Characterization and preparation of bio-tubular scaffolds for fabricating artificial vascular grafts by combining electrospinning and a 3D printing system.

The last decade has seen artificial blood vessels composed of natural polymer nanofibers grafted into human bodies to facilitate the recovery of damaged blood vessels. However, electrospun nanofibers (ENs) of biocompatible materials such as chitosan (CTS) suffer from poor mechanical properties. This study describes the design and fabrication of artificial blood vessels composed of a blend of CTS and PCL ENs and coated with PCL strands using rapid prototyping technology. The resulting tubular vessels exhibited excellent mechanical properties and showed that this process may be useful for vascular reconstruction.

Mechanical and Electrical Characterization of Piezoelectric Artificial Cochlear Device and Biocompatible Packaging.

This paper presents the development of a piezoelectric artificial cochlea (PAC) device capable of analyzing vibratory signal inputs and converting them into electrical signal outputs without an external power source by mimicking the function of human cochlea within an audible frequency range. The PAC consists of an artificial basilar membrane (ABM) part and an implantable packaged part. The packaged part provides a liquid environment through which incoming vibrations are transmitted to the membrane part. The membrane part responds to the transmitted signal, and the local area of the ABM part vibrates differently depending on its local resonant frequency. The membrane was designed to have a logarithmically varying width from 0.97 mm to 8.0 mm along the 28 mm length. By incorporating a micro-actuator in an experimental platform for the package part that mimics the function of a stapes bone in the middle ear, we created a similar experimental environment to cochlea where the human basilar membrane vibrates. The mechanical and electrical responses of fabricated PAC were measured with a laser Doppler vibrometer and a data acquisition system, and were compared with simulation results. Finally, the fabricated PAC in a biocompatible package was developed and its mechanical and electrical characteristics were measured. The experimental results shows successful frequency separation of incoming mechanical signal from micro-actuator into frequency bandwidth within the 0.4 kHz-5 kHz range.

Development of a multi-channel piezoelectric acoustic sensor based on an artificial basilar membrane.

In this research, we have developed a multi-channel piezoelectric acoustic sensor (McPAS) that mimics the function of the natural basilar membrane capable of separating incoming acoustic signals mechanically by their frequency and generating corresponding electrical signals. The McPAS operates without an external energy source and signal processing unit with a vibrating piezoelectric thin film membrane. The shape of the vibrating membrane was chosen to be trapezoidal such that different locations of membrane have different local resonance frequencies. The length of the membrane is 28 mm and the width of the membrane varies from 1 mm to 8 mm. Multiphysics finite element analysis (FEA) was carried out to predict and design the mechanical behaviors and piezoelectric response of the McPAS model. The designed McPAS was fabricated with a MEMS fabrication process based on the simulated results. The fabricated device was tested with a mouth simulator to measure its mechanical and piezoelectrical frequency response with a laser Doppler vibrometer and acoustic signal analyzer. The experimental results show that the as fabricated McPAS can successfully separate incoming acoustic signals within the 2.5 kHz-13.5 kHz range and the maximum electrical signal output upon acoustic signal input of 94 dBSPL was 6.33 mVpp. The performance of the fabricated McPAS coincided well with the designed parameters.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.

An osteogenic bioink composed of alginate, cellulose nanofibrils, and polydopamine nanoparticles for 3D bioprinting and bone tissue engineering.

Bioprinting is an emerging technology for manufacturing cell-laden three-dimensional (3D) scaffolds, which are used to fabricate complex 3D constructs and provide specific microenvironments for supporting cell growth and differentiation. The development of bioinks with appropriate printability and specific bioactivities is crucial for bioprinting and tissue engineering applications, including bone tissue regeneration. Therefore, to produce functional bioinks for osteoblast printing and bone tissue formation, we formulated various nanocomposite hydrogel-based bioinks using natural and biocompatible biomaterials (i.e., alginate, tempo-oxidized cellulose nanofibrils (TOCNF), and polydopamine nanoparticles (PDANPs)). Rheological studies and printability tests revealed that bioinks containing 1.5% alginate and 1.5% TOCNF in the presence or absence of PDANP (0.5%) are suitable for 3D printing. Furthermore, in vitro studies of 3D-printed osteoblast-laden scaffolds indicated that the 0.5% PDANP-incorporated bioink induced significant osteogenesis. Overall, the bioink consisting of alginate, TOCNF, and PDANPs exhibited excellent printability and bioactivity (i.e., osteogenesis).

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering.

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.

Surface modification of a three-dimensional polycaprolactone scaffold by polydopamine, biomineralization, and BMP-2 immobilization for potential bone tissue applications.

Three-dimensional (3D) bioprinting is a free-form fabrication technique enabling fine feature control for tissue engineering applications. Especially, 3D scaffolds capable of supporting cell attachment, proliferation, and osteogenic differentiation are a prerequisite for bone tissue regeneration. Herein, we elaborated this approach to produce a 3D polycaprolactone (PCL) scaffold with long-term osteogenic activity. Specifically, we coated polydopamine (PDA) on 3D PCL scaffolds, subsequently deposited hydroxyapatite (HA) nanoparticles via biomimetic mineralization, and finally immobilized bone morphogenetic protein-2 (BMP-2). Material properties were characterized and compared with various 3D scaffolds, including PCL, PDA-coated PCL (PCL/PDA), and PDA-coated and HA-deposited PCL (PCL/PDA/HA). In vitro cell culture studies with osteoblasts revealed that the PCL/PDA/HA scaffolds immobilized with BMP-2 showed long-term retention of BMP-2 (for up to 21 days) and significantly increased osteoblast proliferation and osteogenic differentiation, as evidenced by metabolic activity, alkaline phosphatase activity, and calcium deposition. We believe that this multifunctional osteogenic 3D scaffold will be useful for bone tissue engineering applications.

Bio-plotted hydrogel scaffold with core and sheath strand-enhancing mechanical and biological properties for tissue regeneration.

Three-dimensional bio-plotted scaffolds constructed from encapsulated biomaterials or so-called "bio-inks" have received much attention for tissue regeneration applications, as advances in this technology have enabled more precise control over the scaffold structure. As a base material of bio-ink, sodium alginate (SA) has been used extensively because it provides suitable biocompatibility and printability in terms of creating a biomimetic environment for cell growth, even though it has limited cell-binding moiety and relatively weak mechanical properties. To improve the mechanical and biological properties of SA, herein, we introduce a strategy using hydroxyapatite (HA) nanoparticles and a core/sheath plotting (CSP) process. By characterizing the rheological and chemical properties and printability of SA and SA/HA-blended inks, we successfully fabricated bio-scaffolds using CSP. In particular, the mechanical properties of the scaffold were enhanced with increasing concentrations of HA particles and SA hydrogel. Specifically, HA particles blended with the SA hydrogel of core strands enhanced the biological properties of the scaffold by supporting the sheath part of the strand encapsulating osteoblast-like cells. Based on these results, the proposed scaffold design shows great promise for bone-tissue regeneration and engineering applications.

3D Printing of Bone-Mimetic Scaffold Composed of Gelatin/ß-Tri-Calcium Phosphate for Bone Tissue Engineering.

3D printed scaffolds composed of gelatin and ß-tri-calcium phosphate (ß-TCP) as a biomimetic bone material are fabricated, thereby providing an environment appropriate for bone regeneration. The Ca2+ in ß-TCP and COO- in gelatin form a stable electrostatic interaction, and the composite scaffold shows suitable rheological properties for bioprinting. The gelatin/ß-TCP scaffold is crosslinked with glutaraldehyde vapor and unreacted aldehyde groups which can cause toxicity to cells is removed by a glycine washing. The stable binding of the hydrogel is revealed as a result of FTIR and degradation rate. It is confirmed that the composite scaffold has compressive strength similar to that of cancellous bone and 60 wt% ß-TCP groups containing 40 wt% gelatin have good cellular activity with preosteoblasts. Also, in the animal experiments, the gelatin/ß-TCP scaffold confirms to induce bone formation without any inflammatory responses. This study suggests that these fabricated scaffolds can serve as a potential bone substitute for bone regeneration.

Bone regeneration by means of a three-dimensional printed scaffold in a rat cranial defect.

Recently, computer-designed three-dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer-designed 3D scaffold to drive new bone formation in a bone defect. Poly-L-lactide (PLLA) and bioactive ß-tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer-by-layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG-63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full-thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG-63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG-63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG-63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.

In situ gold nanoparticle growth on polydopamine-coated 3D-printed scaffolds improves osteogenic differentiation for bone tissue engineering applications: in vitro and in vivo studies.

In this study, we designed scaffolds coated with gold nanoparticles (GNPs) grown on a polydopamine (PDA) coating of a three-dimensional (3D) printed polycaprolactone (PCL) scaffold. Our results demonstrated that the scaffolds developed here may represent an innovative paradigm in bone tissue engineering by inducing osteogenesis as a means of remodeling and healing bone defects.

The use of heparin chemistry to improve dental osteogenesis associated with implants.

In this study, we designed a hybrid Ti by heparin modifying the Ti surface followed by Growth/differentiation factor-5 (GDF-5) loading. After that, products were characterized by physicochemical analysis. Quantitative analysis of functionalized groups was also confirmed. The release behavior of GDF-5 grafted samples was confirmed for up to 21days. The surface modification process was found to be successful and to effectively immobilize GDF-5 and provide for its sustained release behavior. As an in vitro test, GDF-5 loaded Ti showed significantly enhanced osteogenic differentiation with increased calcium deposition under nontoxic conditions against periodontal ligament stem cells (PDLSc). Furthermore, an in vivo result showed that GDF-5 loaded Ti had a significant influence on new bone formation in a rabbit model. These results clearly confirmed that our strategy may suggest a useful paradigm by inducing osseo-integration as a means to remodeling and healing of bone defects for restorative procedures in dentistry.

Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering.

UNLABELLED: For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7±5ng when treatment with 100ng/ml rhBMP2 and 153.3±2.4ng when treated with 500ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. STATEMENT OF SIGNIFICANCE: Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid, solvent-free method is necessary to improve scaffold generation. Recently, 3D printing such as a rapid prototyping technique has several benefits in that manufacturing is a simple process using computer aided design and scaffolds can be generated without using solvents. In this study, we designed a bio-active scaffold using a very simple and direct method to manufacture DOPA coated 3D PCL porous scaffold grafted with rhBMP2 as a means to create bone-tissue regenerative scaffolds. To our knowledge, our approach can allow for the generation of scaffolds which possessed good properties for use as bone-tissue scaffolds.

Evaluation of small intestine submucosa and poly(caprolactone-co-lactide) conduits for peripheral nerve regeneration.

The present study employed nerve guidance conduits (NGCs) only, which were made of small intestine submucosa (SIS) and poly(caprolactone-co-lactide) (PCLA) to promote nerve regeneration in a peripheral nerve injury (PNI) model with nerve defects of 15 mm. The SIS- and PCLA-NGCs were easily prepared by rolling of a SIS sheet and a bioplotter using PCLA, respectively. The prepared SIS- and PCLA-NGCs fulfilled the general requirement for use as artificial peripheral NGCs such as easy fabrication, reproducibility for mass production, suturability, sterilizability, wettability, and proper mechanical properties to resist collapsing when applied to in vivo implantation. The SIS- and PCLA-NGCs appeared to be well integrated into the host sciatic nerve without causing dislocations and serious inflammation. All NGCs stably maintained their NGC shape for 8 weeks without collapsing, which matched well with the nerve regeneration rate. Staining of the NGCs in the longitudinal direction showed that the regenerated nerves grew successfully from the SIS- and PCLA-NGCs through the sciatic nerve-injured gap and connected from the proximal to distal direction along the NGC axis. SIS-NGCs exhibited a higher nerve regeneration rate than PCLA-NGCs. Collectively, our results indicate that SIS- and PCLA-NGCs induced nerve regeneration in a PNI model, a finding that has significant implications in the future with regard to the feasibility of clinical nerve regeneration with SIS- and PCLA-NGCs prepared through an easy fabrication method using promising biomaterials.

A computer-designed scaffold for bone regeneration within cranial defect using human dental pulp stem cells.

A computer-designed, solvent-free scaffold offer several potential advantages such as ease of customized manufacture and in vivo safety. In this work, we firstly used a computer-designed, solvent-free scaffold and human dental pulp stem cells (hDPSCs) to regenerate neo-bone within cranial bone defects. The hDPSCs expressed mesenchymal stem cell markers and served as an abundant source of stem cells with a high proliferation rate. In addition, hDPSCs showed a phenotype of differentiated osteoblasts in the presence of osteogenic factors (OF). We used solid freeform fabrication (SFF) with biodegradable polyesters (MPEG-(PLLA-co-PGA-co-PCL) (PLGC)) to fabricate a computer-designed scaffold. The SFF technology gave quick and reproducible results. To assess bone tissue engineering in vivo, the computer-designed, circular PLGC scaffold was implanted into a full-thickness cranial bone defect and monitored by micro-computed tomography (CT) and histology of the in vivo tissue-engineered bone. Neo-bone formation of more than 50% in both micro-CT and histology tests was observed at only PLGC scaffold with hDPSCs/OF. Furthermore, the PLGC scaffold gradually degraded, as evidenced by the fluorescent-labeled PLGC scaffold, which provides information to tract biodegradation of implanted PLGC scaffold. In conclusion, we confirmed neo-bone formation within a cranial bone defect using hDPSCs and a computer-designed PLGC scaffold.

Enhanced adhesion of preosteoblasts inside 3D PCL scaffolds by polydopamine coating and mineralization.

In tissue engineering, fabrication of 3D scaffolds with well-defined, inter-connected pores followed by culture of mammalian cells is a typical approach. In practice, however, hydrophobicity of scaffold surfaces is not suitable for cells to be adhered because of poor wettability. Especially, infiltration followed by adhesion of cells inside hydrophobic scaffolds remains as a challenge. Thus, hydrophilic conversions of the surfaces regardless of surface location are critical for success. Herein, a method to enhance infiltration and adhesion of preosteoblasts inside hydrophobic poly(ϵ-caprolactone) (PCL) scaffolds by a bio-inspired, hydroxyapatite formation is demonstrated. The approach can be a general method for controlling hydrophilicity of inner surfaces of scaffolds.

Coaxially electrospun micro/nanofibrous poly(epsilon-caprolactone)/eggshell-protein scaffold.

Eggshell membrane (ESM) has potential as a natural scaffold because of its highly porous structure and good biocompatibility. To mimic its structure and surface properties, soluble eggshell membrane protein (SEP) was extracted from ESM and electrospun with a biodegradable polymer, poly(epsilon-caprolactone) (PCL). SEP/PCL micro/nanofibers were fabricated using a coaxial electrospinning process with a dual nozzle and an auxiliary cylindrical electrode. The fiber web was characterized using the water contact angle, pore-size distribution, mechanical properties and cellular behavior. The SEP/PCL web, which demonstrated the feasibility of producing a scaffold with adequate hydrophilicity, suitable pore size and good mechanical properties compared to a pure PCL micro/nanofiber web, exhibits the ability to mimic a natural biomaterial.

Fabrication of a biocomposite reinforced with hydrophilic eggshell proteins.

Soluble eggshell proteins were used as a reinforcing material of electrospun micro/nanofibers for tissue engineering. A biocomposite composed of poly(epsilon-caprolactone) (PCL) micro/nanofibers and soluble eggshell protein was fabricated with a two-step fabrication method, which is an electrospinning process followed by an air-spraying process. To achieve a stable electrospinning process, we used an auxiliary cylindrical electrode connected with a spinning nozzle. PCL biocomposite was characterized in water contact angle and mechanical properties as well as cell proliferation for its application as a tissue engineering material. It showed an improved hydrophilic characteristic compared with that of a micro/nanofiber web generated from a pure PCL solution using a typical electrospinning process. Moreover, the fabricated biocomposite had good mechanical properties compared to a typical electrospun micro/nanofiber mat. The fabricated biocomposite made human dermal fibroblasts grow better than pure PCL. From the results, the reinforced polymeric micro/nanofiber scaffold can be easily achieved with these modified processes.