Je-Hee Jang

Role of subnano-, nano- and submicron-surface features on osteoblast differentiation of bone marrow mesenchymal stem cells.

Subnano, nano and sub-micron surface features can selectively activate integrin receptors and induce osteoblast differentiation of bone marrow mesenchymal stem cells. Although it is widely accepted that nanoscale titanium surface roughness may promote differentiation of various osteoblast lineages, there has been no clear report on the threshold dimension of surface features and the optimized dimensions of surface features for triggering integrin activation and stem cell differentiation. This study systematically controlled titanium surface features from the sub-nano to sub-micron scales and investigated the corresponding effects on stem cell responses, such as integrin activation, cyclins, key transcriptional genes of osteoblast differentiation and osteoblastic phenotype genes. Surface features with sub-nano surface dimensions were insufficient to increase integrin activation compared to pure nanoscale titanium surface features. Although both pure nanoscale and nano-submicron hybrid scales of titanium surface features were sufficient for activating integrin-ligand proteins interactions through the α integrin subunits, only nano-submicron hybrid titanium surface features significantly accelerated subsequent osteoblast differentiation of primary mouse bone marrow stromal cells after 2 weeks. In addition, live cell analysis of human bone marrow mesenchymal stem cells on transparent titanium demonstrated rapid cytoskeletal re-organization on the nanoscale surface features, which ultimately induced higher expression of osteoblast phenotype genes after 3 weeks.

Enhanced osteoblast response to an equal channel angular pressing-processed pure titanium substrate with microrough surface topography

This study investigated the surface characteristics and in vitro biocompatibility of ultrafine-grain pure titanium substrates produced by equal channel angular pressing (ECAP) using MC3T3-E1 pre-osteoblast cells, compared with those of conventional coarse-grain pure titanium (CP) and Ti-6Al-4V (Ti64) substrates. All Ti surfaces were grit-blasted with hydroxyapatite particles to produce microrough surfaces. The surface characteristics were evaluated by electron back-scattered diffractometry, scanning electron microscopy, contact angle and surface energy measurements, and optical profilometry. The morphology of spread cells, cell attachment, viability, alkaline phosphatase (ALP) activity, quantitative analysis of osteoblastic gene expression and mineralization nodule formation on different surfaces were evaluated. ECAP-processed substrates showed a significantly lower water contact angle and higher surface energy compared with coarse-grain CP and Ti64 substrates (p<0.05). They also showed enhanced cell spreading, attachment, viability and ALP activity compared with the CP and Ti64 surfaces (p<0.05). Real-time polymerase chain reaction analysis showed notably higher ALP, osteopontin and osteocalcin mRNA levels in cells grown on the ECAP surfaces than on the CP and Ti64 surfaces, and the ECAP surfaces showed significantly greater mineralization nodule formation compared with the CP and Ti64 substrates (p<0.05). These results demonstrate the superior osteoblast cell compatibility of microroughened Ti surface made of ECAP-processed ultrafine-grain pure Ti substrates over coarse-grain pure Ti and Ti64 substrates.

Osteoblast response and osseointegration of a Ti–6Al–4V alloy implant incorporating strontium

This study investigated the surface characteristics, in vitro and in vivo biocompatibility of Ti-6Al-4V alloy implants incorporating strontium ions (Sr), produced by hydrothermal treatment using a Sr-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, contact angle and surface energy measurement and inductively coupled plasma-mass spectroscopy (ICP-MS). Human osteoblast-like cell (MG63) attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Sr-containing Ti-6Al-4V surfaces were compared with untreated Ti-6Al-4V surfaces. Fifty-six screw implants (28 control and 28 experimental) were placed in the tibiae and femoral condyles of seven New Zealand White rabbits. The osteoconductivity of Sr-containing Ti-6Al-4V implants was evaluated by removal torque testing and histomorphometric analysis after 4weeks implantation. Hydrothermal treatment produced a crystalline SrTiO(3) layer. ICP-MS analysis showed that Sr ions were released from treated surfaces into the solution. Significant increases in ALP activity (P=0.000), mRNA expressions of key osteoblast genes (osterix, bone sialoprotein, and osteocalcin), removal torque values (P<0.05) and bone-implant contact percentages (P<0.05) in both cortical and cancellous bone were observed for Sr-containing Ti-6Al-4V surfaces. The results indicate that the Sr-containing oxide layer produced by hydrothermal treatment may be effective in improving the osseointegration of Ti-6Al-4V alloy implants by enhancing differentiation of osteoblastic cells, removal torque forces and bone apposition in both cortical and cancellous bone.

Osteoblast response to magnesium ion‐incorporated nanoporous titanium oxide surfaces

OBJECTIVE This study investigated the surface characteristics and in vitro osteoconductivity of a titanium (Ti) surface incorporated with the magnesium ions (Mg) produced by hydrothermal treatment for future application as an endosseous implant surface. MATERIAL AND METHODS Mg-incorporated Ti oxide surfaces were produced by hydrothermal treatment using Mg-containing solution on two different microstructured surfaces--abraded minimally rough (Ma) or grit-blasted moderately rough (RBM) samples. The surface characteristics were evaluated using scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, and inductively coupled plasma atomic emission spectroscopy (ICP-AES). MC3T3-E1 pre-osteoblast cell attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Ma, RBM, Mg-incorporated Ma (Mg), and Mg-incorporated grit-blasted (RBM/Mg) Ti surfaces were evaluated. RESULTS Hydrothermal treatment produced an Mg-incorporated Ti oxide layer with nanoporous surface structures. Mg-incorporated surfaces showed surface morphologies and surface roughness values almost identical to those of untreated smooth or micro-rough surfaces at the micron scale. ICP-AES analysis showed Mg ions released from treated surfaces into the solution. Mg incorporation significantly increased cellular attachment (P=0 at 0.5 h, P=0.01 at 1 h) on smooth surfaces, but no differences were found on micro-rough surfaces. Mg incorporation further increased ALP activity in cells grown on both smooth and micro-rough surfaces at 7 and 14 days of culture (P=0). Real-time polymerase chain reaction analysis showed higher mRNA expressions of the osteoblast transcription factor gene (Dlx5), various integrins, and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on micro-rough (RBM) and Mg-incorporated (Mg and RBM/Mg) surfaces than those on Ma surfaces. Mg incorporation further increased the mRNA expressions of key osteoblast genes and integrins (α1, α2, α5, and β1) in cells grown on both the smooth and the micro-rough surfaces. CONCLUSION These results indicate that an Mg-incorporated nanoporous Ti oxide surface produced by hydrothermal treatment may improve implant bone healing by enhancing the attachment and differentiation of osteoblastic cells.

Effects of phosphoric acid treatment of titanium surfaces on surface properties, osteoblast response and removal of torque forces

This study investigated the surface characteristics and biocompatibility of phosphate ion (P)-incorporated titanium (Ti) surfaces hydrothermally treated with various concentrations of phosphoric acid (H(3)PO(4)). The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, contact angle and surface energy measurement and inductively coupled plasma mass spectroscopy (ICP-MS). MC3T3-E1 cell attachment, spreading, proliferation and osteoblastic gene expression on different surfaces were evaluated. The degree of bony integration was biomechanically evaluated by removal torque testing after 4 weeks of healing in rabbit tibiae. The H(3)PO(4) treatment produced micro-rough Ti surfaces with crystalline P-incorporated Ti oxide layers. High concentration H(3)PO(4) treatment (1% and 2%) produced significantly higher hydrophilic surfaces compared with low H(3)PO(4) treatment (0.5%) and untreated surfaces (P<0.01). ICP-MS analysis showed P ions were released from P-incorporated surfaces. Significant increased cell attachment (P<0.05) and notably higher mRNA expressions of Runx2, alkaline phosphatase, osteopontin and osteocalcin were observed in cells grown on P-incorporated surfaces compared with cells on untreated machined surfaces. P-incorporated surfaces showed significantly higher removal torque forces compared with untreated machined implants (P<0.05). Ti surfaces treated with 2% H(3)PO(4) showed increasing tendencies in osteoblastic gene expression and removal torque forces compared with those treated with lower H(3)PO(4) concentrations or untreated surfaces. These results demonstrate that H(3)PO(4) treatment may improve the biocompatibility of Ti implants by enhancing osteoblast attachment, differentiation and biomechanical anchorage.

Osteoconductivity of hydrophilic microstructured titanium implants with phosphate ion chemistry

This study investigated the surface characteristics and bone response of titanium implants produced by hydrothermal treatment using H(3)PO(4), and compared them with those of implants produced by commercial surface treatment methods - machining, acid etching, grit blasting, grit blasting/acid etching or spark anodization. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle measurement and stylus profilometry. The osteoconductivity of experimental implants was evaluated by removal torque testing and histomorphometric analysis after 6 weeks of implantation in rabbit tibiae. Hydrothermal treatment with H(3)PO(4) and subsequent heat treatment produced a crystalline phosphate ion-incorporated oxide (titanium oxide phosphate hydrate, Ti(2)O(PO(4))(2)(H(2)O)(2); TiP) surface approximately 5microm in thickness, which had needle-like surface microstructures and superior wettability compared with the control surfaces. Significant increases in removal torque forces and bone-to-implant contact values were observed for TiP implants compared with those of the control implants (p<0.001). After thorough cleaning of the implants removed during the removal torque testing, a considerable quantity of attached bone was observed on the surfaces of the TiP implants.

Effect of the pore structure of bioactive glass balls on biocompatibility in vitro and in vivo.

We prepared porous bioactive glass (BG) balls with various pore architectures using a modified version of a polymer templating technique which is generally used for the synthesis of mesoporous BG. Sol-gel derived porous BG is an excellent candidate as a graft material for bone tissue regeneration due to its good bone forming bioactivity and biodegradability. The biodegradability is largely related to the pore architecture and affects its biocompatibility. The pore architecture of the BG balls was controllable by changing the reaction time in chloroform. The relationship between the pore architecture of the BG balls and biocompatibility were studied using MC3T3-E1 pre-osteoblast cells in vitro and the rabbit calvarial model in vivo 8 weeks after implantation. The mesoporous BG balls (BG0) and porous BG beads with a hierarchical pore structure on the nano- to microscale (BG0.5 and BG2) showed a good cell proliferation response and differentiation behavior in vitro and in vivo without serious toxicity. These hierarchically porous structures also enhanced osteoconductivity. However, the existence of too many microscale pores in the BG balls (BG24) led to their rapid biodegradation and, consequently, to serious negative effects in vitro and in vivo. The pore architecture of the BG balls greatly influenced their biocompatibility, as well as bone formation, and should be carefully controlled when designing new materials for use in bioapplications. The porous BG balls with hierarchical pores on the nano- to microscale exhibit favorable biocompatibility in vitro and promise excellent potential applications in the field of biomaterials, such as tissue regeneration and drug storage.

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces.

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

Enhanced osteoblast response to hydrophilic strontium and/or phosphate ions‐incorporated titanium oxide surfaces

OBJECTIVE This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces incorporated with strontium ions (Sr) and/or phosphate ions (P) produced by hydrothermal treatment for future applications as endosseous implant surfaces. MATERIAL AND METHODS Sr and/or P-incorporated Ti oxide surfaces were produced by hydrothermal treatment. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle and surface energy measurements, inductively coupled plasma atomic emission spectroscopy, and profilometry. MC3T3-E1 pre-osteoblast cell attachment, morphology of spread cells, viability, and quantitative analysis of osteoblastic gene expression on grit-blasted microrough (RBM), P-incorporated (P), and P- and Sr-incorporated (SrP) Ti surfaces were evaluated. RESULTS Microstructured P and SrP surfaces showed significantly higher wettability and surface energy compared with RBM surfaces (P<0.01). After immersion in Hanks balanced salt solution, considerable apatite deposition was observed on the P and SrP surfaces. Sr incorporation significantly increased cellular attachment and viability compared with other surfaces (P<0.05). Real-time polymerase chain reaction analysis showed markedly higher mRNA expressions of the osteoblast transcription factor gene (Runx2) and the osteoblast phenotype genes (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) in cells grown on the P and SrP surfaces compared with those on the RBM surface. CONCLUSIONS These results demonstrate that Sr- and P-incorporated Ti oxide surfaces may improve implant bone healing by enhancing attachment, viability, and differentiation of osteoblastic cells.

Healing of rabbit calvarial bone defects using biphasic calcium phosphate ceramics made of submicron-sized grains with a hierarchical pore structure

OBJECTIVES This study investigated the efficacy of new bone graft substitutes - biphasic calcium phosphates (BCP) made of submicron-sized grains with fully interconnected wide-range micron-scale pores in two different macrodesigns: donut shaped with a 300-400 microm central macropore (n-BCP-1) or rod-shaped (n-BCP-2)--in the healing of rabbit calvarial defects, and compared their bone-healing properties with those of various commercial bone substitutes, which included substitutes with similar BCP composition (MBCP and Osteon), anorganic bovine bone (Bio-Oss), and beta-TCP (Cerasorb). MATERIAL AND METHODS The surface morphology of the bone substitutes was investigated using scanning electron microscopy (SEM). Defects 8 mm in diameter were created in the calvaria of 30 adult male New Zealand White rabbits and were filled with six types of bone substitutes. The percentage of newly formed bone (NB%) was evaluated histomorphometrically 4 and 8 weeks after implantation. RESULTS SEM observation showed submicron-sized grains with fully interconnected micropore structures in the n-BCP-1 and n-BCP-2 groups; these groups also showed considerable new bone formation in inner micropores as well as on the outer surfaces. The n-BCP-1 group exhibited enhanced new bone formation and direct ingrowth of bone tissue with blood vessels into central pores. Histomorphometric analysis showed significantly greater NB% in the n-BCP-1 group when compared with the other groups at 4 and 8 weeks (P<0.05). CONCLUSION A new BCP ceramics made of submicron-sized grains with a hierarchical pore structure was an effective osteoconductive material for the treatment of osseous defects of rabbit calvaria.