Nanofiber orientation and surface functionalization modulate human mesenchymal stem cell behavior in vitro

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation

Topographically and chemically modified titanium implants are recognized to have improved osteogenic properties; however, the molecular regulation of this process remains unknown. This study aimed to determine the microRNA profile and the potential regulation of osteogenic differentiation following early exposure of osteoprogenitor cells to sand-blasted, large-grit acid-etched (SLA) and hydrophilic SLA (modSLA) surfaces. Firstly, the osteogenic characteristics of the primary osteoprogenitor cells were confirmed using ALP activity and Alizarin Red S staining. The effect of smooth (SMO), SLA and modSLA surfaces on the TGF-β/BMP (BMP2, BMP6, ACVR1) and non-canonical WNT/Ca2+ (WNT5A, FZD6) pathways, as well as the integrins ITGB1 and ITGA2, was determined. It was revealed that the modified titanium surfaces could induce the activation of TGF-β/BMP and non-canonical WNT/Ca2+ signaling genes. The expression pattern of microRNAs (miRNAs) related to cell differentiation was evaluated. Statistical analysis of the differentially regulated miRNAs indicated that 35 and 32 miRNAs were down-regulated on the modSLA and SLA surfaces respectively, when compared with the smooth surface (SMO). Thirty-one miRNAs that were down-regulated were common to both modSLA and SLA. There were 10 miRNAs up-regulated on modSLA and nine on SLA surfaces, amongst which eight were the same as observed on modSLA. TargetScan predictions for the down-regulated miRNAs revealed genes of the TGF-β/BMP and non-canonical Ca2+ pathways as targets. This study demonstrated that modified titanium implant surfaces induce differential regulation of miRNAs, which potentially regulate the TGF-β/BMP and WNT/Ca2+ pathways during osteogenic differentiation on modified titanium implant surfaces.

The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Synthesis and modification of Alumina nanofibres and its applications

This thesis is a forward study of alumina nanofiber material in developing its applications biology field. It demonstrates that by applying proper modification strategy, alumina nanofiber is a promising material in protein purification and enzyme immobilization. The hydrophobic modification has dramatically improved the rejecting of protein molecular in purification system. On the other hand, utilisation of cross-linking agent firmly combined alumina nanofiber and target enzyme for immobilisation purpose. This step of progress could lead to inspiration of alumina nanofiber’s application in various area.

Stimulation of osteogenic and angiogenic ability of cells on polymers by pulsed laser deposition of uniform akermanite-glass nanolayer

Polymer biomaterials have been widely used for bone replacement/regeneration because of their unique mechanical properties and workability. Their inherent low bioactivity makes them lack osseointegration with host bone tissue. For this reason, bioactive inorganic particles have been always incorporated into the matrix of polymers to improve their bioactivity. However, mixing inorganic particles with polymers always results in inhomogeneity of particle distribution in polymer matrix with limited bioactivity. This study sets out to apply the pulsed laser deposition (PLD) technique to prepare uniform akermanite (Ca2MgSi2O7, AKT) glass nanocoatings on the surface of two polymers (non-degradable polysulfone (PSU) and degradable polylactic acid (PDLLA)) in order to improve their surface osteogenic and angiogenic activity. The results show that a uniform nanolayer composed of amorphous AKT particles (∼30nm) of thickness 130nm forms on the surface of both PSU and PDLLA films with the PLD technique. The prepared AKT-PSU and AKT-PDLLA films significantly improved the surface roughness, hydrophilicity, hardness and apatite mineralization, compared with pure PSU and PDLLA, respectively. The prepared AKT nanocoatings distinctively enhance the alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, OPN and Col I) of bone-forming cells on both PSU and PDLLA films. Furthermore, AKT nanocoatings on two polymers improve the attachment, proliferation, VEGF secretion and expression of proangiogenic factors and their receptors of human umbilical vein endothelial cells (HUVEC). The results suggest that PLD-prepared bioceramic nanocoatings are very useful for enhancing the physicochemical, osteogenic and angiogenic properties of both degradable and non-degradable polymers for application in bone replacement/regeneration.

Effect of culture conditions and calcium phosphate coating on ectopic bone formation

This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaPcoated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients.We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation.

Designing atmospheric-pressure plasma sources for surface engineering of nanomaterials

Atmospheric-pressure plasma processing techniques emerge as efficient and convenient tools to engineer a variety of nanomaterials for advanced applications in nanoscience and nanotechnology. This work presents different methods, including using a quasi-sinusoidal high-voltage generator, a radio-frequency power supply, and a uni-polar pulse generator, to generate atmospheric-pressure plasmas in the jet or dielectric barrier discharge configurations. The applicability of the atmospheric-pressure plasma is exemplified by the surface modification of nanoparticles for polymeric nanocomposites. Dielectric measurements reveal that representative nanocomposites with plasma modified nanoparticles exhibit notably higher dielectric breakdown strength and a significantly extended lifetime.

Study of surface evolution in the wheel-rail interface

This paper aims to trace surface evolution in the wheel-rail interface using data obtained from a twin-disc testing machine and the surface replication technique. Changes in the surface profile of the rail testing disc are explicitly analysed according to the wear mechanism, which helps elaborate a better understanding of the attrition of asperities during the wearing-in process of surface modification. The surface profile amplitude was seen to decrease during the initial running-in phase of the experiment cycle, and after reaching a saturation value, the profile amplitude then increased. Ultimately the results show that grinding will roughen the rail surface and the wheel-rail contact conditions will then remove this surface damage to some saturation value of the profile height. The variation in the rail surface profile beyond this point is then only dependant on the contact conditions which exist between the wheel and rail during normal operation.

Infrared spectroscopic investigation of the effects of titania photocatalyst on the degradation of linear low density polyethylene film for commercial applications

There is a need in industry for a commodity polyethylene film with controllable degradation properties that will degrade in an environmentally neutral way, for applications such as shopping bags and packaging film. Additives such as starch have been shown to accelerate the degradation of plastic films, however control of degradation is required so that the film will retain its mechanical properties during storage and use, and then degrade when no longer required. By the addition of a photocatalyst it is hoped that polymer film will breakdown with exposure to sunlight. Furthermore, it is desired that the polymer film will degrade in the dark, after a short initial exposure to sunlight. Research has been undertaken into the photo- and thermo-oxidative degradation processes of 25 ìm thick LLDPE (linear low density polyethylene) film containing titania from different manufacturers. Films were aged in a suntest or in an oven at 50 °C, and the oxidation product formation was followed using IR spectroscopy. Degussa P25, Kronos 1002, and various organic-modified and doped titanias of the types Satchleben Hombitan and Hunstsman Tioxide incorporated into LLDPE films were assessed for photoactivity. Degussa P25 was found to be the most photoactive with UVA and UVC exposure. Surface modification of titania was found to reduce photoactivity. Crystal phase is thought to be among the most important factors when assessing the photoactivity of titania as a photocatalyst for degradation. Pre-irradiation with UVA or UVC for 24 hours of the film containing 3% Degussa P25 titania prior to aging in an oven resulted in embrittlement in ca. 200 days. The multivariate data analysis technique PCA (principal component analysis) was used as an exploratory tool to investigate the IR spectral data. Oxidation products formed in similar relative concentrations across all samples, confirming that titania was catalysing the oxidation of the LLDPE film without changing the oxidation pathway. PCA was also employed to compare rates of degradation in different films. PCA enabled the discovery of water vapour trapped inside cavities formed by oxidation by titania particles. Imaging ATR/FTIR spectroscopy with high lateral resolution was used in a novel experiment to examine the heterogeneous nature of oxidation of a model polymer compound caused by the presence of titania particles. A model polymer containing Degussa P25 titania was solvent cast onto the internal reflection element of the imaging ATR/FTIR and the oxidation under UVC was examined over time. Sensitisation of 5 ìm domains by titania resulted in areas of relatively high oxidation product concentration. The suitability of transmission IR with a synchrotron light source to the study of polymer film oxidation was assessed as the Australian Synchrotron in Melbourne, Australia. Challenges such as interference fringes and poor signal-to-noise ratio need to be addressed before this can become a routine technique.

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.