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1.  Comparing the TiOblast and Osseospeed surfaces. Histomorphometric and histological analysis in humans 
Oral & Implantology  2008;1(1):34-42.
The aim of the present study was to compare two implant surfaces, the TiOblast (Astra Tech) surface, manufactured by blasting the surface and already present in literature and the Osseospeed (Astra Tech) surface, manufactured by blasting and treating the surface with fluoride ions and recently launched onto the market with the modified surfaces of the latest generation. This study is part of a more extensive research project whose protocol required the insertion of 10 couples of implants; thus in the present discussion partial data are being taken into consideration, with an eye at collecting more data in the future, regarding both microscopy and histomorphometric histological analysis on 5 couples of implants. The purpose of the study is to investigate how the modified surfaces of the latest generation can guarantee a greater osseointegration both from a qualitative and quantitative level compared to the surfaces presently used and that they may represent the first example of “bioactivity”, that is, an active interaction with the processes of new bone formation and tissue healing.
PMCID: PMC3476499  PMID: 23285334
sandblasted surface; fluoride; histology; histomorphometry; microthreads; macrothreads
2.  Leakage evaluation of original and compatible implant–abutment connections: In vitro study using Rhodamine B 
Journal of Dental Biomechanics  2014;5:1758736014547143.
Leakage has been addressed as a major contributing factor to inflammatory reactions at the implant–abutment connection, leading to problems such as oral malodor, inflammation, and marginal bone loss. The aim of this study was to investigate in vitro the leakage at implant–abutment interface of OsseoSpeed™ implants connected to original and compatible abutments. A total of 28 OsseoSpeed implants were divided into four groups (n = 7). Each group was connected to four different abutments according to manufacturers’ recommendations: group A (TiDesign™); group B (Natea™); group C (Dual™); and group D (Implanet™) abutments. The inner volume of each implant–abutment combination was calculated and leakage was detected for each group with spectrophotometric analysis at 1 h (D0) and 48 h (D1) of incubation time using Rhodamine B. At 1 h, leakage volume was significantly lower in TiDesign and Dual than in Natea and Implanet (P < 0.001). At 48 h, however, leakage was significantly lower between TiDesign and all other systems (P < 0.005). Compatible abutments do not fit internal connection of OsseoSpeed implants perfectly, which increases the leakage of the final assembly.
PMCID: PMC4206669  PMID: 25342984
Dental implants; abutment connection; implant–abutment interface; leakage; Rhodamine B; compatible abutment
3.  In vitro assay for osteoinductive activity of different demineralized freeze-dried bone allograft 
Various bone graft materials have been used for periodontal tissue regeneration. Demineralized freeze-dried bone allograft (DFDBA) is a widely used bone substitute. The current widespread use of DFDBA is based on its potential osteoinductive ability. Due to the lack of verifiable data, the purpose of this study was to assess the osteoinductive activity of different DFDBAs in vitro.
Sarcoma osteogenic (SaOS-2) cells (human osteoblast-like cells) were exposed to 8 mg/mL and 16 mg/mL concentrations of three commercial types of DFDBA: Osseo+, AlloOss, and Cenobone. The effect of these materials on cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The osteoinductive ability was evaluated using alizarin red staining, and the results were confirmed by evaluating osteogenic gene expression using reverse transcription polymerase chain reaction (RT-PCR).
In the SaOS-2 cells, an 8 mg/mL concentration of Osseo+ and Cenobone significantly increased cell proliferation in 48 hours after exposure (P<0.001); however, in these two bone materials, the proliferation of cells was significantly decreased after 48 hours of exposure with a 16 mg/mL concentration (P<0.001). The alizarin red staining results demonstrated that the 16 mg/mL concentration of all three tested DFDBA induced complete morphologic differentiation and mineralized nodule production of the SaOS-2 cells. The RT-PCR results revealed osteopontin gene expression at a 16 mg/mL concentration of all three test groups, but not at an 8 mg/mL concentration.
These commercial types of DFDBA are capable of decreasing proliferation and increasing osteogenic differentiation of the SaOS-2 cell line and have osteoinductive activity in vitro.
PMCID: PMC3543938  PMID: 23346466
Alizarin red; Bone graft; Cell differentiation; Cell proliferation; Regeneration
4.  Osteoinduction of Human Mesenchymal Stem Cells by Bioactive Composite Scaffolds without Supplemental Osteogenic Growth Factors 
PLoS ONE  2011;6(10):e26211.
The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.
PMCID: PMC3192176  PMID: 22022571
5.  Effect of CGRP-Adenoviral Vector Transduction on the Osteoblastic Differentiation of Rat Adipose-Derived Stem Cells 
PLoS ONE  2013;8(8):e72738.
Calcitonin gene-related peptide (CGRP) promotes osteoblast recruitment and osteogenic activity. However, no evidence suggests that CGRP could affect the differentiation of stem cells toward osteoblasts. In this study, we genetically modified adipose-derived stem cells (ADSCs) by introducing the CGRP gene through adenoviral vector transduction and investigated on cellular proliferation and osteoblast differentiation in vitro and osteogenesis in vivo as well. For the in vitro analyses, rat ADSCs were transducted with adenoviral vectors containing the CGRP gene (Ad-CGRP) and were cultured in complete osteoblastic medium. The morphology, proliferative capacity, and formation of localized regions of mineralization in the cells were evaluated. The expression of alkaline phosphatase (ALP) and special markers of osteoblasts, such as Collagen I, Osteocalcin (BPG) and Osteopontin (OPN), were measured by cytochemistry, MMT, RT-PCR, and Western blot. For the in vivo analyses, the Ad-CGRP-ADSCs/Beta-tricalcium phosphate (β-TCP) constructs were implanted in rat radial bone defects for 12 weeks. Radiography and histomorphology evaluations were carried out on 4 weeks and 12 weeks. Our analyses indicated that heterogeneous spindle-shaped cells and localized regions of mineralization were formed in the CGRP-transduced ADSCs (the transduced group). A higher level of cellular proliferation, a high expression level of ALP on days 7 and 14 (p<0.05), and increased expression levels of Collagen I, BPG and OPN presented in transduced group (p<0.05). The efficiency of new bone formation was dramatically enhanced in vivo in Ad-CGRP-ADSCs/β-TCP group but not in β-TCP group and ADSCs/β-TCP group. Our results reveal that ADSCs transduced with an Ad-CGRP vector have stronger potential to differentiate into osteoblasts in vitro and are able to regenerate a promising new tissue engineering bone in vivo. Our findings suggest that CGRP-transduced ADSCs may serve as seed cells for bone tissue engineering and provide a potential way for treating bone defects.
PMCID: PMC3758345  PMID: 24023640
6.  A comparative study on the initial stability of different implants placed above the bone level using resonance frequency analysis 
This study evaluated the initial stability of different implants placed above the bone level in different types of bone.
As described by Lekholm and Zarb, cortical layers of bovine bone specimens were trimmed to a thickness of 2 mm, 1 mm or totally removed to reproduce bone types II, III, and IV respectively. Three Implant system (Brånemark System® Mk III TiUnite™, Straumann Standard Implant SLA®, and Astra Tech Microthread™-OsseoSpeed™) were tested. Control group implants were placed in level with the bone, while test group implants were placed 1, 2, 3, and 4 mm above the bone level. Initial stability was evaluated by resonance frequency analysis. Data was statistically analyzed by one-way analysis of variance in confidence level of 95%. The effective implant length and the Implant Stability Quotient (ISQ) were compared using simple linear regression analysis.
In the control group, there was a significant difference in the ISQ values of the 3 implants in bone types III and IV (P<.05). The ISQ values of each implant decreased with increased effective implant length in all types of bone. In type II bone, the decrease in ISQ value per 1-mm increase in effective implant length of the Brånemark and Astra implants was less than that of the Straumann implant. In bone types III and IV, this value in the Astra implant was less than that in the other 2 implants.
The initial stability was much affected by the implant design in bone types III, IV and the implant design such as the short pitch interval was beneficial to the initial stability of implants placed above the bone level.
PMCID: PMC3259444  PMID: 22259702
Effective implant length; Initial stability; Implant design; Resonance frequency analysis
7.  A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice 
This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair.
PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry.
We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP.
We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.
PMCID: PMC3706764  PMID: 23290259
8.  Tricalcium phosphate/hydroxyapatite (TCP-HA) bone scaffold as potential candidate for the formation of tissue engineered bone 
The Indian Journal of Medical Research  2013;137(6):1093-1101.
Background & objectives:
Various materials have been used as scaffolds to suit different demands in tissue engineering. One of the most important criteria is that the scaffold must be biocompatible. This study was carried out to investigate the potential of HA or TCP/HA scaffold seeded with osteogenic induced sheep marrow cells (SMCs) for bone tissue engineering.
HA-SMC and TCP/HA-SMC constructs were induced in the osteogenic medium for three weeks prior to implantation in nude mice. The HA-SMC and TCP/HA-SMC constructs were implanted subcutaneously on the dorsum of nude mice on each side of the midline. These constructs were harvested after 8 wk of implantation. Constructs before and after implantation were analyzed through histological staining, scanning electron microscope (SEM) and gene expression analysis.
The HA-SMC constructs demonstrated minimal bone formation. TCP/HA-SMC construct showed bone formation eight weeks after implantation. The bone formation started on the surface of the ceramic and proceeded to the centre of the pores. H&E and Alizarin Red staining demonstrated new bone tissue. Gene expression of collagen type 1 increased significantly for both constructs, but more superior for TCP/HA-SMC. SEM results showed the formation of thick collagen fibers encapsulating TCP/HA-SMC more than HA-SMC. Cells attached to both constructs surface proliferated and secreted collagen fibers.
Interpretation & conclusions:
The findings suggest that TCP/HA-SMC constructs with better osteogenic potential compared to HA-SMC constructs can be a potential candidate for the formation of tissue engineered bone.
PMCID: PMC3734714  PMID: 23852290
Bone; ceramics phosphate; fibrin; scaffold; tissue engineering
9.  The Dependence of MG63 Osteoblast Responses to (Meth)Acrylate-based Networks on Chemical Structure and Stiffness 
Biomaterials  2010;31(24):6131-6141.
The cell response to an implant is regulated by the implant’s surface properties including topography and chemistry, but less in known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene gycol) dimethacrylate (PEGDMA; Mn~750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-β1, and VEGF-A) was assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry.
PMCID: PMC2896827  PMID: 20510445
Surface Stiffness; Osteoblasts; Hydroxyethyl methacrylate; Polyethylene glycol dimethacrylate; In vitro; mineralized and demineralized bone
10.  Osteoblastic behavior to zirconium coating on Ti-6Al-4V alloy 
The purpose of this study was to assess the surface characteristics and the biocompatibility of zirconium (Zr) coating on Ti-6Al-4V alloy surface by radio frequency (RF) magnetron sputtering method.
The zirconium films were developed on Ti-6Al-4V discs using RF magnetron sputtering method. Surface profile, surface composition, surface roughness and surface energy were evaluated. Electrochemical test was performed to evaluate the corrosion behavior. Cell proliferation, alkaline phosphatase (ALP) activity and gene expression of mineralized matrix markers were measured.
SEM and EDS analysis showed that zirconium deposition was performed successfully on Ti-6Al-4V alloy substrate. Ti-6Al-4V group and Zr-coating group showed no significant difference in surface roughness (P>.05). Surface energy was significantly higher in Zr-coating group than in Ti-6Al-4V group (P<.05). No difference in cell morphology was observed between Ti-6Al-4V group and Zr-coating group. Cell proliferation was higher in Zr-coating group than Ti-6Al-4V group at 1, 3 and 5 days (P<.05). Zr-coating group showed higher ALP activity level than Ti-6Al-4V group (P<.05). The mRNA expressions of bone sialoprotein (BSP) and osteocalcin (OCN) on Zr-coating group increased approximately 1.2-fold and 2.1-fold respectively, compared to that of Ti-6Al-4V group.
These results suggest that zirconium coating on Ti-6Al-4V alloy could enhance the early osteoblast responses. This property could make non-toxic metal coatings on Ti-6Al-4V alloy suitable for orthopedic and dental implants.
PMCID: PMC4279051  PMID: 25551012
Biocompatible materials; Surface-coated materials; Zirconium; Titanium; Surface properties; Cell proliferation
11.  Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells 
Journal of periodontal research  2011;47(3):10.1111/j.1600-0765.2011.01433.x.
Background and Objectives
Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway.
Materials and Methods
SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin.
Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling.
These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.
PMCID: PMC3865600  PMID: 22150562
dental follicle cells; Wnt; cementoblast; maturation; BMP
12.  Osteogenic and angiogenic potentials of monocultured and co-cultured hBMSCs and HUVECs on 3D porous β-TCP scaffold 
Acta biomaterialia  2012;9(1):4906-4915.
The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study was to investigate cell proliferation and differentiation of mono- or co-cultured human bone marrow-derived mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) on a 3D porous, biodegradable β-TCP scaffold. In co-culture studies, the ratio of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs, and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of dsDNA whereas hBMSCs and HUVECs differentiation were assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs, and hBMSCs/HUVECs adhered and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time PCR results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at day 7 and 14, but alp gene expression was significantly increased in hBMSCs co-cultures in absence of distinguishing the contribution of individual cell type.
PMCID: PMC3508299  PMID: 22902820
β-TCP; hBMSCs; HUVECs; Osteogenesis; Angiogenesis
13.  Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production on titanium alloy substrates than on poly-ether-ether-ketone 
Background Context
Multiple biomaterials are clinically available to spine surgeons for performing interbody fusion. Poly-ether-ether-ketone (PEEK) is used frequently for lumbar spine interbody fusion, but alternative materials are also used, including titanium (Ti) alloys. Previously, we showed that osteoblasts exhibit a more differentiated phenotype when grown on machined or grit-blasted titanium aluminum vanadium (Ti6Al4V) alloys with micron-scale roughened surfaces than when grown on smoother Ti6Al4V surfaces or on tissue culture polystyrene (TCPS). We hypothesized that osteoblasts cultured on rough Ti alloy substrates would present a more mature osteoblast phenotype than cells cultured on PEEK, suggesting that textured Ti6Al4V implants may provide a more osteogenic surface for interbody fusion devices.
The aim of the present study was to compare osteoblast response to smooth Ti6Al4V (sTiAlV) and roughened Ti6Al4V (rTiAlV) with their response to PEEK with respect to differentiation and production of factors associated with osteogenesis.
Study Design
This in vitro study compared the phenotype of human MG63 osteoblast-like cells cultured on PEEK, sTiAlV, or rTiAlV surfaces and their production of bone morphogenetic proteins (BMPs).
Surface properties of PEEK, sTiAlV, and rTiAlV discs were determined. Human MG63 cells were grown on TCPS and the discs. Confluent cultures were harvested, and cell number, alkaline phosphatase–specific activity, and osteocalcin were measured as indicators of osteoblast maturation. Expression of messenger RNA (mRNA) for BMP2 and BMP4 was measured by real-time polymerase chain reaction. Levels of BMP2, BMP4, and BMP7 proteins were also measured in the conditioned media of the cell cultures.
Although roughness measurements for sTiAlV (Sa=0.09±0.01), PEEK (Sa=0.43±0.07), and rTiAlV (Sa= 1.81±0.51) varied, substrates had similar contact angles, indicating comparable wettability. Cell morphology differed depending on the surface. Cells cultured on Ti6Al4V had lower cell number and increased alkaline phosphatase specific activity, osteocalcin, BMP2, BMP4, and BMP7 levels in comparison to PEEK. In particular, roughness significantly increased the mRNA levels of BMP2 and BMP4 and secreted levels of BMP4.
These data demonstrate that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment without addition of exogenous factors, which may induce better and faster bone during interbody fusion.
PMCID: PMC3618467  PMID: 22424980
Ti6Al4V; PEEK; Osteoblast; BMP; Roughness
14.  Epigenetic Regulation of Early Osteogenesis and Mineralized Tissue Formation by a HOXA10-PBX1-Associated Complex 
Cells, Tissues, Organs  2011;194(2-4):146-150.
Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.
PMCID: PMC3178072  PMID: 21597276
Chromatin remodeling during osteogenesis; Osterix histone acetylation; Pbx inhibition of osteoblastogenesis
15.  Ceramic Identity Contributes to Mechanical Properties and Osteoblast Behavior on Macroporous Composite Scaffolds  
Implants formed of metals, bioceramics, or polymers may provide an alternative to autografts for treating large bone defects. However, limitations to each material motivate the examination of composites to capitalize on the beneficial aspects of individual components and to address the need for conferring bioactive behavior to the polymer matrix. We hypothesized that the inclusion of different bioceramics in a ceramic-polymer composite would alter the physical properties of the implant and the cellular osteogenic response. To test this, composite scaffolds formed from poly(lactide-co-glycolide) (PLG) and either hydroxyapatite (HA), β-tricalcium phosphate (TCP), or bioactive glass (Bioglass 45S®, BG) were fabricated, and the physical properties of each scaffold were examined. We quantified cell proliferation by DNA content, osteogenic response of human osteoblasts (NHOsts) to composite scaffolds by alkaline phosphatase (ALP) activity, and changes in gene expression by qPCR. Compared to BG-PLG scaffolds, HA-PLG and TCP-PLG composite scaffolds possessed greater compressive moduli. NHOsts on BG-PLG substrates exhibited higher ALP activity than those on control, HA-, or TCP-PLG scaffolds after 21 days, and cells on composites exhibited a 3-fold increase in ALP activity between 7 and 21 days versus a minimal increase on control scaffolds. Compared to cells on PLG controls, RUNX2 expression in NHOsts on composite scaffolds was lower at both 7 and 21 days, while expression of genes encoding for bone matrix proteins (COL1A1 and SPARC) was higher on BG-PLG scaffolds at both time points. These data demonstrate the importance of selecting a ceramic when fabricating composites applied for bone healing.
PMCID: PMC4047943  PMID: 24955539
bioceramic; hydroxyapatite; bioactive glass; composite; scaffold; bone
16.  Adult Stem Cells Properties in Terms of Commitment, Aging and Biological Safety of Grit-Blasted and Acid-Etched Ti Dental Implants Surfaces 
Titanium (Ti) is one of the most widely used biomaterials for manufacturing dental implants. The implant surface properties strongly influence osseointegration. The aim of the present study was to in vitro investigate the characteristics of Ti dental implants in terms of mutagenicity, hemocompatibility, biocompatibility, osteoinductivity and biological safety. The Ames test was used to test the mutagenicity of the Ti dental implants, and the hemolysis assay for evaluating their hemocompatibility. Human adipose - derived stem cells (ADSCs) were then seeded onto these implants in order to evaluate their cytotoxicity. Gene expression analyzing with real-time PCR was carried out to investigate the osteoinductivity of the biomaterials. Finally, the genetic stability of the cells cultured onto dental implants was determined by karyotyping. Our results demonstrated that Ti dental implants are not mutagenic, do not cause hemolysis, and are biocompatible. The MTT assay revealed that ADSCs, seeded on Ti dental implants, proliferate up to 30 days in culture. Moreover, ADSCs loaded on Ti dental implants show a substantial expression of some osteoblast specific markers, such as COL1A1, OPN, ALPL, and RUNX2, as well as chromosomal stability after 30 days of culture in a medium without osteogenic factors. In conclusion, the grit-blasted and acid-etched treatment seems to favor the adhesion and proliferation of ADSCs and improve the osteoinductivity of Ti dental implant surfaces.
PMCID: PMC4293610  PMID: 25635249
Titanium dental implants; surface properties; adipose- derived stem cells; biocompatibility; osteogenic differentiation
17.  Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro 
Osteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).
Osteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.
BCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.
Together these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.
PMCID: PMC3691632  PMID: 23767824
Biofilm; Osteoblast; Osteogenic differentiation; Staphylococcus aureus; Receptor activator of NF-kB ligand; Osteoprotegrin
18.  Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate 
The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO2). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.
The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO2 and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO2 implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO2 implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO2 implants.
The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.
PMCID: PMC4077223  PMID: 24987458
Implants; Porous titanium; Hydroxyapatite; Silicatitanate; Osteoblasts; Cell adhesion; Differentiation; Mineralization
19.  Culture Human Mesenchymal Stem Cells With Calcium Phosphate Cement Scaffolds for Bone Repair 
Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean ± SD; n = 5) of (19.5 ± 1.4) MPa, higher than (8.0 ± 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 ± 1.3)% at 8 days, matching (90.7 ± 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 ± 11)% at 1 day to (90 ± 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 ± 0.93) [(mM pNpp/min)/(μg DNA)] versus control media (3.62 ± 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 ± 19.7)% compared with (6.1 ± 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering.
PMCID: PMC2989678  PMID: 20091907
calcium phosphate cement; chitosan; strength and toughness; human mesenchymal stem cells; bone tissue engineering
20.  The effect of polystyrene sodium sulfonate grafting on polyethylene terephthalate artificial ligaments on in vitro mineralisation and in vivo bone tissue integration 
Biomaterials  2013;34(29):7048-7063.
This study investigates the impact of polystyrene sodium sulfonate (PolyNaSS) grafting onto the osseointegration of a polyethylene terephthalate artificial ligament (Ligament Advanced Reinforcement System, LARS™) used for Anterior Cruciate Ligament (ACL). The performance of grafted and non-grafted ligaments was assessed in vitro by culturing human osteoblasts under osteogenic induction and this demonstrated that the surface modification was capable of up-regulating the secretion of ALP and induced higher level of mineralisation as measured 6 weeks post-seeding by Micro-Computed Tomography. Grafted and non-grafted LARS™ were subsequently implanted in an ovine model for ACL reconstruction and the ligament-to-bone interface was evaluated by histology and biomechanical testing 3 and 12 months post-implantation. The grafted ligaments exhibited more frequent direct ligament-to-bone contact and bone formation in the core of the ligament at the later time point than the nongrafted specimens, the grafting also significantly reduced the fibrous encapsulation of the ligament 12 months post-implantation. However, this improved osseo-integration was not translated into a significant increase in the biomechanical pull-out loads. These results provide evidences that PolyNaSS grafting improved the osseo-integration of the artificial ligament within the bone tunnels. This might positively influence the outcome of the surgical reconstructions, as higher ligament stability is believed to limit micro-movement and therefore permits earlier and enhanced healing.
PMCID: PMC3779617  PMID: 23790438
21.  Magnesium vs. machined surfaced titanium - osteoblast and osteoclast differentiation 
This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source.
40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs).
MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM.
Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.
PMCID: PMC4085238  PMID: 25006378
Magnesium; Ion implantation; Titanium surface; MC3T3-E1; Mouse bone marrow-derived macrophages (BMMs)
22.  Physical properties and biological/odontogenic effects of an experimentally developed fast-setting α-tricalcium phosphate-based pulp capping material 
BMC Oral Health  2014;14:87.
Recently, fast-setting α-tricalcium-phosphate (TCP) cement was developed for use in the pulp capping process. The aim of this study was to investigate the physical properties and biological effects of α-TCP cement in comparison with mineral trioxide aggregate (MTA).
We measured the setting time, pH values, compressive strength, and solubility of the two materials. We evaluated biocompatibility on the basis of cell morphology and a viability test using human dental pulp cells (hDPCs). Chemical composition of each material was analyzed by energy dispersive x-ray spectroscopic (EDS) analysis. The expression of odontogenic-related genes was evaluated by Western blotting and immunofluorescence. The calcified nodule formation was measured by Alizarin red staining. We performed the pulp capping procedure on rat teeth for histological investigation. The data were analyzed by an independent t-test for physical properties, one-way ANOVA for biological effects, and the Mann-Whitney U test for tertiary dentin formation. A P value of less than 0.05 was considered statistically significant for all tests.
The setting time, pH values, and compressive strength of α-TCP was lower than that of MTA (P < 0.05); however, the solubility of α-TCP was higher than that of MTA (P < 0.05). The resultant cell viability observed with the two materials was similar (P > 0.05). Scanning electron microscopy (SEM) revealed that cells attached to both materials were flat and had cytoplasmic extensions. The expression of odontogenic-related markers and mineralized nodule formation were higher in the two experimental groups compared to the control group (P < 0.05). Continuous tertiary dentin was formed underneath the capping materials in all samples of the tested groups.
Our study demonstrated that the α-TCP exhibited biocompatibility and odontogenicity comparable to MTA, whereas it had a quicker setting time.
PMCID: PMC4105101  PMID: 25015173
Calcium phosphate; Fast-setting; Mineral trioxide aggregate; Odontogenic; Pulp capping; Tertiary dentin
23.  Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function▿  
Infection and Immunity  2010;78(9):3726-3735.
Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.
PMCID: PMC2937457  PMID: 20584977
24.  Characteristics and osteogenic effect of zirconia porous scaffold coated with β-TCP/HA 
The purpose of this study was to evaluate the properties of a porous zirconia scaffold coated with bioactive materials and compare the in vitro cellular behavior of MC3T3-E1 preosteoblastic cells to titanium and zirconia disks and porous zirconia scaffolds.
Titanium and zirconia disks were prepared. A porous zirconia scaffold was fabricated with an open cell polyurethane disk foam template. The porous zirconia scaffolds were coated with β-TCP, HA and a compound of β-TCP and HA (BCP). The characteristics of the specimens were evaluated using scanning electron microscopy (SEM), energy dispersive x-ray spectrometer (EDX), and x-ray diffractometry (XRD). The dissolution tests were analyzed by an inductively coupled plasma spectrometer (ICP). The osteogenic effect of MC3T3-E1 cells was assessed via cell counting and reverse transcriptase-polymerase chain reaction (RT-PCR).
The EDX profiles showed the substrate of zirconia, which was surrounded by the Ca-P layer. In the dissolution test, dissolved Ca2+ ions were observed in the following decreasing order; β-TCP > BCP > HA (P<.05). In the cellular experiments, the cell proliferation on titanium disks appeared significantly lower in comparison to the other groups after 5 days (P<.05). The zirconia scaffolds had greater values than the zirconia disks (P<.05). The mRNA level of osteocalcin was highest on the non-coated zirconia scaffolds after 7 days.
Zirconia had greater osteoblast cell activity than titanium. The interconnecting pores of the zirconia scaffolds showed enhanced proliferation and cell differentiation. The activity of osteoblast was more affected by microstructure than by coating materials.
PMCID: PMC4146729  PMID: 25177472
Zirconia porous scaffold; Osteogenic effect; Cellular response; β-TCP; HA
25.  Circulating Osteogenic Cells: Characterization and Relationship to Rates of Bone Loss in Postmenopausal Women 
Bone  2010;47(1):83-92.
There is increasing evidence that osteogenic cells are present not only in bone marrow (BM) but also in peripheral blood (PB). Since staining for alkaline phosphatase (AP) identifies osteoprogenitor cells in BM, we sought to further characterize BM versus PB hematopoietic lineage negative (lin−)/AP+ cells and to compare gene expression in PB lin−/AP+ cells from postmenopausal women undergoing rapid versus slow bone loss. PB lin−/AP+ cells were smaller than their BM counterparts, and both were negative for the pan-hematopoietic marker, CD45. BM and PB lin−/AP+ cells were capable of mineralization in vitro. Using whole genome linear amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we found that relative to the BM cells, PB lin−/AP+ cells expressed similar levels of a number of key osteoblast marker genes (runx2, osterix, osteopontin, OPG, periostin), consistent with the PB cells being in the osteoblastic lineage. Importantly, however, compared to the BM cells, PB lin−/AP+ cells expressed lower levels of mRNAs for AP, type I collagen, and for a panel of proliferation markers, but higher levels of osteocalcin, osteonectin, and PTHR1 mRNAs, as well as those for RANKL and ICAM-1, both of which are important in supporting osteoclastogenesis. Using microarray followed by QPCR analysis, we further demonstrated that, compared to postmenopausal women undergoing slow bone loss, PB lin−/AP+ cells from women undergoing rapid bone loss expressed lower levels of mRNAs for hydroxyprostaglandin dehydrogenase, interferon regulator factor 3, Wnt1-induced secreted protein 1, and TGFβ2, but higher levels of the Smad3 interacting protein, zinc finger DHHC-type containing 4 and col1α2. These data thus demonstrate that while PB lin−/AP+ cells express a number of osteoblastic genes and are capable of mineralization, they are a relatively quiescent cell population, both in terms of cell proliferation and matrix synthesis. However, their higher expression of RANKL and ICAM-1 mRNAs as compared to BM lin−/AP+ cells suggests a role for the PB lin−/AP+ cells in regulating osteoclastogenesis that warrants further investigation. Our study also provides “proof-of-concept” for the use of PB lin−/AP+ cells in clinical-investigative studies, and identifies several pathways that could potentially regulate rates of bone loss in postmenopausal women.
PMCID: PMC2891408  PMID: 20362080
osteoporosis; bone loss; menopause; osteoblast; progenitors

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