Comparison of Bone Marrow Stromal Cell Behaviors on Poly(caprolactone) with or without Surface Modification: Studies on Cell Adhesion,Survival and Proliferation

Poly(caprolactone) (PCL) is a promising biodegradable polymer for tissue engineering. However, intrinsically poor cell-adhesive properties of PCL may limit its application. In this study, the PCL film surface was modified with RGDC peptide by a chemical immobilization procedure. Furthermore, bone marrow stromal cell (BMSC) behaviors including attachment, spreading, focal adhesion formation, focal adhesion kinase (FAK) activation, apoptosis and proliferation when cultured on the modified PCL films were investigated. Our results demonstrated that PCL with RGD modification promoted initial BMSC attachment, spreading and focal adhesion formation. At a later time point (12 h), BMSC attachment on both RGD peptide-modified PCL and PCL-NH₂ films significantly increased compared to untreated PCL films. Importantly, FAK phosphorylation was significantly increased only on the films with RGD-modified films, not on the PCL-NH₂ films, demonstrating that PCL with RGD modification had an advantage in initiating the specific integrin-mediated signal transduction and might play an important role in the subsequent retardation in cell death and enhancement in cell proliferation. The present results provide more evidence that functionalizing PCL with RGD peptides may be a feasible way to improve the interaction between BMSC and PCL substrate, which is important in tissue engineering.     

Osteoblasts on rod shaped hydroxyapatite nanoparticles incorporated PCL film provide an optimal osteogenic niche for stem cell differentiation

After the clinical insertion of a bone biomaterial, the surrounding osteoblasts would migrate and attach to the implant surface and foster a microenvironment that largely determines the differentiation fate of the comigrated mesenchymal stem cells. Whether the fostered microenvironment is suitable for osteogenic differentiation of mesenchymal stem cells is critical for the subsequent osseointegration. In this study, we determined (1) how the spherical or rod-shaped hydroxyapatite nanoparticles (nHA) incorporated poly(?-caprolactone) (PCL) films (PCL-spherical nHA, PCL-rod nHA) interact with primary human osteoblasts (HOBs); (2) how the microenvironment rendered by their interaction affects osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs). HOBs were seeded on PCL, PCL-spherical nHA, and PCL-rod nHA films, respectively. When cultured alone, the HOBs on PCL-rod nHA films showed most efficient osteoblastic differentiation compared with those on PCL or PCL-spherical nHA films. When cocultured with ASCs in an indirect coculture system, only the HOBs on PCL-rod nHA films up-regulated the gene expression of Runx2, bone sialoprotein, and osteocalcin of ASCs. Additionally, the HOBs on PCL-rod nHA films showed significant up-regulation of bone morphogenic protein 2 gene and protein expression and induced highest phosphorylated Smad1/5 protein level in ASCs. Treatment of the coculture medium with bone morphogenic protein 2 inhibitor (Noggin) largely abolished the osteogenic differentiation of the ASCs induced by the HOBs on PCL-rod nHA films. In conclusion, HOBs can not only best display their osteoblastic phenotype by culturing on PCL-rod nHA films but also render an optimal osteogenic niche for the differentiation of stem cells.     

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(epsilon-caprolactone) surfaces

This study was aimed to investigate whether the activation of poly-(epsilon-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine-glycine-aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5 x 10(15)He(+)/cm(2) (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm(2)). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed alpha1, alpha2, alpha3, alpha5, beta1, and alphaVbeta3; after 24h, cells cultured on tissue-plastic expressed high levels of beta1 and alphaVbeta3, while alpha1 showed a low intensity and alpha2, alpha3, and alpha5 were negative. beta1 and alphaVbeta3 were selected to evaluate the interaction between cells and PCL samples. The beta1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in alphaVbeta3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.     

The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone

Using techniques of tissue engineering, synthetic substitutes can be applied for the repair and regeneration of damaged bone. It has been found that material surface properties are crucial for cell adhesion and spreading, i.e. cell activities that are related directly to the ability of osteoblasts to proliferate. This fact has promoted the strategy of creating an ECM-like layer onto materials, so as to influence the cell response. In this study human bone-derived osteoblasts have been used to test the effects of surface modification by low energy ion beams of a poly epsilon-caprolactone (PCL) substrate and subsequent RGD adsorption. Osteoblasts were seeded and grown onto untreated and irradiated poly epsilon-caprolactone films, with or without RGD-adsorption step, and viability, morphology, and spreading of the osteoblasts were studied at different time endpoints. Differences were observed in the organization of cytoskeleton within cells: stress fibers were more evident in irradiated samples vs. untreated and total cell adhesion was higher. Surface characterization by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and surface free energy measurements showed that the polar character of PCL, i.e., the acid-base term, was increased following irradiation treatment. Moreover the irradiated PCL had a nano-sized topography, which also could improve osteoblasts adhesion. We found that the treatment of the surface with ion beam is per se improving osteoblasts adhesion and spreading onto PCL. Furthermore, also if a significant RGD adsorption was obtained for irradiated PCL surfaces, it was found that in the investigated conditions it seems to have only a minor effect on the cell response. This study suggests that new strategies involving irradiation-based treatments can be adopted to promote the initial steps of bone deposition onto synthetic surfaces, exploiting the surface-induced reorganization of the ECM matrix.     

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic-co-glycolic acid)/bioactive glass substrate for tissue engineering

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.     

Evaluation of cell affinity on poly(L-lactide) and poly(epsilon-caprolactone) blends and on PLLA-b-PCL diblock copolymer surfaces

An evaluation of cell proliferation and adhesion on biocompatible film supports was performed. A series of films were compression molded from commercially available poly (L-lactide), PLLA, and poly(epsilon-caprolactone), PCL, and from their melt mixed blends (PLLA/PCL blends). These were compared with compression molded films of PLLA-b-PCL model diblock copolymers. The samples were analyzed by differential scanning calorimetry (DSC), contact angle measurements, and scanning force microscopy (SFM). Cell adhesion and proliferation were performed with monkey derived fibroblasts (VERO) and with osteoblastic cells obtained either enzymatically or from explants cultures of Sprague-Dawley rat calvaria. Migration studies were performed with bone explants of the same origin. The results obtained indicate that although all materials tested were suitable for the support of cellular growth, a PLLA-b-PCL diblock copolymer sample with 93% PLLA was significantly more efficient. This sample exhibited a unique surface morphology with long range ordered domains (of the order of 2-3 mum) of edge-on PLLA lamellae that can promote "cell contact guidance." The influence of other factors such as chemical composition, degree of crystallinity, and surface roughness did not play a major role in determining cell preference toward a specific surface for the materials employed in this work.     

Electrosprayed Hydroxyapatite on Polymer Nanofibers to Differentiate Mesenchymal Stem Cells to Osteogenesis

Abstract

Electrospraying of hydroxyapatite (HA) nanoparticles onto the surface of polymer nanofibers provides a potentially novel substrate for the adhesion, proliferation and differentiation of mesenchymal stem cells (MSCs) into bone tissue regeneration. HA nanoparticles (4%) were electrosprayed on the surface of electrospun polycaprolactone (PCL) nanofibers (420 ± 15 nm) for bone tissue engineering. PCL/HA nanofibers were comparatively characterized with PCL/Collagen (275 ± 56 nm) nanofibers by FT-IR analysis to confirm the presence of HA. Fabricated PCL/HA and PCL/Collagen nanofibers and TCP (control) were used for the differentiation of equine MSC into osteogenic lineages in the presence of DMEM/F12 medium supplemented with β-glycerophosphate, ascorbic acid and dexamethasone. Cell proliferation and differentiation into an osteogenic lineage was evaluated by MTS assay, SEM observation, ALP activity, ARS staining, quantification of mineral deposition and expression of osteocalcin. Proliferation of MSCs increased significantly (P ? 0.05) up to 12% in PCL/Collagen (day 15) compared to PCL/HA nanofibrous substrate. ALP activity was increased 20% in PCL/HA by day 10 confirming the direction of osteogenic lineage from MSCs differentiation. PCL/HA stimulated an increased mineral secretion up to 26% by day 15 on ARS staining compared to PCL/Collagen nanofibers and showing cuboidal morphology by expressing osteocalcin. These results confirmed that the specifically fabricated PCL/HA composite nanofibrous substrate enhanced the differentiation of MSCs into osteogenesis.     

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin,and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.     

Effect of hydrogel porosity on marrow stromal cell phenotypic expression

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.     

Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films

Direct surface modification of biodegradable polycaprolactone (PCL) was performed without the necessity of synthesis of functionisable co-polymers. An easy-to-perform three-step procedure consisting of amination, reaction with hetero-bifunctional cross-linkers and conjugation of an RGD-motif-containing peptide was used to modify polymer films and improve the attachment of endothelial cells. The biological activity of modified surfaces was assessed by estimating microvascular endothelial cell attachment. Covalent coating with RGD resulted in an approximately 11-fold increase of endothelial cell attachment on modified PCL surfaces compared with untreated polymer. The specificity of the attachment enhancement was confirmed by using a control peptide. It is concluded that chemical surface modification is an appropriate method of rendering degradable polymers, such as PCL, cell-adhesive.     

PLGA doping of PCL affects the plastic potential of human mesenchymal stem cells, both in the presence and absence of biological stimuli

A range of poly epsilon-caprolactone (PCL) films mixed/doped with poly(lactide-co-glycolide) (PLGA) (65:35) in 0, 10, 20, and 30 wt % were produced, sterilized using ethylene oxide, and analyzed using FTIR. Characterized human mesenchymal stem cells (hMSCs) were cultured in contact with the materials in basal, chondrogenic, and osteogenic medium for time periods up to 28 days, to determine if the materials could induce differentiation of MSC both in the presence and absence of biological stimuli. Viable cell adhesion was analyzed under all conditions. Collagen I, collagen II, sox-9, osteocalcin, osteopontin, osteonectin, and CBFA1 were evaluated at both the mRNA (real-time PCR) and protein production levels (fluorescent immunohistochemistry) and used to identify cell differentiation. Pure PCL and PCL mixed with PLGA demonstrated a chondrogenic potential. Only PCL 8 (80 wt % PCL, 20 wt % PLGA) facilitated osteogenic differentiation of MSCs under osteogenic conditions. This was attributed to the increased hydrophilic nature of the surface allowing sufficient homogeneous cell attachment and the formation of filamentous F-actin in the cells, allowing osteogenic differentiation. Of all materials tested, PCL 7 (70 wt % PCL, 30 wt % PLGA) demonstrated the greatest chondrogenic differentiation potential under basal and stimulated conditions at both the mRNA and protein production level.     

Tailoring surface properties of biomedical polymers by implantation of Ar and He ions

Ion implantation at 25 and 100 keV has been used as a tool for the modification of the surface properties of two biomedical polymers. The modulation induced by the different energy dispersion mechanisms of Ar and He have allowed satisfactory modifications for both the activation of the surfaces of chemically functional polycaprolactone (PCL) and the stabilization of anti-fouling poly(ethylene glycol) (PEG). In both cases the implantations have been performed at doses of 10(14) cm(-2) by taking into account the effect of different current densities, which are shown to distinctly influence the fragmentation-crosslinking of the target polymers. The resultant films were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, time of flight secondary ion mass spectroscopy and atomic force microscopy. Both shifts in zeta potential versus pH curves and the alteration of the polar components of the surface free energy (contact angle measurements) were correlated with the composition analysis. The response of the modified surfaces towards biomolecular interaction is demonstrated by the induction of preferential adsorption on irradiated PCL and the inhibited adsorption onto implanted PEG regions for selected oligopeptides and proteins.     

A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix

In this study, nanofibrous matrices of polycaprolactone (PCL) and PCL/collagen with immobilized epidermal growth factor (EGF) were successfully fabricated by electrospinning for the purpose of damaged skin regeneration. Nanofiber diameters were found to be 284 ± 48 nm for PCL and 330 ± 104 nm for PCL/collagen matrices. The porosities were calculated as 85% for PCL and 90% for PCL/collagen matrices. The covalent immobilization of EGF onto the nanofibrous matrices was verified by the increase of surface atomic nitrogen ratio from 1.0 to 2.4% for PCL and from 3.7 to 4.7% for PCL/collagen. Moreover, EGF immobilization efficiencies of PCL and PCL/collagen matrices were determined as 98.5 and 99.2%, respectively. Human dermal keratinocytes (HS2) were cultivated on both neat and EGF immobilized PCL and PCL/collagen matrices to investigate the effects of matrix chemical composition and presence of EGF on cell proliferation and differentiation. EGF immobilized PCL/collagen matrices exerted early cell spreading and rapid proliferation. Statistically high expression levels of loricrin in HS2 cells cultivated on EGF immobilized PCL/collagen matrices were (p < 0.001) regarding superior differentiation ability of these cells compared to HS2 cells cultured on neat PCL and PCL/collagen matrices. In conclusion, this novel EGF immobilized PCL/collagen nanofibrous matrix could potentially be considered as an alternative dermal substitutes and wound healing material for skin tissue engineering applications.     

Surface properties and biocompatibility of solvent-cast poly[-caprolactone] films

Poly(epsilon-caprolactone) (PCL) was dissolved in four solvent systems, chloroform, tetrahydrofuran, acetone and ethyl acetate, and cast onto glass Petri dishes. The surface properties of the resulting films were investigated. The extent to which their properties were determined by the solvent used in each case was quantified in terms of contact angle, surface morphology, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and the adhesion and proliferation of fibroblasts by direct contact. The surface of the PCL film in contact with glass was denoted the SG surface, and the other, which was exposed to the gas phase, a mixture of air and residual solvent vapour, was denoted the SA surface. In the case of hydrophobic solvent systems, the advancing contact angle of the SG surface was always lower than that of the SA surface. With hydrophilic solvent systems, on the other hand, the advancing contact angle of the SG film surface was higher when the contact angle of the Petri dish was higher than that of the gaseous mixture of the air and solvent vapour, otherwise it was lower or equal to that of the surface on which it was cast. The surface morphology was dictated by the solubility of PCL in the respective solvent systems: high dissolution solvents such as chloroform and tetrahydrofuran produced films that comprised PCL aggregates, the particles being larger in the case of chloroform, whereas the less efficient solvents (acetone and ethyl acetate) resulted in a filamentous structure. The ATR-FTIR results confirmed that the chemistry of the SA surfaces differed according to the solvent system used. Preliminary cell culture experiments carried out with the PCL films established that murine (L929) fibroblasts grew well on all surfaces regardless of the solvent used, although the rates of adhesion and proliferation were not as great as on tissue culture plastic controls. Of all the surfaces examined in this study, the cells favoured the SG aspect of ethyl acetate cast PCL films, the surface of which had the finest pore size and relatively low contact angle.     

Solution-mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSCs) on PLGA by promoting the formation of a calcium-phosphate-rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells was tested. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSCs, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell-derived factors that promoted distant MSC differentiation. Thus PLGA-BG composites demonstrate significant potential as a bone-replacement material.     

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.     

Effects of calcium ion incorporation on osteoblast gene expression in MC3T3-E1 cells cultured on microstructured titanium surfaces

The surface characteristics of a calcium ion (Ca)-incorporated titanium (Ti) surface, produced by hydrothermal treatment using an alkaline Ca-containing solution, and its effects on osteoblastic differentiation were investigated. MC3T3-E1 pre-osteoblastic cells were cultured on machined or grit-blasted Ti surfaces with and without Ca incorporation. The MTT assay was used to determine cell proliferation, and real-time PCR was used for quantitative analysis of osteoblastic gene expression. Hydrothermal treatment with a Ca-containing solution produced a crystalline CaTiO(3) nanostructure of approximately 100 nm in dimension, preserving original micron-scaled surface topographies and microroughness caused by machining, blasting, or blasting and etching treatments. After immersion in Hank's balanced salt solution, considerable apatite formation was observed on all surfaces of the Ca-incorporated samples. Significantly more cell proliferation was found on Ca-incorporated Ti surfaces than on untreated Ti surfaces (p < 0.001). Quantitative real-time PCR analysis showed notably higher alkaline phosphatase, osteopontin, and osteocalcin mRNA levels in cells grown on Ca-incorporated blasted surfaces than on other surfaces at an early time point. Thus, Ca incorporation may have a beneficial effect on osseointegration of microstructured Ti implants by accelerating osteoblast proliferation and differentiation during the early healing phase following implantation.     

Physico-chemical and biological evaluation of excimer laser irradiated polyethylene terephthalate (pet) surfaces

The aim of this work was to investigate the consequences of excimer laser irradiation on the physico-chemical and biological properties of polyethylene terephthalate (PET) films, currently used for medical devices. Three PET films from different origins were studied in the present work, chosen with respect to their chemical and physical properties, which are of high importance for ulterior medical application as vascular prostheses. Multiple assays were carried out to characterize the physical and chemical effects of the laser irradiation: surface morphology tests (light microscopy, Dektak profilometer and confocal laser scanning microscopy) showed the strong transformation of the surface with the laser treatment. Contact angle measurements revealed a significant increase of the surface energy for each PET depending on the applied fluency. Finally XPS characterization of the surface demonstrated the appearance of new chemical species favorable for cell attachment. This aspect had to be strongly considered regarding to the multiple biological effects of laser irradiated surfaces on living cells. Different cell culture experiments were carried out with L132 human epithelial cells after 6-days culture: proliferation and vitality rate, cell adhesion and cell morphology. Results clearly revealed that laser treatment improved cell proliferation (up to 140% with respect to controls), vitality (10% higher than controls), morphology and adhesion kinetics (more than 16% of control). A significant correlation (R2=0.906) was also established on one PET between the fluencies of laser treatment and the cellular response. These results emphasized high importance of the choice of the PET material for a medical application: only one of the three considered PET films showed really improved cellular response.     

Fabrication of Zn containing apatite cement and its initial evaluation using human osteoblastic cells.

Recently, the effects of Zn2+ on osteogenesis stimulation have become major topics in the research fields of bone formation and organism essential elements. Based on the fundamental finding of Zn2+ with respect to osteogenesis stimulation, Ito et al. have prepared Zn doped beta-tricalcium phosphate (ZnTCP) and have reported that ZnTCP enhances the proliferation of MC3T3-E1 cells. In this investigation, we studied the effects of ZnTCP added to apatite cement (AC) with respect to its setting reaction and proliferation of human osteoblastic cells as an initial evaluation for the feasibility of AC containing ZnTCP. Compositional analysis using powder X-ray diffractometer revealed that ZnTCP shows no reactivity with the setting reaction of AC. As a result, the mechanical strength of set AC decreased increasing amounts of added ZnTCP as if ZnTCP acts as a pore in AC. The setting time of AC was not affected by addition of ZnTCP up to 10%. When AC containing ZnTCP was immersed in alpha-MEM containing 10% bovine serum, Zn2+ was released from AC. Larger amounts of Zn2+ were released from AC containing larger amounts of ZnTCP. When human osteoblastic cells were incubated on the surface of AC discs, proliferation of human osteoblastic cells was significantly increased on the surface of AC that contained 5% ZnTCP when compared with that containing no ZnTCP. In contrast, proliferation of human osteoblastic cells decreased on the surface of AC that contained 10% ZnTCP when compared with that free from ZnTCP; indicating cytotoxicity. We concluded therefore, that addition of ZnTCP to AC is useful to enhance the osteoconductivity of AC when release of Zn2+ can be carefully regulated.