Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues.

Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations.

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.

Development, characterisation and biocompatibility testing of a cobalt-containing titanium phosphate-based glass for engineering of vascularized hard tissues.

There is a continuing need to develop scaffold materials that can promote vascularisation throughout the tissue engineered construct. This study investigated the effect of cobalt oxide (CoO) doped into titanium phosphate glasses on material properties, biocompatibility and vascular endothelial growth factor (VEGF) secretion by osteoblastic MG63 cells. Glasses composed of (P2O5)45(Na2O)20(TiO2)05(CaO)30-x(CoO)x(x=0, 5, 10, and 15 mol%) were fabricated and the effect of Co on physicochemical properties including density, glass transition temperature (Tg), degradation rate, ion release, and pH changes was assessed. The results showed that incorporation of CoO into the glass system produced an increase in density with little change in Tg. It was then confirmed that the pH did not change significantly when CoO was incorporated in the glass, and stayed constant at around 6.5-7.0 throughout the dissolution study period of 336 h. Ion release results followed a specific pattern with increasing amounts of CoO. In general, although incorporation of CoO into a titanium phosphate glass increased its density, other bulk and surface properties of the glass did not show any significant changes. Cell culture studies performed using MG63 cells over a 7-day period indicated that the glasses provide a stable surface for cell attachment and are biocompatible. Furthermore, VEGF secretion was significantly enhanced on all glasses compared with standard tissue culture plastic and Co doping enhanced this effect further. In conclusion, the developed Co-doped glasses are stable and biocompatible and thus offer enhanced potential for engineering vascularized tissue.

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.

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

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.

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 ß1 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.

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 * 5P(2)O(5) 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.

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.

Development of robotic dispensed bioactive scaffolds and human adipose-derived stem cell culturing for bone tissue engineering.

Bioactive and degradable scaffolds made from bioactive glass-polycaprolactone with a mineralized surface and a well-defined three-dimensional (3D) pore configuration were produced using a robotic dispensing technique. Human adipose-derived stem cells (hASCs) were cultured on the 3D scaffolds, and the osteogenic development of cells within the scaffolds was addressed under a dynamic flow perfusion system for bone tissue engineering. The bioactive glass component introduced within the composite assisted in the surface mineralization of the 3D scaffolds. The hASCs initially adhered well and grew actively over the mineralized surface, and migrated deep into the channels of the 3D scaffold. In particular, dynamic perfusion culturing helped the cells to proliferate better on the 3D structure compared to that under static culturing condition. After 4 weeks of culturing by dynamic perfusion, the cells not only covered the scaffold surface completely but also filled the pore channels bridging the stems. The osteogenic differentiation of the hASCs with the input of osteogenic factors was stimulated significantly by the dynamic perfusion flow, as determined by alkaline phosphate expression. Overall, the culturing of hASCs upon the currently developed 3D scaffold in conjunction with the dynamic perfusion method may be useful for tissue engineering of bone.

FGF2-adsorbed macroporous hydroxyapatite bone granules stimulate in vitro osteoblastic gene expression and differentiation.

Hydroxyapatite bone granules with a macroporous structure were produced and then adsorbed with basic fibroblast growth factor (FGF2). The in vitro scaffolding role of the granules in cell population and osteogenic differentiation was investigated. The FGF2-adsorbed porous granules allowed the MC3T3-E1 cells to adhere well and then proliferate actively. While the cell growth level on the FGF2-treated granules was observed to be similar to that on the untreated granules, the expression of genes associated with bone, including collagen type I, alkaline phosphatase, and osteocalcin was significantly upregulated by the FGF2 treatment, particularly at the early stage. Moreover, the production of alkaline phosphatase with prolonged culturing was greatly enhanced on the FGF2-adsorbed granules. Taken together, the FGF2 treatment of the hydroxyapatite granules was effective in the osteogenic development and the FGF2-adsorbed bone granules may be useful in bone regeneration area.

Evacuated calcium phosphate spherical microcarriers for bone regeneration.

Microparticulates are an effective three-dimensional (3D) matrix for the culture of stem cells to be used in tissue engineering of bone. Herein, bioactive calcium phosphate microparticles with an evacuated morphology were prepared, and their potential to support stem cells for bone tissue engineering was addressed. Spherical particles with sizes of hundreds of micrometers were produced using the emulsification method, during which the internal portion was evacuated with the aid of solvent evaporation. The evacuated portion of the microspheres, which is considered to enhance cell population and be replaced with new bone, was found to comprise approximately 65-70% of the total volume. Stem cells derived from human adipose and rat bone marrow were isolated and cultured on the evacuated microspheres. When compared to a two-dimensional culture dish, the 3D spherical substrate provided cells with more space to adhere and populate, which became more evident as the cell seeding quantity increased. Moreover, better cell proliferation was observed on the evacuated microspheres than on the conventional filled microspheres, suggesting that evacuation of the internal part of the microspheres was useful for generating a large cell population. The differentiation of cells cultured on the 3D evacuated microspheres into osteoblasts with appropriate osteogenic cues was also more effective when compared to cells cultured on a two-dimensional dish. When implanted within a rabbit calvarium, the evacuated microspheres induced rapid bone formation at 6 weeks with a typical lamella pattern. Based on the results, the evacuated calcium phosphate microspheres are considered an effective 3D matrix for direct filling of bone defects as well as for bone tissue engineering using stem cells.

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells.

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(epsilon-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA-PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA-PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA-PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA-PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering.

Nanofibrous membrane of collagen-polycaprolactone for cell growth and tissue regeneration.

Nanofibrous substrates of synthetic polymers including polycaprolactone (PCL) have shown considerable potential in tissue regeneration. This paper reports the use of PCL/collagen nanofibers to improve the in vitro osteoblastic responses for the applications in bone regeneration area. Collagen and PCL were dissolved in a co-solvent, and the resulting solution was electrospun into a nanofibrous web. Nonwoven fibrous matrices were successfully produced at various compositional ratios (PCL/collagen = 1/3, 1 and 3 by weight). Although the PCL nanofiber was hydrophobic, the presence of collagen significantly improved the water affinity, such as the water contact angle and water uptake capacity. Tensile mechanical tests showed that the collagen-PCL nanofiber had a significantly higher extension rate (approximately 2.8-fold) than the PCL while maintaining the maximum tensile load in a similar range. The osteoblastic cells cultured on the collagen-PCL nanofibrous substrate showed better initial adhesion and a higher level of growth than those cultured on the PCL nanofiber. Furthermore, real-time RT-PCR revealed the expression of a series of bone-associated genes, including osteopontin, collagen type I and alkaline phosphatase. The expression of these genes was significantly higher on the collagen-PCL nanofiber than on the PCL nanofiber. When subcutaneously implanted in mouse the collagen-PCL membrane facilitated tissue cells to well penetrate into the nanofibrous structure at day 7, whilst no such cell penetration was noticed in the pure PCL nanofiber. Overall, the presence of collagen within the PCL nanofiber improves the water affinity, tensile extension rate, and the tissue cell responses, such as initial adhesion, growth, penetration and the expression of bone-associated genes. Therefore, the collagen-PCL nanofibrous membrane may have potential applications in the cell growth and bone tissue regeneration.

Tissue engineering polymeric microcarriers with macroporous morphology and bone-bioactive surface.

PCL microspheres featuring a macroporous morphology and a bone-bioactive surface have been prepared. 'Camphene' was introduced to generate pores within the microsphere network. The pore size was variable from a few to tens to hundreds of microm depending on the Camphene/PCL ratio. Macropores (with sizes >50 microm) could be obtained with a Camphene/PCL ratio exceeding 6. The microsphere surface was further tailored with apatite mineral phase through solution-mediated precipitation, to endow the interface with bone bioactivity. Rat bone marrow stromal cells attached and spread actively on microspheres and populated well within their macropores. The developed microspheres may be potentially applicable as a cell delivery scaffold for bone tissue engineering.

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(epsilon-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.

Nanofibrous matrices of poly(lactic acid) and gelatin polymeric blends for the improvement of cellular responses.

Substrates with a nanofibrous morphology are considered as a prospective matrix to populate and support cells in the tissue regeneration area. Although the nanofibers made of synthetic degradable polymers, including poly(lactic acid) (PLA), have been well studied, their poor cell affinity has restricted wider applications. Herein, we produced blending nanofibers made of PLA and gelatin to improve the cellular responses of PLA. For this, both PLA and gelatin were dissolved in an organic solvent, varying the compositions of PLA:gelatin at 1:3 and 1:1 by weight, and the solutions were electrospun into nanofibers. At all compositions, nanofibers could be successfully generated with diameters of approximately hundreds of nanometers. The addition of gelatin into PLA markedly improved the wettability of the nanofibrous substrate. The osteoblastic cells attached and spread well on all the blending nanofibers and pure PLA. In particular, the cellular growth was significantly higher on the gelatin-blended PLA than on the pure PLA nanofiber. On the basis of this study, the PLA/gelatin blending polymeric nanofibers are considered to be useful as a bone cell supporting matrix in the tissue regeneration area.