Hye-Sun Yu

Apatite-mineralized polycaprolactone nanofibrous web as a bone tissue regeneration substrate

Degradable synthetic polymers with a nanofibrous structure have shown great promise in populating and recruiting cells for the reconstruction of damaged tissues. However, poor cell affinity and lack of bioactivity have limited their potential usefulness in bone regeneration. We produced polymeric nanofiber poly(epsilon-caprolactone) (PCL) with its surface mineralized with bone-like apatite for use as bone regenerative and tissue engineering matrices. PCL was first electrospun into a nanofibrous web, and the surface was further mineralized with apatite following a series of solution treatments. The surface of the mineralized PCL nanofiber was observed to be almost fully covered with nanocrystalline apatites. Through mineralization, the wettability of the nanofiber matrix was greatly improved. Moreover, the murine-derived osteoblastic cells were shown to attach and grow actively on the apatite-mineralized nanofibrous substrate. In particular, the mineralized PCL nanofibrous substrate significantly stimulated the expression of bone-associated genes, including Runx2, collagen type I, alkaline phosphatase, and osteocalcin, when compared with the pure PCL nanofiber substrate without mineralization. The currently developed polymer nanofibrous web with the bioactive mineralized surface is considered to be potentially useful as bone regenerative and tissue engineering matrices.

Bioactivity improvement of poly(ε-caprolactone) membrane with the addition of nanofibrous bioactive glass

Nanofibrous glass with a bioactive composition was added to a degradable polymer poly(epsilon-caprolactone) (PCL) to produce a nanocomposite in thin membrane form ( approximately 260 microm). The bioactivity and osteoblastic responses of the nanocomposite membrane were examined and compared with those of a pure PCL membrane. Glass nanofibers with diameters in the range of hundreds of nanometers were added to a PCL solution at 20 wt.%, and the mixture was stirred vigorously and air dried. The obtained nanocomposite membrane showed that many chopped glass nanofibers formed by the mixing step were embedded uniformly into the PCL matrix. The nanocomposite membrane induced the rapid formation of apatite-like minerals on the surface when immersed in a simulated body fluid. Murine-derived osteoblastic cells (MC3T3-E1) grew actively over the nanocomposite membrane with cell viability significantly improved compared with those on the pure PCL membrane. Moreover, the osteoblastic activity, as assessed by the expression of alkaline phosphatase, was significantly higher on the nanocomposite membrane than on the pure PCL membrane. The currently developed nanocomposite of the bioactive glass-added PCL might find applications in the bone regeneration areas such as the guided bone regeneration (GBR) membrane.

Collagen gel three-dimensional matrices combined with adhesive proteins stimulate neuronal differentiation of mesenchymal stem cells

Three-dimensional gel matrices provide specialized microenvironments that mimic native tissues and enable stem cells to grow and differentiate into specific cell types. Here, we show that collagen three-dimensional gel matrices prepared in combination with adhesive proteins, such as fibronectin (FN) and laminin (LN), provide significant cues to the differentiation into neuronal lineage of mesenchymal stem cells (MSCs) derived from rat bone marrow. When cultured within either a three-dimensional collagen gel alone or one containing either FN or LN, and free of nerve growth factor (NGF), the MSCs showed the development of numerous neurite outgrowths. These were, however, not readily observed in two-dimensional culture without the use of NGF. Immunofluorescence staining, western blot and fluorescence-activated cell sorting analyses demonstrated that a large population of cells was positive for NeuN and glial fibrillary acidic protein, which are specific to neuronal cells, when cultured in the three-dimensional collagen gel. The dependence of the neuronal differentiation of MSCs on the adhesive proteins containing three-dimensional gel matrices is considered to be closely related to focal adhesion kinase (FAK) activation through integrin receptor binding, as revealed by an experiment showing no neuronal outgrowth in the FAK-knockdown cells and stimulation of integrin β1 gene. The results provided herein suggest the potential role of three-dimensional collagen-based gel matrices combined with adhesive proteins in the neuronal differentiation of MSCs, even without the use of chemical differentiation factors. Furthermore, these findings suggest that three-dimensional gel matrices might be useful as nerve-regenerative scaffolds.

Preparation of porous bioactive ceramic microspheres and in vitro osteoblastic culturing for tissue engineering application

Microparticulates are useful for directly filling defective tissues as well as for delivering cells and bioactive molecules in regenerative medicine. This paper reports on the production of bioactive ceramic microspheres with an interconnected macropore structure. The sol-gel derived calcium silicate powder was homogenized with an oligomeric Camphene melt, which was used as a novel porogen, and spherical-shaped microparticulates were obtained by an oil-in-water emulsion method. A porous structure was generated through the sublimation of Camphene within the calcium silicate-Camphene solidified blend under ambient conditions. The microspheres retained the crystalline phase of apatite and wollastonite during heat treatment and induced calcium phosphate precipitation under a body-simulating medium, showing the characteristics of bone-bioactive materials. Osteoblastic cells were observed to anchor to and spread well over the surface of the porous microspheres, and further to proliferate actively with culturing time. The bioactive and porous microspheres developed are considered potentially useful in the regeneration of hard tissues as a matrix for tissue engineering as well as a direct filling material.

Construction of mesenchymal stem cell-containing collagen gel with a macrochanneled polycaprolactone scaffold and the flow perfusion culturing for bone tissue engineering.

Abstract A novel bone tissue-engineering construct was developed by using poly(ɛ-caprolactone) (PCL)-macrochanneled scaffolds combined with stem cell-seeded collagen hydrogels and then applying flow perfusion culture. Rat mesenchymal stem cells (MSCs) were loaded into collagen hydrogels, which were then combined with macrochanneled PCL scaffolds. Collagen hydrogels were demonstrated to provide favorable growth environments for MSCs and to foster proliferation. Cell number determination identified retention of substantially fewer (50–60%) cells when they were seeded directly onto macrochanneled PCL than of cells engineered within collagen hydrogels. Additionally, the cells actively proliferated within the combined scaffold for up to 7 days. MSC-loaded collagen–PCL scaffolds were subsequently cultured under flow perfusion to promote proliferation and osteogenic differentiation. Cells proliferated to levels significantly higher in flow perfusion culture than that under static conditions during 21 days. A quantitative polymerase chain reaction (QPCR) assay revealed significant alterations in the transcription of bone-related genes such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP), such as 8-, 2.5-, and 3-fold induction, respectively, after 10 days of flow perfusion relative to those in static culture. OPN and OCN protein levels, as determined by Western blot, increased under flow perfusion. Cellular mineralization was significantly enhanced by the flow perfusion during 21 and 28 days. Analyses of mechanosensitive gene expression induced by flow perfusion shear stress revealed significant upregulation of c-fos and cyclooxygenase-2 (COX-2) during the initial culture period (3–5 days), suggesting that osteogenic stimulation was possible as a result of mechanical force-driven transduction. These results provide valuable information for the design of a new bone tissue-engineering system by combining stem cell-loaded collagen hydrogels with macrochanneled scaffolds in flow perfusion culture.

Novel Scaffolds of Collagen with Bioactive Nanofiller for the Osteogenic Stimulation of Bone Marrow Stromal Cells

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.

Macrochanneled bioactive ceramic scaffolds in combination with collagen hydrogel: A new tool for bone tissue engineering†

New tissue-engineering tool for bone regeneration is described to facilitate homogeneous cell seeding and effective osteogenic development. Calcium phosphate (CaP) scaffolds with macrochanneled and well-defined pore structure was developed, however, a large portion of the cells seeded directly within the scaffold easily penetrates without good adhesion to the scaffold surface. To overcome this, a method was exploited to dispense cells evenly throughout the CaP scaffold using collagen hydrogel. Rat bone marrow-derived mesenchymal stem cells (MSCs) were mixed within a neutralized collagen solution, which was then infiltrated into the macrochanneled pore space and gelled to result in macrochanneled bioceramic scaffold combined with MSCs-hydrogel. MSCs contained within the hydrogel-CaP scaffolds were highly viable, with similar growth pattern to those in the collagen hydrogel. Cells seeded by this approach were initially almost double in number compared with those seeded directly onto the CaP scaffold and had an active proliferation more than 14 days. Assessments of the MSCs showed significantly higher alkaline phosphatase levels in the combined scaffold, which was accompanied by enhanced osteogenesis including the expression of genes [collagen type I, bone sialoprotein, and osteopontin (OPN)] and proteins (OPN and osteocalcin). Extracellular calcium was also elevated significantly in the combined scaffold compared to the CaP scaffold. In addition, mechanical strength of the constructs was improved significantly in the combined scaffold compared to the CaP scaffold. Based on these, the cell culturing and tissue engineering strategy within the macrochanneled bioactive ceramic scaffolds could be improved greatly by the combinatory approach of using collagen hydrogel.

Bone Cell Responses of Titanium Blasted with Bioactive Glass Particles

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<0.05) with respect to the G-Ti and Ti groups. In particular, the alkaline phosphatase (AP) activity of the cells was significantly higher on the BG-Ti than on the other groups after culturing for 14 days. Moreover, the mineralization behavior of the cells, as assessed by Alizarin S Red, was superior on the BG-Ti to that observed on the other groups after culturing for 14 and 28 days. Overall, the blasting of Ti with a bioactive glass composition is considered beneficial for producing substrates with enhanced osteogenic potential.

Feasibility of silica-hybridized collagen hydrogels as three-dimensional cell matrices for hard tissue engineering

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.