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Many finite element (FE) models have been designed based on geometric information from computed tomography (CT) data, and validated via comparison with experimental results for human cadaver ossicular bones. Here, we describe a novel method for developing and analyzing the biomimetic ceramic ossicles (BCO) in combination with 3D printing technology, and we establish an FE model of the BCO for analyzing vibration performance. Novel biomimetic ceramic ossicles (BCO) made of hydroxyapatite (HA) were fabricated using 3D printing technology, and their vibration properties were measured. We created a 3D model of the BCO using computer-aided design, which corresponds to the ossicular structure and geometry, and created an FE model of the human ossicles via a comparison of experimental and simulated vibrations to investigate the characteristics of the ossicular chain. The FE model was established based on the displacements of the malleus, incus, and stapes, which was analyzed using an externally applied vibrational force.

INTRODUCTION AND OBJECTIVE:Organ-on-a-chip is a microengineered biomimetic system containing microfluidic channels and tissue-specific cell types, which replicate functional units of living organs ...

Summary Bioprinting technology has emerged as a powerful tool for building tissue and organ structures in the field of tissue engineering. This technology allows precise placement of cells, biomaterials and biomolecules in spatially predefined locations within confined three-dimensional (3D) structures. Various bioprinting technologies have been developed and utilized for applications in life sciences, ranging from studying cellular mechanisms to constructing tissues and organs for implantation, including heart valve, myocardial tissue, trachea and blood vessels. In this article, we introduce the general principles and limitations of the most widely used bioprinting technologies, including jetting- and extrusion-based systems. Application-based research focused on tissue regeneration is presented, as well as the current challenges that hamper clinical utility of bioprinting technology.

The regeneration of articular cartilage consisting of hyaline cartilage and hydrogel scaffolds has been generally used in tissue engineering. However, success in in vivo studies has been rarely reported. The hydrogel scaffolds implanted into articular cartilage defects are mechanically unstable and it is difficult for them to integrate with the surrounding native cartilage tissue. Therefore, it is needed to regenerate cartilage and bone tissue simultaneously. We developed hybrid scaffolds with hydrogel scaffolds for cartilage tissue and with ceramic scaffolds for bone tissue. For in vivo study, hybrid scaffolds were press-fitted into osteochondral tissue defects in a rabbit knee joints and the cartilage tissue regeneration in blank, hydrogel scaffolds, and hybrid scaffolds was compared. In 12th week after implantation, the histological and immunohistochemical analyses were conducted to evaluate the cartilage tissue regeneration. In the blank and hydrogel scaffold groups, the defects were filled with fibrous tissues and the implanted hydrogel scaffolds could not maintain their initial position; in the hybrid scaffold group, newly generated cartilage tissues were morphologically similar to native cartilage tissues and were smoothly connected to the surrounding native tissues. This study demonstrates hybrid scaffolds containing hydrogel and ceramic scaffolds can provide mechanical stability to hydrogel scaffolds and enhance cartilage tissue regeneration at the defect site.

Pretreatment using various types of biophysical stimuli could provide appropriate potential to cells during construction of the engineered tissue in vitro. We hypothesized that multiple combinations of these biophysical stimuli could enhance osteogenic differentiation in vitro and bone formation in vivo. Cyclic strain, an electromagnetic field, and ultrasound were selected and combined as effective stimuli for osteogenic differentiation using a developed bioreactor. Here we report the experimental evaluation of the osteogenic effects of various combinations of three different biophysical stimuli in vitro and in vivo using human adipose-derived stem cells (ASCs). Osteogenic differentiation of ASCs was accelerated by multiple-combination biophysical stimulation in vitro. However, both single stimulation and double-combination stimulation were sufficient to accelerate bone regeneration in vivo, while the osteogenic marker expression of those groups was not as high as that of triple-combination stimulation in vitro. We inferred from these data that ASCs appropriately differentiated into the osteogenic lineage by biophysical stimulation could be a better option for accelerating bone formation in vivo than relatively undifferentiated or completely differentiated ASCs. Although many questions remain about the mechanisms of combined effects of various biophysical stimuli, this approach could be a more powerful tool for bone tissue regeneration.

Hydrogels offer interesting possibilities in various biomedical applications, including tissue regeneration, drug delivery, and cell therapy, due to excellent biocompatibility and good nutrient and oxygen transportation abilities. However, most of these applications require improvements in mechanical properties and functionalization. In this study, a novel technique for fabricating 3D nanofiber-reinforced hydrogel composites is described as a means of enhancing the strength and durability of hydrogels. The method is based on the layer-by-layer electrospinning of nanofibers on an evenly spread, thin hydrogel solution. A coaxial nozzle was introduced for electrospinning highly wettable ethanol-treated nanofibers. This process enhanced the compatibility between the nanofiber reinforcements and the hydrogel matrix. The compressive strength and stiffness of the resulting nanofiber-reinforced hydrogel composites were enhanced to ∼221% and ∼434% compared to the pure hydrogel, respectively. Moreover, the equilibrium modulus was increased by a factor of nearly 1.73 when the volume fraction of nanofibers was 0.085. It was demonstrated that a 3D nanofiber-reinforced hydrogel composite could be fabricated without the cumbersome stacking of hydrogel-coated fiber meshes. Furthermore, the mechanical properties of the reinforced composites can be modulated by adjusting the volume fraction of nanofibers.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells in adult tissues. Current challenges for the clinical application of MSCs include donor site morbidity, which underscores the need to identify alternative sources of MSCs. This study aimed to explore potential new sources of multipotent MSCs for use in tissue regeneration and the functional restoration of organs.Mixed methods research.Tertiary care center.The authors isolated MSCs from human inferior turbinate tissues discarded during turbinate surgery of 10 patients for nasal obstruction. The expression of surface markers for MSCs was assessed by fluorescence-activated cell sorting. The differentiation potential of human turbinate mesenchymal stromal cells (hTMSCs) was analyzed by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and Western blot analysis.Surface epitope analysis revealed that hTMSCs were negative for CD14, CD19, CD34, and HLA-DR and positive for CD29, CD73, and CD90, representing a characteristic phenotype of MSCs. Extracellular matrices with characteristics of cartilage, bone, and adipose tissue were produced by inducing the chondrogenic, osteogenic, and adipogenic differentiation of hTMSCs, respectively. The expression of neuron-specific markers in hTMSCs was confirmed immunocytochemically.The hTMSCs represent a new source of multipotent MSCs that are potentially applicable to tissue engineering and regenerative medicine. The availability of differentiated adult cells will allow the development of an effective tissue regeneration method.

An important component in tissue engineering is the three-dimensional (3D) scaffold, which guides cells to form target tissue, maintains tissue volume, and provides sufficient structural support during tissue regeneration. However, until recently, conventional scaffold fabrication methods have not satisfied the requirements for tissue regeneration. The development of additive fabrication technologies, known as solid freeform fabrication (SFF), has made it possible to fabricate scaffolds with very fine structures and complex geometries using computer-aided design (CAD) data acquired from medical images of patients. Due to the advantages of SFF technology, it is rapidly becoming the technique of choice for scaffold fabrication. Moreover, recent research has demonstrated that a variety of biomaterials are suitable for use in various SFF systems. This paper reviews the application, advancement, and potential of SFF technologies in the fabrication of scaffolds for tissue regeneration.

Calcium phosphates are very interesting materials for use as scaffolds for bone tissue engineering. These materials include hydroxyapatite (HA) and tricalcium phosphate (TCP), which are inorganic components of human bone tissue and are both biocompatible and osteoconductive. Although these materials have excellent properties for use as bone scaffolds, many researchers have used these materials as additives to synthetic polymer scaffolds for bone tissue regeneration, because they are difficult to manufacture three-dimensional (3D) scaffolds. In this study, we fabricated 3D calcium phosphate scaffolds with the desired inner and outer architectures using solid freeform fabrication technology. To fabricate the scaffold, the sintering behavior was evaluated for various sintering temperatures and slurry concentrations. After the fabrication of the calcium phosphate scaffolds, in-vitro cell proliferation and osteogenic differentiation tests were carried out.

Recently, many groups have researched the reconstruction of bone tissue and the development of bone scaffolds using solid freeform fabrication technology. However, the capacity to produce three-dimensional hydroxyapatite (HA) scaffolds with a very accurate architecture is limited by difficulties in the manufacturing process. In this study, a HA scaffold with an accurate pore size of 300+/[email protected] was fabricated using a microstereolithography (MSTL) system and molding technology. In addition, an agar-overlay test was performed to investigate the cytotoxicity of the fabricated scaffold.

Three-dimensional (3D) bioprinting technology has been utilized as a method to engineer complex tissues and organs. This rapidly growing technology allows for precise placement of multiple types of cells, biomaterials, and biomolecules in spatially predefined locations within 3D structures. Many researchers are focusing on the further development of bioprinting technology and its applications. In this chapter, we introduce the general principles and limitations of widely used bioprinting systems and applications for tissue and organ regeneration. In addition, the current challenges facing the clinical applications of bioprinting technology are addressed.

To regenerate the bone tissue, the fabrication of scaffolds for better tissue regeneration has attracted a great deal of attention. In fact, growth factors are already used in clinical practice and are being investigated for enhancing the capacity for bone tissue regeneration. However, despite their strong osteoinductive activity, these growth factors have several limitations: safety issues, high treatment costs, and the potential for ectopic bone formation. The aim of this study was therefore to develop ceramic scaffolds that could promote the capacity for bone regeneration without growth factors. Three-dimensional ceramic scaffolds were successfully fabricated from hydroxyapatite (HA) and tricalcium phosphate (TCP) using projection-based microstereolithography, which is an additive manufacturing technology. The effects of calcium ions released from ceramic scaffolds on osteogenic differentiation and bone regeneration were evaluated in vitro and in vivo. The osteogenesis-related gene expression and area of new bone formation in the HA/TCP scaffolds was higher than those in the HA scaffolds. Moreover, regenerated bone tissue in HA/TCP scaffolds were more matured than that in HA scaffolds. Through this study, we were able to enhance the bone regeneration capacity of scaffolds not by growth factors but by calcium ions released from the scaffolds. Ceramic scaffolds developed in this study might be useful for enhancing the capacity for regeneration in complex bone defects.

The regeneration of articular cartilage consisting of hyaline cartilage and hydrogel scaffolds has been generally used in tissue engineering. However, success in in vivo studies has been rarely reported. The hydrogel scaffolds implanted into articular cartilage defects are mechanically unstable and it is difficult for them to integrate with the surrounding native cartilage tissue. Therefore, it is needed to regenerate cartilage and bone tissue simultaneously. We developed hybrid scaffolds with hydrogel scaffolds for cartilage tissue and with ceramic scaffolds for bone tissue. For in vivo study, hybrid scaffolds were press-fitted into osteochondral tissue defects in a rabbit knee joints and the cartilage tissue regeneration in blank, hydrogel scaffolds, and hybrid scaffolds was compared. In 12th week after implantation, the histological and immunohistochemical analyses were conducted to evaluate the cartilage tissue regeneration. In the blank and hydrogel scaffold groups, the defects were filled with fibrous tissues and the implanted hydrogel scaffolds could not maintain their initial position; in the hybrid scaffold group, newly generated cartilage tissues were morphologically similar to native cartilage tissues and were smoothly connected to the surrounding native tissues. This study demonstrates hybrid scaffolds containing hydrogel and ceramic scaffolds can provide mechanical stability to hydrogel scaffolds and enhance cartilage tissue regeneration at the defect site.

An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration.

The online version of the original article can be found at http://dx.doi.org/10.1007/s12541-016-0198-2

Fabrication of three-dimensional (3D) scaffolds with appropriate mechanical properties and desired architecture for promoting cell growth and new tissue formation is one of the most important efforts in tissue engineering field. Scaffolds fabricated from bioactive ceramic materials such as hydroxyapatite and tricalcium phosphate show promise because of their biological ability to support bone tissue regeneration. However, the use of ceramics as scaffold materials is limited because of their inherent brittleness and difficult processability. The aim of this study was to create robust ceramic scaffolds, which have a desired architecture. Such scaffolds were successfully fabricated by projection-based microstereolithography, and dilatometric analysis was conducted to study the sintering behavior of the ceramic materials. The mechanical properties of the scaffolds were improved by infiltrating them with a polycaprolactone solution. The toughness and compressive strength of these ceramic/polymer scaffolds were about twice those of ceramic scaffolds. Furthermore, the osteogenic gene expression on ceramic/polymer scaffolds was better than that on ceramic scaffolds. Through this study, we overcame the limitations of previous research on fabricating ceramic scaffolds and these new robust ceramic scaffolds may provide a much improved 3D substrate for bone tissue regeneration.

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell-based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose-derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor β, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three-dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short-term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment.

Scaffolds play an important role in the regeneration of artificial tissues or organs. A scaffold is a porous structure with a micro-scale inner architecture in the range of several to several hundreds of micrometers. Therefore, computer-aided construction of scaffolds should provide sophisticated functionality for porous structure design and a tool path generation strategy that can achieve micro-scale architecture. In this study, a new unit cell-based computer-aided manufacturing (CAM) system was developed for the automated design and fabrication of a porous structure with micro-scale inner architecture that can be applied to composite tissue regeneration. The CAM system was developed by first defining a data structure for the computing process of a unit cell representing a single pore structure. Next, an algorithm and software were developed and applied to construct porous structures with a single or multiple pore design using solid freeform fabrication technology and a 3D tooth/spine computer-aided design model. We showed that this system is quite feasible for the design and fabrication of a scaffold for tissue engineering.

Poly(propylene fumarate) (PPF) is an ultraviolet-curable and biodegradable polymer with potential applications for bone regeneration. In this study, we designed and fabricated three-dimensional (3D) porous scaffolds based on a PPF polymer network using micro-stereolithography (MSTL). The 3D scaffold was well fabricated with a highly interconnected porous structure and porosity of 65%. These results provide a new scaffold fabrication method for tissue engineering. Surface modification is a commonly used and effective method for improving the surface characteristics of biomaterials without altering their bulk properties that avoids the expense and long time associated with the development of new biomaterials. Therefore, we examined surface modification of 3D scaffolds by applying accelerated biomimetic apatite and arginine-glycine-aspartic acid (RGD) peptide coating to promote cell behavior. The apatite coating uniformly covered the scaffold surface after immersion for 24 h in 5-fold simulated body fluid (5SBF) and then the RGD peptide was applied. Finally, the coated 3D scaffolds were seeded with MC3T3-E1 pre-osteoblasts and their biologic properties were evaluated using an MTS assay and histologic staining. We found that 3D PPF/diethyl fumarate (DEF) scaffolds fabricated with MSTL and biomimetic apatite coating can be potentially used in bone tissue engineering.

Three-dimensional integrated organ printing (IOP) technology seeks to fabricate tissue constructs that can mimic the structural and functional properties of native tissues. This technology is particularly useful for complex tissues such as those in the musculoskeletal system, which possess regional differences in cell types and mechanical properties. Here, we present the use of our IOP system for the processing and deposition of four different components for the fabrication of a single integrated muscle-tendon unit (MTU) construct. Thermoplastic polyurethane (PU) was co-printed with C2C12 cell-laden hydrogel-based bioink for elasticity and muscle development on one side, while poly(ϵ-caprolactone) (PCL) was co-printed with NIH/3T3 cell-laden hydrogel-based bioink for stiffness and tendon development on the other. The final construct was elastic on the PU-C2C12 muscle side (E = 0.39 ± 0.05 MPa), stiff on the PCL-NIH/3T3 tendon side (E = 46.67 ± 2.67 MPa) and intermediate in the interface region (E = 1.03 ± 0.14 MPa). These constructs exhibited >80% cell viability at 1 and 7 d after printing, as well as initial tissue development and differentiation. This study demonstrates the versatility of the IOP system to create integrated tissue constructs with region-specific biological and mechanical characteristics for MTU engineering.

Scaffold fabrication using solid freeform fabrication (SFF) technology is a hot topic in tissue engineering. Here, we present a new indirect SFF technology based on microstereolithography (MSTL), which has the highest resolution of all SFF methods, to construct a three-dimensional (3D) porous scaffold by combining SFF with molding technology. To realize this indirect method, we investigated and modified a water-soluble photopolymer. We used MSTL technology to fabricate a high-resolution 3D porous mold composed of the modified polymer. The mold can be removed using an appropriate solvent. We tested two materials, polycaprolactone and calcium sulfate hemihydrate, using the molding process, and developed a lost-mold shape forming process by dissolving the mold. This procedure demonstrated that the proposed method can yield scaffold pore sizes as small as 60–70 µm. In addition, cytotoxicity test results indicated that the proposed process is feasible for producing 3D porous scaffolds.