Evaluation of microleakage and push-out bond strength of various composite resins for sealing the screw-access channel in implant-supported restorations.

STATEMENT OF PROBLEM: Microleakage and loss of the composite resin sealing the screw-access channel are frequent complications of screw-retained implant-supported prostheses. How the screw-access channel should be best restored to reduce such complications is unclear. PURPOSE: The purpose of this in vitro study was to evaluate the microleakage and bond strength of 3 types of composite resins (flowable, packable, and bulk-fill) with or without a bonding agent treatment to seal the screw-access channel of 2 types of restorative materials (zirconia and Co-Cr alloy) with or without thermocycling. MATERIAL AND METHODS: In total, 240 yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) specimens (IPS e.max ZirCAD) and 240 Co-Cr alloy (Vera PDS) specimens were prepared with a Ø3×3-mm cylindrical cavity at the center to simulate the screw-access channel. Three types of composite resins (flowable, packable, and bulk-fill resin) (Filtek Z350 XT Flowable Restorative, Filtek Z350 XT Universal Restorative, and Filtek One Bulk Fill Restorative) were applied to restore the access channel of the zirconia and Co-Cr specimens with or without a bonding agent (Single Bond Universal Adhesive). Microleakage and push-out bond strength were determined and compared by dividing the specimens into experimental groups with or without thermocycling (1000 times with 30 seconds at 5 ±2 °C and 55 ±2 °C). The results were analyzed by using a 1-way ANOVA and 4-way ANOVA. Adjustment for multiple comparisons was made with the Tukey Honestly Significant Difference (HSD) test. RESULTS: The specimens subjected to thermocycling showed a lower bond strength (P<.001) and higher microleakage (P<.001) than specimens stored in a constant-temperature water bath. Specimens treated with bonding agents showed a higher bond strength (P<.001) and lower microleakage (P<.001) than specimens not treated with a bonding agent. Higher bond strengths were observed in the order of bulk-fill resin, packable resin, and flowable resin (P<.001). Packable resin showed higher microleakage than flowable resin and bulk-fill resin (P<.05). No significant difference in microleakage was found between the flowable resin and bulk-fill resin (P>.05). CONCLUSIONS: Higher bond strengths were observed in the order of bulk-fill resin, packable resin, and flowable resin. Less microleakage was observed in the flowable resin and bulk-fill resin than in the packable composite resin. Bonding agent treatment was effective in increasing bond strength and decreasing microleakage. Zirconia and Co-Cr showed a bond strength similar to that of composite resins, but zirconia showed higher microleakage than Co-Cr. Restoring the screw-access channel with the bulk-fill resin should increase bond strength and reduce microleakage.

Mechanoluminescent, Air-Dielectric MoS2 Transistors as Active-Matrix Pressure Sensors for Wide Detection Ranges from Footsteps to Cellular Motions.

Tactile pressure sensors as flexible bioelectronic devices have been regarded as the key component for recently emerging applications in electronic skins, health-monitoring devices, or human-machine interfaces. However, their narrow range of sensible pressure and their difficulty in forming high integrations represent major limitations for various potential applications. Herein, we report fully integrated, active-matrix arrays of pressure-sensitive MoS2 transistors with mechanoluminescent layers and air dielectrics for wide detectable range from footsteps to cellular motions. The inclusion of mechanoluminescent materials as well as air spaces can increase the sensitivity significantly over entire pressure regimes. In addition, the high integration capability of these active-matrix sensory circuitries can enhance their spatial resolution to the level sufficient to analyze the pressure distribution in a single cardiomyocyte. We envision that these wide-range pressure sensors will provide a new strategy toward next-generation electronics at biomachine interfaces to monitor various mechanical and biological phenomena at single-cell resolution.

Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering.

Bioactive nanocomposite scaffolds with cell-adhesive surface have excellent bone regeneration capacities. Fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-nBG/PCL) scaffolds with an open pore architecture were generated by a robotic-dispensing technique. The surface immobilization level of FN was significantly higher on the nBG/PCL scaffolds than on the PCL scaffolds, mainly due to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds significantly improved cell responses, including initial anchorage and subsequent cell proliferation. Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate for bone regeneration.

Guided Bone Regeneration with a Nitric-Oxide Releasing Polymer Inducing Angiogenesis and Osteogenesis in Critical-Sized Bone Defects.

Synthetic scaffolds, as bone grafts, provide a favorable environment for the repair and growth of new bone tissue at defect sites. However, the lack of angio- and osteo-induction limits the usefulness of artificial scaffolds for bone regeneration. Nitric oxide (NO) performs essential roles in healing processes, such as regulating inflammation and addressing incomplete revascularization. In this study, a polymer capable of controlled NO release is developed to promote the osteogenic capacity in artificial scaffolds. The biological efficiency of the NO compound is assessed by its effect on pre-osteoblasts and macrophages in vitro and the extent of vascularization and bone formation in the calvaria defect model in vivo. The compound does not inhibit cell adhesion or proliferation. NO treatment significantly increases both alkaline phosphatase activity and mineralization in pre-osteoblasts. Macrophages treated with NO secrete high levels of anti-inflammatory factors and adopt the pro-regenerative phenotype. In the critical-sized defect model, the collagen scaffold containing the NO compound enhances neovascularization and bone formation. The developed NO-releasing system promotes osteogenesis and regeneration of damaged bone tissue. As the multiple functions of NO involve macrophage modulation and angiogenesis, such release systems may be valuable for guiding bone regeneration in critical-sized defects.

Alginate combined calcium phosphate cements: mechanical properties and in vitro rat bone marrow stromal cell responses.

Here, we prepared self-setting calcium phosphate cements (CPCs) based on α-tricalcium phosphate with the incorporation of sodium alginate, and their mechanical properties and in vitro cellular responses were investigated. The addition of alginate enhanced the hardening reaction of CPCs showing shorter setting times within a range of powder-to-liquid ratios. When immersed in a body simulating fluid the alginate-CPCs fully induced a formation of an apatite crystalline phase similar to that of bare CPCs. The compressive and tensile strengths of the CPCs were found to greatly improve during immersion in the fluid, and this improvement was more pronounced in the alginate-CPCs. As a result, the alginate-CPCs retained significantly higher strength values than the bare CPCs after 3-7 days of immersion. The rat bone marrow derived stromal cells (rBMSCs) cultured on the alginate-CPCs initially adhered to and then spread well on the cements surface, showed an on-going increase in the population with culture time, and differentiated into osteoblasts expressing bone-associated genes (collagen type I, osteopontin and bone sialoprotein) and synthesizing alkaline phosphatase. However, the stimulated level of osteogenic differentiation was not confirmative with the incorporation of alginate into the CPC composition based on the results. One merit of the use of alginate was its usefulness in forming CPCs into a variety of scaffold shapes including microspheres and fibers, which is associated with the cross-link of alginate under the calcium-containing solution.

Multi-functional effects of a nitric oxide-conjugated copolymer for accelerating palatal wound healing.

The damaged site of a palatal wound is difficult to repair and often remains unclosed due to failure of the healing process, which occurs in inadequate environments of the oral cavity. Nitric oxide (NO) has effective functions in repairing damaged tissues, but it has a limitation due to short lifetime and rapid diffusion. Here, we synthesize a donor to deliver exogenous NO gas and verify its therapeutic effect for the palatal wound healing, which is known to take longer for healing due to the poor environment of warm saliva containing millions of microbes. NO was incorporated into the synthetic polymer and the NO-donors were characterized based upon their ability to release NO. The NO donor not only reduced cytotoxicity but also increased migration and proliferation in gingival fibroblasts. Moreover, the angiogenic capacity was improved by NO-donor treatment. In the palatal wound model, the NO-treatment was involved in enhancing the biological responses associated with wound healing. This strategy suggests that treatment involving controlled NO release may have beneficial effects on palatal wound healing.

Smart contact lens and transparent heat patch for remote monitoring and therapy of chronic ocular surface inflammation using mobiles.

Wearable electronic devices that can monitor physiological signals of the human body to provide biomedical information have been drawing extensive interests for sustainable personal health management. Here, we report a human pilot trial of a soft, smart contact lens and a skin-attachable therapeutic device for wireless monitoring and therapy of chronic ocular surface inflammation (OSI). As a diagnostic device, this smart contact lens enables real-time measurement of the concentration of matrix metalloproteinase-9, a biomarker for OSI, in tears using a graphene field-effect transistor. As a therapeutic device, we also fabricated a stretchable and transparent heat patch attachable on the human eyelid conformably. Both diagnostic and therapeutic devices can be incorporated using a smartphone for their wireless communications, thereby achieving instantaneous diagnosis of OSI and automated hyperthermia treatments. Furthermore, in vivo tests using live animals and human subjects confirm their good biocompatibility and reliability as a noninvasive, mobile health care solution.

Smart, soft contact lens for wireless immunosensing of cortisol.

Despite various approaches to immunoassay and chromatography for monitoring cortisol concentrations, conventional methods require bulky external equipment, which limits their use as mobile health care systems. Here, we describe a human pilot trial of a soft, smart contact lens for real-time detection of the cortisol concentration in tears using a smartphone. A cortisol sensor formed using a graphene field-effect transistor can measure cortisol concentration with a detection limit of 10 pg/ml, which is low enough to detect the cortisol concentration in human tears. In addition, this soft contact lens only requires the integration of this cortisol sensor with transparent antennas and wireless communication circuits to make a smartphone the only device needed to operate the lens remotely without obstructing the wearer's view. Furthermore, in vivo tests using live rabbits and the human pilot experiment confirmed the good biocompatibility and reliability of this lens as a noninvasive, mobile health care solution.

Hierarchical microchanneled scaffolds modulate multiple tissue-regenerative processes of immune-responses, angiogenesis, and stem cell homing.

Recapitulating the in vivo microenvironments of damaged tissues through modulation of the physicochemical properties of scaffolds can boost endogenous regenerative capacity. A series of critical events in tissue healing including immune-responses, angiogenesis, and stem cell homing and differentiation orchestrate to relay the regeneration process. Herein, we report hierarchically structured ('microchanneled') 3D printed scaffolds (named 'µCh'), in contrast to conventional 3D printed scaffolds, induce such cellular responses in a unique way that contributes to accelerated tissue repair and remodeling. The µCh reduced the extracellular trap formation of anchored neutrophils at the very beginning (24 h) of implantation while increasing the number of live cells. Among the macrophages covered the surface of µCh over 7 days a major population polarized toward alternativelly activated phase (M2) which contrasted with control scaffolds where classically activated phase (M1) being dominant. The mesenchymal stem cells (MSCs) recruited to the µCh were significantly more than those to the control, and the event was correlated with the increased level of stem cell homing cytokine, stromal derived factor 1 (SDF1) sequestered to the µCh. Furthermore, the neo-blood vessel formation was more pronounced in the µCh, which was in line with the piling up of angiogenic factor, vascular endothelial growth factor (VEGF) in the µCh. Further assays on the protein sequestration to the µCh revealed that a set of chemokines involved in early pro-inflammatory responses were less found whereas representative adhesive proteins engaged in the cell-matrix interactions were significantly more captured. Ultimately, the fibrous capsule formation on the µCh was reduced with respect to the control, when assessed for up to 21 days, indicating less severe foreign body reaction. The tissue healing and regenerative capacity of the µCh was then confirmed in a critically sized bone model, where those series of events observed are essential to relay bone regeneration. The results over 6 weeks showed that the µCh significantly enhanced the early bone matrix deposition and accelerated bone regeneration. While more in-depth studies remain as to elucidate the underlying mechanisms for each biological event, the molecular, cellular and tissue reactions to the µCh were coherently favorable for the regeneration process of tissues, supporting the engineered scaffolds as potential therapeutic 3D platforms.

Nucleotide Analysis in Korean Dairy Products Using High- Performance Liquid Chromatography with Diode Array Detector.

Nucleotides play important roles in numerous intracellular biochemical processes and are used in infant formulas and other dairy products. However, domestic analytical methods for assessing nucleotide content in products have not yet been established, and therefore, methods for determining nucleotide content are urgently required. A rapid and simple analytical method for determining the content of five types of nucleotides in dairy products was improved using solid phase extraction clean-up and high-performance liquid chromatography with diode array detector. The extraction solvent used in the AOAC method was not well dissolved and was changed to hydrophilic EDTA-Na. In addition, the results obtained using the isocratic elution method and a single wavelength were similar to those obtained using the AOAC method, and the time taken for analysis was shortened from 40 min to 25 min. The process of method validation revealed the following parameters: accuracy (84.69%-102.72%), precision (1.51%-6.82%), linearity (0.999), and limit of detection (cytidine 5'-monophosphate, 0.09 mg/L; uridine 5'-monophosphate, 0.11 mg/L; adenosine 5'-monophosphate, 0.12 mg/L; guanosine 5'-monophosphate, 0.11 mg/L; and inosine 5'-monophosphate, 0.14 mg/L). The method was also used to determine the nucleotide concentration in 25 samples (infant formulas, 1.99-29.39 mg/100 g; and cow milk, 0.28-0.83 mg/100 g). The newly improved method was appropriate for analyzing nucleotides in infant formulas and other dairy products faster when compared to conventional methods.

Development of a three-dimensionally printed scaffold grafted with bone forming peptide-1 for enhanced bone regeneration with in vitro and in vivo evaluations.

Defects in bone are some of the most difficult injuries to treat. Biomimetic scaffolds represent a promising approach for successful bone tissue regeneration. In this study, a three-dimensional (3D) scaffold with osteo-inductive functionality was designed and assayed both in-vitro and in-vivo. Bone formation peptide-1 (BFP1), an osteo-promoting specific peptide, was covalently bound to a 3D printed polycaprolactone (PCL) scaffold using polydopamine (DOPA). The amount of BFP1 immobilized on the surface was found to increase depending on the BFP1 concentration of the loading solution. To observe the biological effects of the 3D scaffolds, human tonsil-derived mesenchymal stem cells (hTMSCs) were isolated. The cells were cultured on the scaffolds and observed to rapidly differentiate into osteoblast-like cells with osteo-promoting capabilities. The scaffolds were implanted in a rabbit calvarial defect model for 8 weeks and successfully stimulated both vessel and bone regeneration. Osteo-promoting 3D scaffolds may provide a safer and more efficient approach for bone repair and remodelling in regenerative medicine.

The Application of Fibrin/Hyaluronic Acid-Poly(l-Lactic-co-Glycolic Acid) Construct in Augmentation Rhinoplasty.

Although many graft materials have been used for augmentation rhinoplasty, an ideal graft has not yet been developed. As the field of tissue engineering has been developing, it has been applied to the reconstruction of many organs, but its application in the rhinoplasty field is still limited. This study evaluated the utility of allogenic chondrocytes with fibrin/hyaluronic acid (HA)-poly(l-lactic-co-glycolic acid) (PLGA) constructs in augmentation rhinoplasty. Chondrocytes from rabbit auricular cartilage were isolated and cultured with fibrin/HA hydrogels and implanted into PLGA scaffolds. After 8 weeks of in vitro culture, the scaffolds were implanted in the nasal dorsum of six rabbits. Eight weeks postoperatively, the implanted sites were evaluated with gross, radiologic, and histologic analysis. In vitro, more than 90% of the seeded chondrocytes in the PLGA scaffolds survived for 2 weeks, and they produced a large amount of extracellular matrix and were well differentiated. The grafts maintained their initial shape for 8 weeks after implantation. Radiological and histological evaluations showed that the structure was well maintained with minimal inflammatory response and appropriate elevation levels. However, the formation of neo-chondrocytes was not observed. PLGA scaffolds seeded with fibrin/HA and allogenic chondrocytes can be a biocompatible augmentation material in rhinoplasty in the future.

Elevated contractile responses to acetylcholine in organ cultured rabbit carotid artery.

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum- free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.

Multifunctional and stable bone mimic proteinaceous matrix for bone tissue engineering.

Biomaterial surface design with biomimetic proteins holds great promise for successful regeneration of tissues including bone. Here we report a novel proteinaceous hybrid matrix mimicking bone extracellular matrix that has multifunctional capacity to promote stem cell adhesion and osteogenesis with excellent stability. Osteocalcin-fibronectin fusion protein holding collagen binding domain was networked with fibrillar collagen, featuring bone extracellular matrix mimic, to provide multifunctional and structurally-stable biomatrices. The hybrid protein, integrated homogeneously with collagen fibrillar networks, preserved structural stability over a month. Biological efficacy of the hybrid matrix was proven onto tethered surface of biopolymer porous scaffolds. Mesenchymal stem cells quickly anchored to the hybrid matrix, forming focal adhesions, and substantially conformed to cytoskeletal extensions, benefited from the fibronectin adhesive domains. Cells achieved high proliferative capacity to reach confluence rapidly and switched to a mature and osteogenic phenotype more effectively, resulting in greater osteogenic matrix syntheses and mineralization, driven by the engineered osteocalcin. The hybrid biomimetic matrix significantly improved in vivo bone formation in calvarial defects over 6 weeks. Based on the series of stimulated biological responses in vitro and in vivo the novel hybrid proteinaceous composition will be potentially useful as stem cell interfacing matrices for osteogenesis and bone regeneration.

Gelatin-apatite bone mimetic co-precipitates incorporated within biopolymer matrix to improve mechanical and biological properties useful for hard tissue repair.

Synthetic biopolymers are commonly used for the repair and regeneration of damaged tissues. Specifically targeting bone, the composite approach of utilizing inorganic components is considered promising in terms of improving mechanical and biological properties. We developed gelatin-apatite co-precipitates which mimic the native bone matrix composition within poly(lactide-co-caprolactone) (PLCL). Ionic reaction of calcium and phosphate with gelatin molecules enabled the co-precipitate formation of gelatin-apatite nanocrystals at varying ratios. The gelatin-apatite precipitates formed were carbonated apatite in nature, and were homogeneously distributed within the gelatin matrix. The incorporation of gelatin-apatite significantly improved the mechanical properties, including tensile strength, elastic modulus and elongation at break, and the improvement was more pronounced as the apatite content increased. Of note, the tensile strength increased to as high as 45 MPa (a four-fold increase vs. PLCL), the elastic modulus was increased up to 1500 MPa (a five-fold increase vs. PLCL), and the elongation rate was ~240% (twice vs. PLCL). These results support the strengthening role of the gelatin-apatite precipitates within PLCL. The gelatin-apatite addition considerably enhanced the water affinity and the acellular mineral-forming ability in vitro in simulated body fluid; moreover, it stimulated cell proliferation and osteogenic differentiation. Taken together, the GAp-PLCL nanocomposite composition is considered to have excellent mechanical and biological properties, which hold great potential for use as bone regenerative matrices.

A novel therapeutic design of microporous-structured biopolymer scaffolds for drug loading and delivery.

Three-dimensional (3-D) open-channeled scaffolds of biopolymers are a promising candidate matrix for tissue engineering. When scaffolds have the capacity to deliver bioactive molecules the potential for tissue regeneration should be greatly enhanced. In order to improve drug-delivery capacity, we exploit 3-D poly(lactic acid) (PLA) scaffolds by creating microporosity within the scaffold network. Macroporous channeled PLA with a controlled pore configuration was obtained by a robotic dispensing technique. In particular, a room temperature ionic liquid (RTIL) bearing hydrophilic counter-anions, such as OTf and Cl, was introduced to the biopolymer solution at varying ratios. The RTIL-biopolymer slurry was homogenized by ultrasonication, and then solidified through the robotic dispensing process, during which the biopolymer and RTIL formed a bicontinuous interpenetrating network. After ethanol wash-out treatment the RTIL was completely removed to leave highly microporous open channels throughout the PLA network. The resultant pore size was observed to be a few micrometers (average 2.43 µm) and microporosity was determined to be ∼ 70%. The microporous surface was also shown to favor initial cell adhesion, stimulating cell anchorage on the microporous structure. Furthermore, in vivo tissue responses assessed in rat subcutaneous tissue revealed good tissue compatibility, with minimal inflammatory reactions, while gathering a larger population of fibroblastic cells than the non-microporous scaffolds, and even facilitating invasion of the cells within the microporous structure. The efficacy of the micropore networks generated within the 3-D scaffolds in loading and releasing therapeutic molecules was addressed using antibiotic sodium ampicillin and protein cytochrome C as model drugs. The microporous scaffolds exhibited significantly enhanced drug loading capacity: 4-5 times increase in ampicillin and 9-10 times increase in cytochrome C compared to the non-microporous scaffolds. The release of ampicillin loaded within the microporous scaffolds was initially fast (∼ 85% for 1 week), and was then slowed down, showing a continual release up to a month. On the other hand, cytochrome C was shown to release in a highly sustainable manner over a month, without showing an initial burst release effect. This study provides a novel insight into the generation of 3-D biopolymer scaffolds with high performance in loading and delivery of biomolecules, facilitated by the creation of microporous channels through the scaffold network. The capacity to support tissue cells while in situ delivering drug molecules makes the current scaffolds potentially useful for therapeutic tissue engineering.

Naturally and synthetic smart composite biomaterials for tissue regeneration.

The development of smart biomaterials for tissue regeneration has become the focus of intense research interest. More opportunities are available by the composite approach of combining the biomaterials in the form of biopolymers and/or bioceramics either synthetic or natural. Strategies to provide smart capabilities to the composite biomaterials primarily seek to achieve matrices that are instructive/inductive to cells, or that stimulate/trigger target cell responses that are crucial in the tissue regeneration processes. Here, we review in-depth, recent developments concerning smart composite biomaterials available for delivery systems of biofactors and cells and scaffolding matrices in tissue engineering. Smart composite designs are possible by modulating the bulk and surface properties that mimic the native tissues, either in chemical (extracellular matrix molecules) or in physical properties (e.g. stiffness), or by introducing external therapeutic molecules (drugs, proteins and genes) within the structure in a way that allows sustainable and controllable delivery, even time-dependent and sequential delivery of multiple biofactors. Responsiveness to internal or external stimuli, including pH, temperature, ionic strength, and magnetism, is another promising means to improve the multifunctionality in smart scaffolds with on-demand delivery potential. These approaches will provide the next-generation platforms for designing three-dimensional matrices and delivery systems for tissue regenerative applications.

Robocasting nanocomposite scaffolds of poly(caprolactone)/hydroxyapatite incorporating modified carbon nanotubes for hard tissue reconstruction.

Nanocomposite scaffolds with tailored 3D pore configuration are promising candidates for the reconstruction of bone. Here we fabricated novel nanocomposite bone scaffolds through robocasting. Poly(caprolactone) (PCL)-hydroxyapatite (HA) slurry containing ionically modified carbon nanotubes (imCNTs) was robotic-dispensed and structured layer-by-layer into macrochanneled 3D scaffolds under adjusted processing conditions. Homogeneous dispersion of imCNTs (0.2 wt % relative to PCL-HA) was achieved in acetone, aiding in the preparation of PCL-HA-imCNTs slurry with good mixing property. Incorporation of imCNTs into PCL-HA composition significantly improved the compressive strength and elastic modulus of the robotic-dispensed scaffolds (~1.5-fold in strength and ~2.5-fold in elastic modulus). When incubated in simulated body fluid (SBF), PCL-HA-imCNT nanocomposite scaffold induced substantial mineralization of apatite in a similar manner to the PCL-HA scaffold, which was contrasted in pure PCL scaffold. MC3T3-E1 cell culture on the scaffolds demonstrated that cell proliferation levels were significantly higher in both PCL-HA-imCNT and PCL-HA than in pure PCL, and no significant difference was found between the nanocomposite scaffolds. When the PCL-HA-imCNT scaffold was implanted into a rat subcutaneous tissue for 4 weeks, soft fibrous tissues with neo-blood vessels formed well in the pore channels of the scaffolds without any significant inflammatory signs. Tissue reactions in PCL-HA-imCNT scaffold were similar to those in PCL-HA scaffold, suggesting incorporated imCNT did not negate the beneficial biological roles of HA. While more long-term in vivo research in bone defect models is needed to confirm clinical availability, our results suggest robotic-dispensed PCL-HA-imCNT nanocomposite scaffolds can be considered promising new candidate matrices for bone regeneration.

Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold.

A reliable source of osteogenic cells is an essential factor for bone tissue engineering. In this study, human-induced pluripotent stem cells (hiPSCs) without an embryoid body step were cultured in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique, after which osteogenesis was promoted by the addition of exogenous osteogenic factors. The osteogenesis of the hiPSCs was demonstrated based on the detection of osteogenic molecules, such as osteopontin, using flow cytometry analysis, quantitative polymerase chain reaction and western blotting. Thereafter, the cell-scaffold constructs were transplanted into the subcutaneous site of male athymic mice. At 4 weeks after implantation, histological assays (hematoxylin & eosin staining, Alizarin red staining, and osteocalcin immunostaining) were conducted to determine the bone induction of hiPSCs. The results indicated a production of pronounced levels of extracellular matrices and their mineral deposition within the cell-scaffold implant, suggesting possible in vivo bone induction by the hiPSCs-based tissue engineering approach. The results presented here provide useful information regarding the tissue engineering of bone utilizing hiPSCs in conjunction with cell-supporting scaffolds.