesity to Impaired Osteoblast Differentiation (P11-034-19)

ObjectivesNutritional status during intrauterine and/or early postnatal life has substantial influences on adult offspring health. However, evidence on the impact of high fat diet (HFD)-induced maternal obesity on regulation of fetal bone development is sparse. Thus, we investigated the effects of maternal obesity in rodent and human cells on epigenetic regulation of osteoblast differentiation. MethodsFirst, female Sprague-Dawley rats were fed either a low-fat AIN-93G control diet or a high fat diet (HFD) (45% fat calories) for 10 wk starting at 6 wk of age. Lean (from control diet) and obese (from HFD) female rats were then time-impregnated (n = 6 per group) by control diet fed male rats. At gestational day 18.5 (E18.5), all fetuses were taken and embryonic osteogenic calvarial cells (EOCCs) were isolated. Second, human osteo-progenitors of mesenchymal stem cells were isolated from umbilical cord following delivery from pregnant mothers. ResultsWe found epigenetic regulation of polycomb-regulated gene Ezh2 (Enhancer of zeste homolog 2) in embryonic rats from HFD obese rat dams. Increased enrichment of repressive histone mark H3K27me3 on the gene body of SATB2 (ChIP Seq analysis) was associated with aberrant differentiation of EOCCs to mature osteoblasts. Knocking down Ezh2 in EOCCs and ST2 cells increased SATB2 expression; on the other hand, Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with ChIP experimental results showing strong association between H3K27me3, Ezh2 and SATB2. Second, human mesenchymal stem cells (MSCs) from umbilical cord (UC) were isolated following delivery from obese/overweight (pre-pregnancy BMI ≥ 25 kg/m2) and control (pre-pregnancy BMI between 19–25 kg/m2) pregnant mothers. We found: 1) UC-MSCs from pregnant obese/overweight mothers showed increased Ezh2 expression and decreased SATB2 mRNA expression, which was concurrent lower osteoblastogenesis potential in EOCCs; 2) ChIP experiments using H3K27me3 IP (immune-precipitation) showed significant association between H3K27me3, Ezh2 and SATB2. ConclusionsThese findings indicate maternal HFD-induced obesity-associated decrease of fetal pre-osteoblastic cell differentiation is under epigenetic control through SATB2 expression. Funding SourcesSupported by USDA-ARS Project.

Role of nesfatin-1 in the metabolism of skeletal tissues

NUCB2/nesfatin-1, a member of the adipokine family, is a peptide hormone with pleiotropic action. It has been found in different tissues, including cartilage and bone cells. Nesfatin-1 is produced by chondrocytes, and its synthesis increases with the degree of cell differentiation and upon stimulation by pro-inflammatory cytokines, as shown in an in vitro study. An increase in serum levels of nesfatin-1 has been observed in humans with osteoarthritis, which indicates the influence of pro-inflammatory cytokines on nesfatin-1 release. On the other hand, nesfatin-1 stimulates the synthesis of pro-inflammatory cytokines by chondrocytes, which suggests its participation, together with other adipokines, in the pathogenesis and/or progression of inflammatory complications of cartilage degenerative diseases. Nesfatin-1 also promotes pre-osteoblastic cell differentiation and mineralization and inhibits macrophage differentiation towards osteoclasts. Moreover, exogenous nesfatin-1 given to ovariectomized rats reduces osteopenic changes. Therefore, it seems that nesfatin-1 may play a protective role in cartilage and bone diseases. However, further studies are required to determine whether nesfatin-1 can be used for monitoring and treatment of cartilage and bone diseases..

Macrolactin F inhibits RANKL-mediated osteoclastogenesis by suppressing Akt, MAPK and NFATc1 pathways and promotes osteoblastogenesis through a BMP-2/smad/Akt/Runx2 signaling pathway

The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts. In the current study, macrolactin F (MF) was investigated for novel biological activity on the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages (BMMs). We found that RANKL-induced osteoclast formation and differentiation from BMMs was significantly inhibited by MF in a dose-dependent manner without cytotoxicity. RANKL-induced F-actin ring formation and bone resorption activity in BMMs which was attenuated by MF. In addition, MF suppressed the expression of osteoclast-related genes, including c-myc, RANK, tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T cells c1 (NFATc1), cathepsin K and matrix metalloproteinase 9 (MMP9). Furthermore, the protein expression NFATc1, c-Fos, MMP9, cathepsin K and phosphorylation of Jun N-terminal kinase (JNK), p38 and Akt were also down-regulated by MF treatment. Interestingly, MF promoted pre-osteoblast cell differentiation on Alizarin Red-mineralization activity, alkaline phosphatase (ALP) activity, and the expression of osteoblastogenic markers including Runx2, Osterix, Smad4, ALP, type I collagen alpha 1 (Col1α), osteopontin (OPN), and osteocalcin (OCN) via activation of the BMP-2/smad/Akt/Runx2 pathway on MC3T3-E1. Taken together, these results indicate that MF may be useful as a therapeutic agent to enhance bone health and treat osteoporosis.

Icaritin induces MC3T3-E1 subclone14 cell differentiation through estrogen receptor-mediated ERK1/2 and p38 signaling activation

Icaritin (ICT), a hydrolytic product of icariin from the genus Epimedium, has many indicated pharmacological and biological activities. Several studies have shown that ICT has potential osteoprotective effects, including stimulation of osteoblast differentiation and inhibition of osteoclast differentiation. However, the molecular mechanism for this anabolic action of ICT remains largely unknown. Here, we found that ICT could enhance MC3T3-E1 subclone 14 preosteoblastic cell differentiation associated with increased mRNA levels and protein expression of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), osteoponin (OPN) and runt-related transcription factor 2 (RUNX2), and improved mineralization, confirmed by bone nodule formation and collagen synthesis. To characterize the underlying mechanisms, we examined the effect of ICT on estrogen receptor (ER) and mitogen-activated protein kinase (MAPK) signaling. ICT treatment induced p38 kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, but it demonstrated at the same time point no effect on activation of c-Jun N-terminal kinase (JNK). ER antagonist ICI182780, p38 antagonist SB203580 and ERK1/2 antagonist PD98059 markedly inhibited the ICT-induced the mRNA expression of ALP, COL1, OC and OPN. ICI182780 attenuated the ICT-induced phosphorylation of p38 and ERK1/2. These observations indicate a potential mechanism of osteogenic effects of ICT involving the ERK1/2 and p38 pathway activation through the ER.

Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

Titanium (Ti) nanorods fabricated using selective corrosion of Ti substrate by anodic technology show better biocompatibility with pre-osteoblast cells. The current study investigated the response of the murine pre-osteoblast cell MC3T3-E1 on Ti nanorod topography and untreated Ti surfaces by means of examination of the morphology and osteogenic differentiation responsible for the pre-osteoblast reaction. The morphology of MC3T3-E1 cells was observed using scanning electron microscopy, and alkaline phosphatase (ALP) activity was measured using a colorimetric assay after incubation for 7, 14, and 21 days. The expression of three osteogenic differentiation markers including ALP, osteocalcin (OCN), and collagen type 1A1 (COL1A1) and two transcription factors including runt related transcription factor 2 (Runx2) and osterix (Osx) at different time points was detected using real-time polymerase chain reaction analysis in both groups. Osx was used to confirm the protein level. The results showed that Ti nanorod surfaces provided prolonged higher levels of ALP activity compared with unmodified Ti surface on the 14th and 21st days. Gene expression analysis of ALP, OCN, and COL1A1 showed significant upregulation with modified nanorod topography after incubation for 14 and 21 days. Osteogenic transcription factors of Runx2 and Osx exhibited changes consistent with the osteogenic differentiation markers, and this may contribute to the persistently active differentiation of MC3T3-E1 cells in the Ti nanorod group. These results demonstrated that the current nanostructured surface may be considered bioadaptive topography to control cellular behaviors and osteoblast differentiation. The in vivo performance and applicability are further required to investigate osseointegration between implant and host bone in the early stages for prevention of aseptic implant loosening.

Oxygen and nitrogen plasma etching of three-dimensional hydroxyapatite/chitosan scaffolds fabricated by additive manufacturing

Three-dimensional (3D) chitosan and hydroxyapatite (HAp)/chitosan (CH) scaffolds were fabricated by additive manufacturing, then their surfaces were etched with oxygen (O2) and nitrogen (N2) plasma. O2 and N2 plasma etching was performed to increase surface properties such as hydrophilicity, roughness, and surface chemistry on the scaffolds. After etching, hydroxyapatite was exposed on the surface of 3D HAp/CH scaffolds. The surface morphology and chemical properties were characterized by contact angle measurement, scanning electron microscopy, X-ray diffraction, and attenuated total reflection Fourier infrared spectroscopy. The cell viability of 3D chitosan scaffolds was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The differentiation of preosteoblast cells was evaluated by alkaline phosphatase assay. The cell viability was improved by O2 and N2 plasma etching of 3D chitosan scaffolds. The present fabrication process for 3D scaffolds might be applied to a potential tool for preparing biocompatible scaffolds.

Prostate cancer cells induce osteoblastic differentiation via semaphorin 3A.

Prostate cancer metastasis to bone is the second most commonly diagnosed malignant disease among men worldwide. Such metastatic disease is characterized by the presence of osteoblastic bone lesions, and is associated with high rates of mortality. However, the various mechanisms involved in prostate cancer-induced osteoblastic differentiation have not been fully explored. Semaphorin 3A (Sema 3A) is a newly identified regulator of bone metabolism which stimulates differentiation of pre-osteoblastic cells under physiological conditions. We investigated in this study whether prostate cancer cells can mediate osteoblastic activity through Sema 3A. We cultured osteoprogenitor MC3T3-E1 cells in prostate cancer-conditioned medium, and analyzed levels of Sema 3A protein in diverse prostate cancer cell lines to identify cell lines in which Sema 3A production showed a positive correlation with osteo-stimulation. C4-2 cells were stably transfected with Sema 3A short hairpin RNA to further determine whether Sema 3A contributes to the ability of C4-2 cells to induce osteoblastic differentiation. Down-regulation of Sema 3A expression decreased indicators of C4-2 CM-induced osteoblastic differentiation, including alkaline phosphatase production and mineralization. Additionally, silencing or neutralizing Sema 3A in C4-2 cells resulted in diminished β-catenin expression in osteogenitor MC3T3-E1 cells. Our results suggest that prostate cancer-induced osteoblastic differentiation is at least partially mediated by Sema 3A, and may be regulated by the β-catenin signalling pathway. Sema 3A may represent a novel target for treatment of prostate cancer-induced osteoblastic lesions.

Mechanical properties and in vitro biological response to porous titanium alloys prepared for use in intervertebral implants

The generation of titanium foams is a promising strategy for modifying the mechanical properties of intervertebral reinforcements. Thus, the aim of this study was to compare the in vitro biological response of Ti6Al4V alloys with different pore sizes for use in intervertebral implants in terms of the adhesion, proliferation, and differentiation of pre-osteoblastic cells. We studied the production of Ti6Al4V foams by powder metallurgy and the biological responses to Ti6Al4V foams were assessed in terms of different pore interconnectivities and elastic moduli. The Ti6Al4V foams obtained had similar porosities of approximately 34%, but different pore sizes (66µm for fine Ti6Al4V and 147µm for coarse Ti6Al4V) due to the sizes of the microsphere used. The Ti6Al4V foams had a slightly higher Young׳s modulus compared with cancellous bone. The dynamic mechanical properties of the Ti6Al4V foams were slightly low, but these materials can satisfy the requirements for intervertebral prosthesis applications. The cultured cells colonized both sizes of microspheres near the pore spaces, where they occupied almost the entire area of the microspheres when the final cell culture time was reached. No statistical differences in cell proliferation were observed; however, the cells filled the pores on fine Ti6Al4V foams but they only colonized the superficial microspheres, whereas the cells did not fill the pores on coarse Ti6Al4V foams but they were distributed throughout most of the material. In addition, the microspheres with wide pores (coarse Ti6Al4V) stimulated higher osteoblast differentiation, as demonstrated by the Alcaline Phosphatase (ALP) activity. Our in vitro results suggest that foams with wide pore facilitate internal cell colonization and stimulate osteoblast differentiation.

Isolation of Organic Matrix Nacreous Proteins fromHaliotis diversicolorand Their Effect OnIn VitroOsteoinduction

The organic matrix proteins of molluscan nacre are known to engage biocompatible and osteoinductive properties. In this study, we aimed to extract Haliotis diversicolor nacreous proteins and assayed their effect on pre-osteoblastic cell differentiation. We found that acid extracted nacreous proteins (AEP) consisted primarily of a major protein band of 25 kDa and three other minor proteins. AEP enhanced alkaline phosphatase (ALP) activity of MC3T3-E1 cells both in time- and concentration-dependent manners. Transcriptional up-regulation of osteogenic markers, including collagen type I (COL-I), osteopontin (OPN) and osteocalcin (OCN), was also apparent in days 7 and 14 upon AEP treatments. At the translational level, higher protein expression of COL-I was evident in AEP treated cells, and the protein was presumably laid down as extracellular matrix. Further de novo sequencing of an AEP major protein revealed a match with the abalone mantle protein sometsuke. Conclusively, we demonstrated that H. diversicolor AEP contains a factor, potentially the mantle protein sometsuke, which may impart in the osteoblastic cell differentiation.

Effect of icariin on cell proliferation and the expression of bone resorption/formation-related markers in human periodontal ligament cells

Periodontitis is a common destructive inflammatory disease that leads to changes in the tooth-supporting tissues. Human periodontal ligament cells are essential in periodontal tissue regeneration. The traditional Chinese medicine icariin promoted bone formation, stimulated the osteogenic differentiation of preosteoblastic cells and inhibited osteoclast differentiation and bone resorption. Thus, in the present study, the effect of icariin on cell proliferation and the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfa1) and osteocalcin (OC) was investigated in human periodontal ligament cells, by an MTT assay, qPCR and western blot analysis. The results demonstrated that icariin promoted cell proliferation in a dose- and time-dependent manner, upregulated OPG, Cbfa1 and OC expression, and downregulated RANKL production and the RANKL/OPG expression ratio. This suggested the potential value of icariin in treating alveolar bone resorption and promoting periodontal tissue regeneration, due to its ability to stimulate the proliferation and osteogenic differentiation of human periodontal ligament cells and inhibit osteoclast differentiation.

The effect of calcium phosphate cement–silica composite materials on proliferation and differentiation of pre-osteoblast cells

Calcium phosphate cement (CPC) is increasingly being used for bone and tissue engineering in the medical and dental fields. In the current study, we investigated the ability of a CPC–silica composite to enhance the activity of cells by analyzing its effects on the proliferation and differentiation of MC3T3-E1, an osteoblast precursor cell line. We found that the CPC–silica composite materials with low silica content (5wt%) promoted good initial cell adhesion and proliferation. In addition, the CPC–silica composite materials exhibited a higher capacity for stimulating osteogenic differentiation, which was assayed using alkaline phosphatase (ALP) activity and RT-PCR, than CPC alone. Therefore, we propose that 5wt% silica content may serve as a useful component in calcium phosphate-based bone cements owing to its ability to promote osteoblast differentiation.

Fabrication of novel poly(lactic acid)/amorphous magnesium phosphate bionanocomposite fibers for tissue engineering applications via electrospinning

Fibrous bionanocomposites consisting of amorphous magnesium phosphate (AMP) nanospheres and polylactic acid (PLA) were fabricated by electrospinning. There are two important signatures of this paper. First, AMP, as an alternative to well-known calcium phosphate (CaP) materials, is added to PLA as the second phase. To the best of our knowledge, it is the first attempt to fabricate magnesium phosphate (MgP)/biopolymer composite. This is made possible by our previously reported research on the successful synthesis of AMP nanospheres via microwave processing. Second, the sustained release of magnesium and phosphate ions from PLA matrix can stimulate a series of cell responses. The structure of the composites and their bone-like apatite-forming abilities in simulated body fluid (SBF) were examined. Additionally, the effects on the proliferation and differentiation of preosteoblast cells were evaluated by performing in vitro cell culture and monitoring markers such as Osteocalcin (OCN), Osteopontin (OPN), Alkaline phosphatase (ALP) and Collagen type-I (Col I) using real-time polymerase chain reaction (PCR). For better dispersion of AMP in the fibers, a surfactant, 12-hydroxysteric acid (HSA), as previously reported in the literature, was used. However, HSA significantly inhibited the proliferation and differentiation of preosteoblast cells, indicating the potential risk in using HSA in the combination of AMP or MgP in tissue engineering applications.

Porous surface modified bioactive bone cement for enhanced bone bonding.

BackgroundPolymethylmethacrylate bone cement cannot provide an adhesive chemical bonding to form a stable cement-bone interface. Bioactive bone cements show bone bonding ability, but their clinical application is limited because bone resorption is observed after implantation. Porous polymethylmethacrylate can be achieved with the addition of carboxymethylcellulose, alginate and gelatin microparticles to promote bone ingrowth, but the mechanical properties are too low to be used in orthopedic applications. Bone ingrowth into cement could decrease the possibility of bone resorption and promote the formation of a stable interface. However, scarce literature is reported on bioactive bone cements that allow bone ingrowth. In this paper, we reported a porous surface modified bioactive bone cement with desired mechanical properties, which could allow for bone ingrowth.Materials and MethodsThe porous surface modified bioactive bone cement was evaluated to determine its handling characteristics, mechanical properties and behavior in a simulated body fluid. The in vitro cellular responses of the samples were also investigated in terms of cell attachment, proliferation, and osteoblastic differentiation. Furthermore, bone ingrowth was examined in a rabbit femoral condyle defect model by using micro-CT imaging and histological analysis. The strength of the implant–bone interface was also investigated by push-out tests.ResultsThe modified bone cement with a low content of bioactive fillers resulted in proper handling characteristics and adequate mechanical properties, but slightly affected its bioactivity. Moreover, the degree of attachment, proliferation and osteogenic differentiation of preosteoblast cells was also increased. The results of the push-out test revealed that higher interfacial bonding strength was achieved with the modified bone cement because of the formation of the apatite layer and the osseointegration after implantation in the bony defect.ConclusionsOur findings suggested a new bioactive bone cement for prosthetic fixation in total joint replacement.

X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24h after irradiation at 2 and 4Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.

Well-defined hydroxyapatite–polycation nanohybrids via surface-initiated atom transfer radical polymerization for biomedical applications

The ability to manipulate and control the surface properties of hydroxyapatite (HA) nanoparticles is of crucial importance for the design of HA-based carriers of therapeutic agents. In this work, surface-initiated atom transfer radical polymerization (ATRP) of (2-dimethyl amino)ethyl methacrylate (DMAEMA) is first employed to tailor the functionality of HA surfaces in a well-controlled manner and to produce a series of new cationic hybrids (termed as HA-PDM). The HA parts of HA-PDM were coated by different lengths of PDMAEMA chains. The HA-PDM exhibited a good ability to condense plasmid DNA (pDNA) with suitable particle size and a zeta potential for gene transfection. Most importantly, in comparison with PDMAEMA homopolymers, the HA-PDM displayed considerably enhanced buffering capacity, and exhibited much higher gene transfection efficiencies in different cell lines, including osteoblast MC3T3 and osteosarcoma MG63 cells. In addition, the HA-PDM/pDNA complexes also could largely enhance the differentiation of preosteoblast cells. Such well-defined HA-PDM nanohybrids possess great potential applications as new drug-delivery vectors in bone tissue engineering.

Controlling apatite microparticles formation by calcining electrospun sol–gel derived ultrafine silica fibers

Electrospun ultrafine silica fibers were calcined at 150–800 °C. The relation of calcination temperature to the ability to form biomimetic apatite in a simulated body fluid solution (SBF) was evaluated. The largest apatite particles, formed on non-calcined fibers after 1 week of soaking in SBF, were 10 μm in diameter, had a narrow size distribution (coefficient of variation 9%), and were similar to pearls on string. The particles size decreased with increasing calcination temperature below 250 °C and the particles formed on the fibers calcined at 250 °C were 4.5 μm in diameter. No particles were found on those calcined above 500 °C. By using a concentrated SBF at 1.5-times higher ionic concentrations than SBF, the size of apatite microparticles increased about 50%. The fibrous substrate covered with apatite particles was effective for osteoblastic differentiation of pre-osteoblastic cells.

Abstract B106: Role of TGF-beta superfamily ligand and estrogen signaling in mediating osteoblastic tumorigenesis of prostate cancer cells.

Abstract Prostate cancer metastasizes to bone in 80% of patients suffering from advanced disease. The nature of bone lesions is predominantly osteoblastic resulting in severe bone pain, hypocalcemia and nerve compression syndromes. Transforming growth factor-beta (TGFβ), which is a multifunctional cytokine, is a critical regulator of osteogenesis. Our group has recently shown that TGFβ signaling helps in promoting osteoblastic metastasis by a novel human prostate cancer cell line, PacMetUT1. TGFβ has been suggested to be one of the physiological upregulatory factors of bone aromatase in human osteoblast-like cells. Recent data further implicates an aberrant aromatase expression and estrogen signaling in the development of prostate malignancy. However, the effect of TGFβ superfamily ligands on aromatase expression and estrogen production and the mechanism by which osteoblastic lesions are formed have not been intensively investigated in prostate cancer cells. We hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would in turn lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ and BMP-4/7 on aromatase expression and osteogenic differentiation of pre-osteoblastic cells in vitro. Treatment with TGFβ 1 and BMP-4/7 resulted in an increase in aromatase expression in both mouse pre-osteoblasts and human bone marrow-derived mesenchymal stem cells. Treatment with letrozole (aromatase inhibitor) as well as an anti-estrogen (ICI 182, 780) resulted in a decrease in osteoblastic differentiation of mesenchymal stem cells as measured by alkaline phosphatase activity assay and expression of osteogenic markers by quantitative real-time PCR analysis. Furthermore, 17-β estradiol treatment of PacMetUT1 cells resulted in an enhanced anchorage-dependent and independent cell growth in vitro. This effect was very significantly inhibited by tamoxifen treatment, which is a selective estrogen receptor modulator. 17-β estradiol also induced a change in cell morphology of PacMetUT1 cells with a reduction in E-cadherin expression. Our results demonstrate an aromatase-mediated induction in osteogenesis by TGFβ superfamily ligands in the pre-osteoblastic cells and a stimulatory effect of estrogen signaling on PacMetUT1 cell tumorigencity in vitro. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis of prostate cancer cells in vivo and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy, to alleviate bone metastasis in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B106.

Osteoconductive protamine-based polyelectrolyte multilayer functionalized surfaces

The integration of orthopedic implants with host bone presents a major challenge in joint arthroplasty, spinal fusion and tumor reconstruction. The cellular microenvironment can be programmed via implant surface functionalization allowing direct modulation of osteoblast adhesion, proliferation, and differentiation at the implant–bone interface. The development of layer-by-layer assembled polyelectrolyte multilayer (PEM) architectures has greatly expanded our ability to fabricate intricate nanometer to micron scale thin film coatings that conform to complex implant geometries. The in vivo therapeutic efficacy of thin PEM implant coatings for numerous biomedical applications has previously been reported. We have fabricated protamine-based PEM thin films that support the long-term proliferation and differentiation of pre-osteoblast cells on non-cross-linked film-coated surfaces. These hydrophilic PEM functionalized surfaces with nanometer-scale roughness facilitated increased deposition of calcified matrix by osteoblasts in vitro, and thus offer the potential to enhance implant integration with host bone. The coatings can make an immediate impact in the osteogenic culture of stem cells and assessment of the osteogenic potential of new therapeutic factors.