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Microstructured titanium regulates interleukin production by osteoblasts, an effect modulated by exogenous BMP-2.

ABSTRACT: Microtextured implant surfaces increase osteoblast differentiation in vitro and enhance bone-to-implant contact in vivo and clinically. These implants may be used in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2) to enhance peri-implant bone formation. However, the effect of surface modifications alone or in combination with rhBMP-2 on the osteoblast-produced inflammatory microenvironment is unknown. MG63 cells were cultured on tissue culture polystyrene or titanium substrates: smooth pretreated (PT, Ra=0.2?m), sandblasted/acid-etched (SLA, Ra=3.2?m) or hydrophilic-SLA (modSLA). Expression and protein production of pro-inflammatory interleukins (IL1b, IL6, IL8, IL17) and anti-inflammatory interleukins (IL10) were measured in cells with or without rhBMP-2. To determine which BMP signaling pathways were involved, cultures were incubated with BMP pathway inhibitors to blockSmad (dorsomorphin), TAB/TAK1 ((5Z)-7-oxozeaenol) or PKA (H-8) signaling. Culture on rough SLA and modSLA surfaces decreased pro-inflammatory interleukins and increased anti-inflammatory IL10. This effect was negated in cells treated with rhBMP-2, which caused an increase in pro-inflammatory interleukins and a decrease in anti-inflammatory interleukins through TAB/TAK signaling. The results suggest that surface microtexture modulates the inflammatory process during osseointegration, an effect that may enhance healing. However, rhBMP-2 in combination with microtextured titanium implants can influence the effect of cells on these surfaces, and may adversely affect cells involved in osseointegration.

SUBMITTER: Hyzy SL

PROVIDER: S-EPMC3618455 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Microstructured titanium regulates interleukin production by osteoblasts, an effect modulated by exogenous BMP-2.

Hyzy S L SL Olivares-Navarrete R R Hutton D L DL Tan C C Boyan B D BD Schwartz Z Z

Acta biomaterialia 20121031 3

Microtextured implant surfaces increase osteoblast differentiation in vitro and enhance bone-to-implant contact in vivo and clinically. These implants may be used in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2) to enhance peri-implant bone formation. However, the effect of surface modifications alone or in combination with rhBMP-2 on the osteoblast-produced inflammatory microenvironment is unknown. MG63 cells were cultured on tissue culture polystyrene or titanium su ...[more]

PMID: 23123301

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Project description:BACKGROUND:Osseointegration is dependent on the implant surface, surrounding bone quality, and the systemic host environment, which can differ in male and female patients. Titanium (Ti) implants with microstructured surfaces exhibit greater pullout strength when compared to smooth-surfaced implants and exhibit enhanced osteogenic cellular responses in vitro. Previous studies showed that 1?,25-dihydroxyvitamin D3 [1?,25(OH)2D3] has a greater effect on rat osteoblast differentiation on microstructured Ti compared to smooth Ti surfaces and tissue culture polystyrene (TCPS). The stimulatory effect of 17?-estradiol (E2) on differentiation is observed in female osteoblasts on micro-rough Ti, but it is not known if male osteoblasts behave similarly in response to E2 and microtopography. This study assessed whether human male and female osteoblasts exhibit sex-specific differences in response to E2 and 1?,25(OH)2D3 when cultured on microstructured Ti surfaces. METHODS:Osteoblasts from three male and three female human donors were cultured on Ti discs with varying surface profiles: a smooth pretreatment (PT), a coarse grit-blasted/acid-etched (SLA), and an SLA surface having undergone modification in a nitrogen environment and stored in saline to maintain hydrophilicity (modSLA). Cells cultured on these surfaces were treated with E2 or 1?,25(OH)2D3. RESULTS:Male and female human osteoblasts responded similarly to microstructure although there were donor-specific differences; cell number decreased, and osteocalcin (OCN), osteoprotegerin (OPG), and latent and active transforming growth factor 1 increased on SLA and modSLA compared to TCPS. Female osteoblasts had higher alkaline phosphatase activity and OCN production than male counterparts but produced less OPG. Both sexes responded similarly to 1?,25(OH)2D3. E2 treatment reduced cell number and increased osteoblast differentiation and factor production only in female cells. CONCLUSIONS:Male and female human osteoblasts respond similarly to microstructure and 1?,25(OH)2D3 but exhibit sexual dimorphism in substrate-dependent responses to E2. E2 affected female osteoblasts, suggesting that signaling is sex-specific and surface-dependent. Donor osteoblasts varied in response, demonstrating the need to test multiple donors when examining human samples. Understanding how male and female cells respond to orthopedic biomaterials will enable greater predictability post-implantation as well as therapies that are more patient-specific.

Project description:Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding ?5?1 and collagen-binding ?1?1 and ?2?1. Knockdown of ?2 or ?1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGF?1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits ?1, ?2, ?5, and ?1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin ?2, ?5, and ?1 silenced cells but decreased in ?1-silenced cells. FGF-2 decreased on modSLA substrates in both ?1 and ?2-silenced cells but was unchanged in response to silencing of either ?5 or ?1. In integrin ?1, ?2, and ?1-silenced cells, Ang-1 increased on modSLA but ?5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. STATEMENT OF SIGNIFICANCE: Successful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

Project description:Peri-implant bone formation depends on the ability of mesenchymal stem cells (MSCs) to colonize implant surfaces and differentiate into osteoblasts, but the precise mechanisms controlling this process remain unclear. In vitro, MSCs undergo osteoblastic differentiation on microstructured titanium (Ti) surfaces in the absence of exogenous media supplements and produce factors that promote osteogenesis while regulating osteoclast activity, including semaphorins. The goal of this study was to evaluate the role of semaphorin 3A (Sema3A) on surface-mediated osteoblastic differentiation and determine the hierarchy of this signaling cascade. Human MSCs were cultured on 15 mm grade 2 smooth (pretreatment, PT), hydrophobic-microrough (sand blasted/acid etched, SLA), hydrophilic-microrough Ti (mSLA) (Institut Straumann AG, Basel, Switzerland), or tissue culture polystyrene (TCPS). Expression of SEMA3A family proteins increased after 7 days of culture, and the increased expression in response to microstructured Ti was dependent on recognition of the surface by integrin α2β1. Exogenous Sema3A increased differentiation whereas differentiation was decreased in cells treated with a Sema3A antibody. Furthermore, Sema3A influenced the production of osteoprotegerin and osteopontin suggesting it as an important local regulator of bone remodeling. Inhibition of Wnt3A and Wnt5A revealed that activation of Sema3A occurs downstream of Wnt5A and may facilitate the translocation of β-catenin bypassing the canonical Wnt3A initiating signal associated with osteoblastic differentiation. Furthermore, chemical inhibition of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase (CaMKII), phospholipase A2 (PLA2), protein kinase C (PKC), and BMP receptors suggest that Sema3A could serve as a feedback mechanism for both Wnt5A and BMP2. Here, we show novel roles for Sema3A family proteins in the surface-dependent modulation of MSCs as well as important interactions with pathways known to be associated with osteoblastic differentiation. Moreover, their effects on bone remodeling markers have significant implications for peri-implant bone remodeling and downstream modulation of osteoclastic activity. These results suggest that Sema3A aids in peri-implant bone formation through regulation on multiple stages of osseointegration, making it a potential target to promote osseointegration in patients with compromised bone remodeling.

Dataset Information

Microstructured titanium regulates interleukin production by osteoblasts, an effect modulated by exogenous BMP-2.

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Microstructured titanium regulates interleukin production by osteoblasts, an effect modulated by exogenous BMP-2.

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