The coating of highly porous Bioglass® based 3D scaffolds with multi‐walled carbon nanotubes (CNT) was investigated. Foam like Bioglass® scaffolds were fabricated by the replica technique and electrophoretic deposition was used to deposit homogeneous layers of CNT throughout the scaffold pore structure. The optimal experimental conditions were determined to be: applied voltage 15 V and deposition time 20 minutes, utilizing a concentrated aqueous suspension of CNT with addition of a surfactant and iodine. The scaffold pore structure remained invariant after the CNT coating, as assessed by SEM. The incorporation of CNTs induced a nanostructured internal surface of the pores which is thought to be beneficial for osteoblast cell attachment and proliferation. Bioactivity of the scaffolds was assessed by immersion studies in simulated body fluid (SBF) for periods of up to 2 weeks and the subsequent determination of hydroxyapatite (HA) formation. The presence of CNTs can enhance the bioactive behaviour of the scaffolds since CNTs can serve as template for the ordered formation of a nanostructured HA layers, which does not occur on uncoated Bioglass® surfaces.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.

Extracellular matrix (ECM) consists of a complex mixture of macromolecules such as collagens, proteoglycans, glycoproteins, and elastic fibers. ECM is essential to preserving tissue architecture, signaling to cells, and regulating calcification in mineralized tissues. Osteoblasts in culture secrete and assemble an extensive ECM rich in type I collagen, and other noncollagenous proteins that can be mineralized. Three-dimensional matrix models can be used in vitro to most appropriately resemble the geometry and biochemistry of natural ECMs. In the present study, MC3T3-E1 mouse calvarial preosteoblasts were cultured within a dense three-dimensional collagenous ECM-like scaffold produced through the method of plastic compression. Plastic compression rapidly produces scaffolds of collagen density approaching native tissue levels with enhanced biomechanical properties while maintaining the viability of resident cells. The proliferation, morphology, and gene expression of seeded MC3T3s, as well as collagen production and matrix mineralization, were investigated for up to 7 weeks in culture. Soluble collagen secretion ranged in concentration from 5 to 30 microg/mL over a 24-h period, concomitant with a steady rate of collagen mRNA expression. Expression of osteogenic markers such as tissue-nonspecific alkaline phosphatase (Alpl), bone sialoprotein (Bsp), and osteopontin (Opn) examined by biochemical assay and reverse transcription-polymerase chain reaction demonstrated cell differentiation. Pericellular voids of ECM around cells, together with evidence of MMP13 mRNA expression, suggested matrix remodeling. Ultrastructural analyses, X-ray microanalysis, micro-computed tomography, as well as Fourier-transform infrared and imaging all confirmed the formation of a calcium-phosphate mineral phase within the fibrillar collagen matrix. In conclusion, preosteoblastic MC3T3 cells seeded within an ECM-like dense collagen scaffold secrete matrix proteins and induce scaffold mineralization in a manner potentially appropriate for bone tissue engineering uses.

Multiwalled carbon nanotube (CNT) films have been successfully fabricated by electrophoretic deposition (EPD) on stainless steel substrates. Electrophoretic deposition was performed using optimised aqueous suspensions under constant voltage conditions. Triton X-100 was used as a surfactant to disperse CNT bundles, and iodine was added as a particle charger. CNT/SiO 2 composite coatings were prepared by electrophoretic co-deposition. Experimental results show that the CNTs were efficiently mixed with SiO 2 nanoparticles to form a network structure. Layered CNT/SiO 2 porous composites were obtained by sequential EPD experiments alternating the deposition of CNT and SiO 2 nanoparticles. The structure of all films deposited was studied in detail by scanning electron microscopy. Possible applications of CNT and CNT/SiO 2 films are as porous coatings in the biomedical field, thermal management devices, biomedical sensors and other functional applications where the properties of CNTs are required.

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Osteoblast differentiation is regulated by the presence of collagen type I (COL I) extracellular matrix (ECM). We have recently demonstrated that Factor XIIIA (FXIIIA) transglutaminase (TG) is required by osteoblasts for COL I secretion and extracellular deposition, and thus also for osteoblast differentiation. In this study we have further investigated the link between COL I and FXIIIA, and demonstrate that COL I matrix increases FXIIIA levels in osteoblast cultures and that FXIIIA is found as cellular (cFXIIIA) and extacellular matrix (ecmFXIIIA) forms. FXIIIA mRNA, protein expression, cellular localization and secretion were enhanced by ascorbic acid (AA) treatment and blocked by dihydroxyproline (DHP) which inhibits COL I externalization. FXIIIA mRNA was regulated by the MAP kinase pathway. Secretion of ecmFXIIIA, and its enzymatic activity in conditioned medium, were also decreased in osteoblasts treated with the lysyl oxidase inhibitor β-aminopropionitrile, which resulted in a loosely packed COL I matrix. Osteoblasts secrete a latent, inactive dimeric ecmFXIIIA form which is activated upon binding to the matrix. Monodansyl cadaverine labeling of TG substrates in the cultures revealed that incorporation of the label occurred at sites where fibronectin co-localized with COL I, indicating that ecmFXIIIA secretion could function to stabilize newly deposited matrix. Our results suggest that FXIIIA is an integral part of the COL I deposition machinery, and also that it is part of the ECM-feedback loop, both of which regulate matrix deposition and osteoblast differentiation.

Tissue engineering approaches for articular cartilage (AC) repair using collagen type I (Coll)-based hydrogels are limited by their low collagen fibril density (CFD; <0.5 wt%) and their poor capacity to support chondrocyte differentiation. Chitosan (CTS) is a well-characterized polysaccharide that mimics the glycosaminoglycans (GAGs) present in native AC extracellular matrix and exhibits chondroprotective properties. Here dense Coll/CTS hydrogel discs (16 mm diameter, 140-250 μm thickness) with CFD (∼6 wt%) approaching that of AC were developed to investigate the effect of CTS content on the growth and differentiation of three-dimensionally seeded RCJ3.1C5.18 chondroprogenitor cells. Compared to dense Coll alone, cells seeded within Coll/CTS showed increased viability and metabolic activity, as well as a decrease in cell-mediated gel contraction. Immunohistochemistry for collagen type II, in combination with Safranin O staining and GAG quantification, indicated greater chondroprogenitor differentiation within Coll/CTS, compared to cells seeded within Coll alone. The complex interplay between scaffold geometry, microstructure, composition, mechanical properties and cell function was further evaluated by rolling dense planar sheets to prepare cylindrically shaped constructs having clinically relevant diameters (3-5 mm diameter, 9 mm height). The compressive modulus of the cylindrically shaped constructs decreased significantly after 7 days in culture, and remained unchanged up to 21 days for each scaffold composition. Unlike Coll, cells seeded within Coll/CTS showed greater viability along the entire radial extent of the cylindrical rolls and increased GAG production at each time point. While GAG content decreased over time and reduced cell viability was observed within the core region of all cylindrical rolls, the incorporation of CTS diminished both these effects. In summary, these findings provide insight into the challenges involved when scaling up scaffolds designed and optimised in vitro for tissue repair.