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Exploiting hydrogels for the cultivation of stem cells, aiming to provide them with physico-chemical cues suitable for osteogenesis, is a critical demand for bone engineering. Here, we developed hybrid compositions of collagen and silica into hydrogels via a simple sol-gel process. The physico-chemical and mechanical properties, degradation behavior, and bone-bioactivity were characterized in-depth; furthermore, the in vitro mesenchymal stem cell growth and osteogenic differentiation behaviors within the 3D hybrid gel matrices were communicated for the first time. The hydrolyzed and condensed silica phase enabled chemical links with the collagen fibrils to form networked hybrid gels. The hybrid gels showed improved chemical stability and greater resistance to enzymatic degradation. The in vitro apatite-forming ability was enhanced by the hybrid composition. The viscoelastic mechanical properties of the hybrid gels were significantly improved in terms of the deformation resistance to an applied load and the modulus values under a dynamic oscillation. Mesenchymal stem cells adhered well to the hybrid networks and proliferated actively with substantial cytoskeletal extensions within the gel matrices. Of note, the hybrid gels substantially reduced the cell-mediated gel contraction behaviors, possibly due to the stiffer networks and higher resistance to cell-mediated degradation. Furthermore, the osteogenic differentiation of cells, including the expression of bone-associated genes and protein, was significantly upregulated within the hybrid gel matrices. Together with the physico-chemical and mechanical properties, the cellular behaviors observed within 3D gel matrices, being different from the previous approaches reported on 2D substrates, provide new information on the feasibility and usefulness of the silica-collagen system for stem cell culture and tissue engineering of hard tissues.

Strontium- and calcium-releasing, titanium-stabilised phosphate-based glasses with a controlled degradation rate are currently under development for orthopaedic tissue engineering applications. Ca and/or Sr were incorporated at varying concentrations in quaternary phosphate-based glasses, in order to promote osteoinduction. Ti was incorporated at a fixed concentration in order to prolong degradation. Glasses of the general formula (P2O5)–(Na2O)–(TiO2)–(CaO)–(SrO) were prepared via the melt-quench technique. The materials were characterised by energy-dispersive X-ray spectroscopy, X-ray diffraction, 31P magic angle spinning nuclear magnetic resonance, Fourier transform infrared spectroscopy, differential thermal analysis and density determination. The dissolution rate in distilled water was determined by measuring mass loss, ion release and pH change over a two-week period. In addition, the cytocompatibility and alkaline phosphatase activity of an osteoblast-like cell line cultured on the surface of glass discs was assessed. The glasses were shown to be amorphous and contained Q1, Q2 and Q3 species. Fourier transform infrared spectroscopy revealed small changes in the glass structure as Ca was substituted with Sr and differential thermal analysis confirmed a decrease in crystallisation temperature with increasing Sr content. Degradation and ion release studies also showed that mass loss was positively correlated with Sr content. These results were attributed to the lower electronegativity of Sr in comparison to Ca favouring the formation of phosphate-based mineral phases. All compositions supported cell proliferation and survival and induced at least 2.3-fold alkaline phosphatase activity relative to the control. Glass containing 17.5 mol% Sr had 3.6-fold greater alkaline phosphatase activity than the control. The gradual release of Ca and Sr supported osteoinduction, indicating their potential suitability in orthopaedic tissue engineering applications.

The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti–Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue formation and the upregaulation of VEGF production can potentially support vascularization.

In order to develop novel, effective therapies for central nervous system regeneration, it is essential to better understand the role of neurotrophic factors and to design, accordingly, better artificial scaffolds to support both neurite outgrowth and synapse formation. Both nerve growth factor and brain-derived neurotrophic factor are major factors in neural survival, development, synaptogenesis, and synaptic connectivity of primary cultured neurons. As a prime candidate coating material for such neural cultures, carbon nanotubes offer unique structural, mechanical, and electrical properties. In this study, carbon nanotubes coated glass-coverslips were used as the matrix of a primary neural culture system used to investigate the effects of carbon nanotubes on neurite outgrowth and nerve growth factor/brain-derived neurotrophic factor release and expression. For these purposes, we performed comparative analyses of primary cultured neurons on carbon nanotubes coated, non-coated, and Matrigel-coated coverslips. The morphological findings showed definite carbon nanotubes effects on the neurite outgrowths and synaptogenic figures in both cortical and hippocampal neurons when compared with the non-coated negative control. Although the carbon nanotubes did not change neurotrophin expression levels, it stimulated brain-derived neurotrophic factor release into the media from both types of neurons. Accordingly, we suggest a different mechanism of action between carbon nanotubes and Matrigel in relation to the specific neurotrophic factors. Since carbon nanotubes supply long-term extracellular molecular cues for the survival and neurite outgrowths of cultured neurons, the results from this study will contribute to an understanding of carbon nanotubes biological effects and provide new insight into their role in the secretion of neurotrophic factors.

An effective tumor-targeted drug delivery system for photodynamic therapy was developed by designing ligand-mediated nanoparticles with stable formulations of a hydrophobic photosensitizer. Novel folic acid (FA)-conjugated amphiphilic block copolymers of polyethylene glycol (PEG) and poly-β-benzyl-L-aspartate (PBLA) with the potential to act as pH-responsive drug release reservoirs were synthesized. The photosensitizer, 2,4-diacetyl deuteroporphyrin IX dimethyl ether (DD-PpIX), was conjugated to the copolymers through pH-sensitive hydrazone linkage. The syntheses and compositions of all copolymers were confirmed by 1H NMR measurement. Photosensitizer-conjugated amphiphilic copolymeric nanoparticles (FA-PEG-P(Asp-Hyd)-DD-PpIX) were prepared by micelle formation in aqueous solution. The particle sizes of the FA-PEG-PBLA and FA-PEG-P(Asp-Hyd)-DD-PpIX nanoparticles were determined by light-scattering measurements. The range was 105–298 nm, depending on copolymer molecular weight and composition. Field emission scanning electron microscopy showed that the FA-PEG-P(Asp-Hyd)-DD-PpIX copolymeric nanoparticles were submicron in size and spherical in shape. The results of in vitro release tests showed that the release profiles of DD-PpIX from the nanoparticles were strongly pH-dependent and influenced by the amount of photosensitizer that was conjugated. In vitro tests using HeLa cells indicated that the FA-PEG-P(Asp-Hyd)-DD-PpIX nanoparticles had low dark-toxicity and showed more than 97% of cellular uptake. Based on our results, the FA-PEG-P(Asp-Hyd)-DD-PpIX nanoparticle system could be a promising approach for developing novel photosensitizer delivery carriers for photodynamic therapy.

This study aims to produce a degradable and bone-bioactive membrane for guiding bone regeneration by combining a degradable polymer, poly(lactic acid) (PLA), with a bioactive inorganic zinc-containing bioactive glass (ZnBG). The in vitro osteogenic development of rat bone marrow mesenchymal stem cells (rBMSCs) upon different membrane substrates (pure PLA control, PLA–BG, and PLA–ZnBG) was investigated in terms of bone cell phenotype syntheses and mineralization. Results showed significantly stimulated production of alkaline phosphatase and osteocalcin at days 14 and 21 in the membranes containing BG and ZnBG, with more in the samples containing ZnBG. The addition of ZnBG in PLA allowed the rBMSCs to express a high level of bone sialoprotein as confirmed by immunostaining. Cellular mineralization of the secreted extracellular matrix showed a significantly higher Ca level on the BG- and ZnBG-added membrane than on the PLA, and the more so in the ZnBG-added one. Based on the in vitro assessments using rBMSCs, the ZnBG-added PLA is considered to be of potential use in guiding active bone regeneration within the periodontal pocket.

Previously, a strong and bioactive ceramic scaffold consisting of a porous zirconia body coated with apatite double layers (fluorapatite (FA) as an inner layer and hydroxyapatite (HA) as an outer layer) was successfully fabricated. In this contribution, the authors investigate the in vivo performance of the engineered bioceramic scaffolds using a rabbit calvarial defect model. In particular, the porosity and pore size of the scaffolds are varied in order to observe the geometrical effects of the scaffolds on their bone formation behaviors. The scaffolds supported on a zirconia framework can be produced with an extremely high porosity (approximately 84-87%), while retaining excellent compressive strength (approximately 7-8 MPa), which has been unachievable in the case of pure apatite scaffolds (approximately 74% porosity with approximately 2 MPa strength). The experimental groups used in this study include three types of zirconia scaffolds coated with apatite; high porosity (approximately 87%) with large pore size (approximately 500- 700 microm): AZ-HL, high porosity (approximately 84%) with small pore size (approximately 150-200 microm): AZ-HS, and low porosity (approximately 75%) with large pore size (approximately 500-700 microm): AZ-LL, as well as one type of HA porous scaffold: low porosity (approximately 74%) with a large pore size (approximately 500-700 microm) for the purpose of comparison. The scaffolds prepared with dimensions of approximately 10 mm (diameter) x 1.2 mm (thickness) are grafted in rabbit calvaria defects. The histological sections are made at 4 and 12 weeks after surgery and immunohistochemical analyses are performed on the samples. All of the specimens show a good healing response without adverse tissue reactions. Good healing is shown at 4 weeks post-surgery with the ingrowth of new bone into the macropore-channels of the scaffolds. The newly formed bone amounts to approximately 19.9-24.2% of the initial defect area, depending on the scaffold type, but there is no statistical significance between the scaffold groups. However, the defects without the scaffolds (control group) show a significantly lower bone formation ratio (approximately 4.3%). At twelve weeks after surgery, the extent of new bone formation is more pronounced in all of the scaffold groups. All of the scaffold groups show significantly higher bone formation ratios (26.7-46.9%) with respect to the control without the graft. In the comparison between the scaffold groups, those with high porosities (AZ-HL and AZ-HS) exhibit significantly higher bone formation as compared to the scaffold with low porosity (AZ-LL). Based on the present in vivo test performed within a rabbit calvaria defect model, it is concluded that the apatite-coated zirconia scaffolds show good bone forming ability and are considered to be a promising scaffolding material for bone regeneration since they possess a high level of both mechanical and biological properties.

Background The objective of this study was to investigate the effects of bioactive calcium phosphate cements (CPC, α-tricalcium phosphate-based) incorporating zinc-bioglass (ZnBG) on the odontogenic differentiation and angiogenesis of human dental pulp cells (HDPCs). Methods BGs with varying concentrations of Zn (0, 2.5 and 5%) were produced via a sol-gel process. The proliferation of HDPCs on CPC/BGs was determined by MTS assay. Alizarin red staining, RT-PCR, and ALP activity were used to assess odontogenic differentiation, and western blot analysis was used to asses signaling pathways. In vitro angiogenesis was examined via mRNA expression of angiogenic genes and tubule formation. Results All cement formulations showed no cytotoxicity. The CPCs with ZnBG showed increased ALP activity, enhanced formation of mineralized nodules, and upregulated mRNA expression of DMP-1, DSPP, Runx2, and osterix in a time- and dose-dependent manner, relative to CPCs without Zn. ZnBG upregulated integrins α1, α2, β1, and β3 and activated integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, RhoA, MAPK, and NF-κB, as well as canonical and non-canonical Wnt signaling. In addition, ZnBG upregulated VEGF mRNA in HDPCs and increased the tubular structure in endothelial cells. Conclusions Our results demonstrate that ZnBG incorporated within CPCs activates odontogenic differentiation and promotes angiogenesis in vitro through integrin, Wnt, MAPK, and NF-κB pathways. Thus, CPCs incorporating ZnBG are promising matrices in tissue engineering to stimulate endodontic regeneration.

The aim of the present study was to fabricate mineralized polycaprolactone nanofibrous scaffold and investigate its ability to elicit odontogenic differentiation of human dental pulp cells, compared to the pure polycaprolactone scaffold. Polycaprolactone nanofibrous scaffold was produced by electrospinning, and the surface was mineralized with apatite. Cellular behaviors on the mineralized polycaprolactone scaffold were assessed in terms of cell adhesion, growth, and odontoblastic differentiation. To evaluate the signal transduction of human dental pulp cells, mRNA expression was analyzed and Western blotting was performed. Mineralized polycaprolactone showed improved cell proliferation, mineralized nodule formation, and expression of odontoblastic marker genes including alkaline phosphatase, osteopontin, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1, as compared with pure polycaprolactone. Although the cell adhesion on the mineralized polycaprolactone was similar to that of the polycaprolactone, the expression level of proteins including collagen type I and the key adhesion receptor (integrin components α1, α2, and β1) was upregulated in mineralized polycaprolactone compared to polycaprolactone. Especially, cells seeded onto mineralized polycaprolactone scaffolds showed significantly increased levels of phosphorylated focal adhesion kinase, a marker of integrin activation, and downstream pathways, such as phosphor (p)-Akt, p-extracellular signal regulated kinase, p-c Jun N-terminal kinase, nuclear factor-kappa B, c-fos, and c-jun, compared with pure polycaprolactone. The mineralized polycaprolactone scaffold is attractive for dentin tissue engineering by promoting growth and odontogenic differentiation of human dental pulp cells through the integrin-mediated signaling pathway.

Novel microcarriers consisting of calcium phosphate cement and alginate were prepared for use as three-dimensional scaffolds for the culture and expansion of cells that are effective for bone tissue engineering. The calcium phosphate cement-alginate composite microcarriers were produced by an emulsification of the composite aqueous solutions mixed at varying ratios (calcium phosphate cement powder/alginate solution = 0.8–1.2) in an oil bath and the subsequent in situ hardening of the compositions during spherodization. Moreover, a porous structure could be easily created in the solid microcarriers by soaking the produced microcarriers in water and a subsequent freeze-drying process. Bone mineral-like apatite nanocrystallites were shown to rapidly develop on the calcium phosphate cement–alginate microcarriers under moist conditions due to the conversion of the α-tricalcium phosphate phase in the calcium phosphate cement into a carbonate–hydroxyapatite. Osteoblastic cells cultured on the microspherical scaffolds were proven to be viable, with an active proliferative potential during 14 days of culture, and their osteogenic differentiation was confirmed by the determination of alkaline phosphatase activity. The in situ hardening calcium phosphate cement–alginate microcarriers developed herein may be used as potential three-dimensional scaffolds for cell delivery and tissue engineering of bone.

Surface modification of Ti-based metals is an important issue in improving the bone cell responses and bone-implant integration. Blasting Ti with granules (mostly alumina) is commonly used to prepare a clean surface and provide a level of roughness. In this study, glass granules with a bioactive composition were used as the blasting source to improve the surface bioactivity and biocompatibility of a Ti substrate. Bioactive glass particles with a composition of 70SiO 2 · 25CaO · 5P2O5 were prepared using a sol—gel method. A Ti disc was blasted with glass particles using a dental blasting unit (BG-Ti). A Ti disc blasted with commercial spherical-shaped glass (G-Ti) and a disc without blasting (Ti) were also prepared for comparison. The blasted Ti contained a large number of glass particles after the blasting process. The surface roughness of the samples in ascending order was G-Ti>BG-Ti>Ti. Murine-derived preosteoblasts (MC3T3-E1) were seeded on the samples, and the cell growth, differentiation, and mineralization behaviors were observed. The osteoblastic cells attached well and spread actively over all the sample groups with extensive cytoskeletal processes. The level of cell growth on the BG-Ti showed a continual increase with culturing up to 7 days, showing good cell viability. However, there was no significant difference (ANOVA, p

The properties of scaffolds and their roles in regulating functions of tissue cells are considered to be of utmost importance in the successful recovery of damaged tissues. Herein, novel scaffolds of collagen and bioactive inorganic nanofiller were produced for bone tissue engineering. In addition, the in vitro responses of bone marrow-derived stromal cells (BMSCs) on these scaffolds were investigated. Glasses with bioactive compositions were prepared in nanofibrous form and homogenized with a collagen to produce hybridized porous scaffolds. The glass fibrous filaments with diameters of a few hundred nanometers were embedded well within the collagen network, characterizing a typical nanocomposite. The scaffolds showed the characteristics of a hydrogel with remarkable water uptake and swelling degree, which were similar to those of the pure collagen. In addition, the scaffolds induced the precipitation of bone-like minerals on the surface under a body-simulating medium, showing the sign of in vitro bone bioactivity. BMSCs adhered and spread well over the scaffold surface and migrated deep into the scaffold network. The osteogenic marker, alkaline phosphatase, was strongly expressed on the hybrid scaffolds, with the level higher than that on pure collagen. Overall, the collagen—inorganic nanofiller scaffolds are considered to find potential utility in bone tissue engineering.

A hydroxyapatite (HA)-titania (TiO2) hybrid coating is developed to improve the biocompatibility of titanium (Ti) implants. The HA predeposited layer on Ti via electron beam (e-beam) evaporation is subsequently treated by micro-arc oxidation (MAO) to produce an HA-TiO2 hybrid layer on Ti. The e-beam-deposited HA layer has a thickness of ≈1 μm and was highly dense prior to MAO. By means of MAO treatment, a rough and porous TiO2 layer is formed beneath the HA layer with a simultaneous local dissolution of the HA layer. Due to the HA precoating, high concentrations of Ca and P are preserved on the coating surface. The osteoblast-like cells on the hybrid coating layer grow and spread favorably. The cell proliferation rate on the hybrid coatings is not much different from that on pure Ti or simple MAO-treated Ti. However, the alkaline phosphatase (ALP) activity of the cells is significantly higher ( p < 0.05) on the HA-TiO2 hybrid coatings than on either the pure Ti or the simple MAO-treated specimen, suggesting that the cellular activity on the hybrid coatings is improved.