4D Cell-Condensate Bioprinting.

4D bioprinting techniques that facilitate formation of shape-changing scaffold-free cell condensates with prescribed geometries have yet been demonstrated. Here, a simple 4D bioprinting approach is presented that enables formation of a shape-morphing cell condensate-laden bilayer system. The strategy produces scaffold-free cell condensates which morph over time into predefined complex shapes. Cell condensate-laden bilayers with specific geometries are readily fabricated by bioprinting technologies. The bilayers have tunable deformability and microgel (MG) degradation, enabling controllable morphological transformations and on-demand liberation of deformed cell condensates. With this system, large cell condensate-laden constructs with various complex shapes are obtained. As a proof-of-concept study, the formation of the letter "C"- and helix-shaped robust cartilage-like tissues differentiated from human mesenchymal stem cells (hMSCs) is demonstrated. This system brings about a versatile 4D bioprinting platform idea that is anticipated to broaden and facilitate the applications of cell condensation-based 4D bioprinting.

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.

Chitosan/polyurethane blended fiber sheets containing silver sulfadiazine for use as an antimicrobial wound dressing.

Electrospun chitosan (CTS) nanofibers have been well known for use as a wound dressing in the biomedical field. Nevertheless, fatal bacterial infections are still a serious problem when CTS nanofibers are used for wound treatment. In this study, we designed a novel wound dressing based on blending the chitosan with polyurethane (CTS/PU) containing silver sulfadiazine (AgSD) in order to enhance both antibacterial activity and mechanical strength. This fiber sheet was produced using the electrospinning (ELSP) technique. The CTS/PU containing AgSD fiber sheet was characterized by energy-dispersive X-ray spectroscopy (EDX). The physicochemical properties of the CTS/PU/AgSD fiber sheets were also characterized by thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). The electrospun fibers were morphologically characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For an in vitro evaluation, the CTS/PU/AgSD fiber sheets were tested for their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa), gram-positive Staphylococcus aureus (S. aureus) and Methicillin-resistant Staphylococcus aureus (MRSA). The results indicate that CTS/PU/AgSD fiber sheets have strong antimicrobial activity as displayed by inhibition of bacterial growth and prevention of infection during the healing process. These results indicate that this material would be good for use as a wound dressing material.

Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration.

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target-specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor-1α (SDF-1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α-smooth muscle actin, and c-kit demonstrated that this system significantly increased the number of stem cell-like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.

Characteristics of novel root-end filling material using epoxy resin and Portland cement.

OBJECTIVES: The aim of this study was to evaluate the physical properties and cytotoxicity of a novel root-end filling material (EPC) which is made from epoxy resin and Portland cement as a mineral trioxide aggregate (MTA) substitute. MATERIALS AND METHODS: EPC, developed as a root-end filling material, was compared with MTA and a mixture of AH Plus sealer and MTA (AMTA) with regard to the setting time, radio-opacity, and microleakage. Setting times were evaluated using Vicat apparatus. Digital radiographs were taken to evaluate the aluminium equivalent radio-opacity using an aluminium step wedge. Extracted single-rooted teeth were used for leakage test using methylene blue dye. After canal shaping and obturation, the apical 3-mm root was resected, and a root-end cavity with a depth of 3 mm was prepared. The root-end cavities were filled with MTA, AMTA, and EPC for 15 specimens in each of three groups. After setting in humid conditions for 24 h, the specimens were tested for apical leakage. For evaluation of the biocompatibility of EPC, cell (human gingival fibroblast) viability was compared for MTA and Portland cement by MTT assay, and cell morphological changes were compared for MTA and AH Plus by fluorescence microscopy using DAPI and F-actin staining. The setting time, radio-opacity, and microleakage were compared using one-way ANOVA and Scheffe's post hoc comparison, and the cytotoxicity was compared using the nonparametric Kruskal-Wallis rank sum test. Statistical significance was set at 95%. RESULTS: EPC had a shorter setting time and less microleakage compared with MTA (p < 0.05). EPC showed 5-mm aluminium thickness radio-opacity and similar biocompatibility to MTA. CONCLUSIONS: Under the conditions of this study, EPC, a novel composite made from a mixture of epoxy resin and Portland cement, was found to be a useful material for root-end filling, with favourable radio-opacity, short setting time, low microleakage, and clinically acceptable low cytotoxicity. CLINICAL RELEVANCE: The novel root-end filling material would be a potentially useful material for a surgical endodontic procedure with favourable properties.

Translational tissue-engineered vascular grafts: From bench to bedside.

Cardiovascular disease is a primary cause of mortality worldwide, and patients often require bypass surgery that utilizes autologous vessels as conduits. However, the limited availability of suitable vessels and the risk of failure and complications have driven the need for alternative solutions. Tissue-engineered vascular grafts (TEVGs) offer a promising solution to these challenges. TEVGs are artificial vascular grafts made of biomaterials and/or vascular cells that can mimic the structure and function of natural blood vessels. The ideal TEVG should possess biocompatibility, biomechanical mechanical properties, and durability for long-term success in vivo. Achieving these characteristics requires a multi-disciplinary approach involving material science, engineering, biology, and clinical translation. Recent advancements in scaffold fabrication have led to the development of TEVGs with improved functional and biomechanical properties. Innovative techniques such as electrospinning, 3D bioprinting, and multi-part microfluidic channel systems have allowed the creation of intricate and customized tubular scaffolds. Nevertheless, multiple obstacles must be overcome to apply these innovations effectively in clinical practice, including the need for standardized preclinical models and cost-effective and scalable manufacturing methods. This review highlights the fundamental approaches required to successfully fabricate functional vascular grafts and the necessary translational methodologies to advance their use in clinical practice.

The expression kinetics of CD137 in chronic hepatitis C patients treated with pegylated-interferon and ribavirin.

OBJECTIVE: CD137, a member of the tumor necrosis factor receptor family, generates co-stimulatory signals leading to T-cell activation and proliferation for viral eradication. We examined the expression kinetics of CD137 to validate whether it can affect treatment outcomes of chronic hepatitis C (CHC) patients. METHODS: The expression of CD137 on peripheral blood mononuclear cells (PBMC) from 50 CHC patients and 20 healthy controls was analyzed by flow cytometry. CD137 expression levels were examined before treatment, and every 4 weeks during treatment until week 24 or 48, and at the 24-week follow-up. RESULTS: CD137 expression on PBMC was significantly lower in CHC patients than controls (15.5 ± 7.8% vs 23.4 ± 5.2%; p < 0.05). Patients with sustained virological response (SVR) showed higher level of CD137 expression on PBMC than treatment failures at week 4 (20.11% vs 10.97%; p < 0.05) and week 12 (15.48% vs 5.74%; p < 0.01). CD137 expression on CD4 T cells was also higher in patients with SVR at week 8 (7.75% vs 3.29%; p < 0.05). CD137 expression on PBMC from patients with SVR recovered to the control level at the 24-week follow-up. In multivariate analysis, the increased expression of CD137 at week 4 and genotype non-1 were significantly associated with SVR. CONCLUSIONS: The increased expression of CD137 within 12 weeks after the initiation of interferon therapy might be associated with a successful treatment outcome. Modulation to improve expression of CD137 might improve efficacy of CHC treatment.

Characteristics of nano-sized yttria powder synthesized by a polyvinyl alcohol solution route at low temperature.

Nano-sized yttria (Y2O3) powders were successfully synthesized at a low temperature of 400 degrees C by a simple polymer solution route. PVA polymer, as an organic carrier, contributed to an atom-scale homogeneous precursor gel and it resulted in fully crystallized, nano-sized yttria powder with high specific surface area through the low temperature calcination. In this process, the content of PVA, calcination temperature and heating time affected the microstructure and crystallization behavior of the powders. The development of crystalline phase and the final particle size were strongly dependant on the oxidation reaction from the polymer burn-out step and the PVA content. In this paper, the PVA solution technique for the fabrication of nano-sized yttria powders is introduced. The effects of PVA content and holding time on the powder morphology and powder specific surface area are also studied. The characterization of the synthesized powders is examined by using XRD, DTA/TG, SEM, TEM and nitrogen gas adsorption. The yttria powder synthesized from the PVA content of 3:1 ratio and calcined at 400 degrees C had a crystallite size of about 20 nm or less with a high surface areas of 93.95-120.76 m2 g(-1).

The comprehensive on-demand 3D bio-printing for composite reconstruction of mandibular defects.

BACKGROUND: The mandible is a functional bio-organ that supports facial structures and helps mastication and speaking. Large mandible defects, generally greater than 6-cm segment loss, may require composite tissue reconstruction such as osteocutaneous-vascularized free flap which has a limitation of additional surgery and a functional morbidity at the donor site. A 3D bio-printing technology is recently developed to overcome the limitation in the composite reconstruction of the mandible using osteocutaneous-vascularized free flap. REVIEW: Scaffold, cells, and bioactive molecules are essential for a 3D bio-printing. For mandibular reconstruction, materials in a 3D bio-printing require mechanical strength, resilience, and biocompatibility. Recently, an integrated tissue and organ printing system with multiple cartridges are designed and it is capable of printing polymers to reinforce the printed structure, such as hydrogel. CONCLUSION: For successful composite tissue reconstruction of the mandible, biologic considerations and components should be presented with a comprehensive on-demand online platform model of customized approaches.

An open-source bioink database for microextrusion 3D printing.

3D printing has rapidly become a critical enabling technology in tissue engineering and regenerative medicine for the fabrication of complex engineered tissues. 3D bioprinting, in particular, has advanced greatly to facilitate the incorporation of a broad spectrum of biomaterials along with cells and biomolecules of interest forin vitrotissue generation. The increasing complexity of novel bioink formulations and application-dependent printing conditions poses a significant challenge for replicating or innovating new bioprinting strategies. As the field continues to grow, it is imperative to establish a cohesive, open-source database that enables users to search through existing 3D printing formulations rapidly and efficiently. Through the efforts of the NIH/NIBIB Center for Engineering Complex Tissues, we have developed, to our knowledge, the first bioink database for extrusion-based 3D printing. The database is publicly available and allows users to search through and easily access information on biomaterials and cells specifically used in 3D printing. In order to enable a community-driven database growth, we have established an open-source portal for researchers to enter their publication information for addition into the database. Although the database has a broad range of capabilities, we demonstrate its utility by performing a comprehensive analysis of the printability domains of two well-established biomaterials in the printing world, namely poly(ϵ-caprolactone) and gelatin methacrylate. The database allowed us to rapidly identify combinations of extrusion pressure, temperature, and speed that have been used to print these biomaterials and more importantly, identify domains within which printing was not possible. The data also enabled correlation analysis between all the printing parameters, including needle size and type, that exhibited compatibility for cell-based 3D printing. Overall, this database is an extremely useful tool for the 3D printing and bioprinting community to advance their research and is an important step towards standardization in the field.

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin.

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.

Highly sinterable calcium phosphate fabricated by using starfish bone.

Nano-sized calcium phosphate powders were simply synthesized by using dried starfish bone. The calcined bone was mixed with phosphoric acid and the dried mixtures were heated for synthesis. The powder compacts were fully sintered at 1100 degrees C for 1 h. The densified samples showed CaO-free calcium phosphate phase and non-uniform, over sized grains.

Cell-Laden Multiple-Step and Reversible 4D Hydrogel Actuators to Mimic Dynamic Tissue Morphogenesis.

Shape-morphing hydrogels bear promising prospects as soft actuators and for robotics. However, they are mostly restricted to applications in the abiotic domain due to the harsh physicochemical conditions typically necessary to induce shape morphing. Here, multilayer hydrogel actuator systems are developed using biocompatible and photocrosslinkable oxidized, methacrylated alginate and methacrylated gelatin that permit encapsulation and maintenance of living cells within the hydrogel actuators and implement programmed and controlled actuations with multiple shape changes. The hydrogel actuators encapsulating cells enable defined self-folding and/or user-regulated, on-demand-folding into specific 3D architectures under physiological conditions, with the capability to partially bioemulate complex developmental processes such as branching morphogenesis. The hydrogel actuator systems can be utilized as novel platforms for investigating the effect of programmed multiple-step and reversible shape morphing on cellular behaviors in 3D extracellular matrix and the role of recapitulating developmental and healing morphogenic processes on promoting new complex tissue formation.

Transparent, Water-Repellent, Antiviral, Antistatic, and Flexible Cu-Plasma-Polymerized Fluorocarbon Nanocomposite Thin Films.

Polymer thin films containing fluorine are attracting much attention in various high-tech industries owing to their transparency, flexibility, and excellent water repellency. However, the generation of static electricity due to high electrical resistance limits their application. In this study, highly transparent and flexible Cu-plasma-polymerized fluorocarbon (PPFC) nanocomposite thin films that exhibit hydrophobicity and antistatic properties are proposed. These films, obtained using the mid-range frequency sputtering, exhibited a light transmittance of 84.2%, a water contact angle of 94.6°, and a sheet resistance of 1.2 × 1012 Ω/□. Transmission electron microscopy and small angle X-ray scattering confirmed that Cu nanoparticles with an average size of 4-5 nm were distributed uniformly in the PPFC matrix. In repeated fatigue bending tests, the Cu-PPFC nanocomposite thin films exhibited excellent mechanical robustness and flexibility. Antiviral properties of the Cu-PPFC nanocomposite thin films were evaluated against influenza A virus, and the number decreased by 96.9% after 30 min. Carbon nanotube-Cu-polytetrafluoroethylene composite targets are advantageous for large-area coating and mass production because they can be applied in large-area sputtering and roll-to-roll processes. The transparency, charging characteristics, and water repellency can be easily controlled in Cu-PPFC nanocomposite thin films by controlling the sputtering power density according to the required product. Therefore, these films can be applied in various industries such as flexible displays, medical, automobiles, functional textiles, and aerospace.

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.

Self-Assembling Peptide Solution Accelerates Hemostasis.

Objective: One of the leading causes of death following traumatic injury is exsanguination. Biological material-based hemostatic agents such as fibrin, thrombin, and albumin have a high risk for causing infection. Synthetic peptide-based hemostatic agents offer an attractive alternative. The objective of this study is to explore the potential of h9e peptide as an effective hemostatic agent in both in vitro and in vivo models. Approach:In vitro blood coagulation kinetics in the presence of h9e peptide was determined as a function of gelation time using a dynamic rheometer. In vivo hemostatic effects were studied using the Wistar rat model. Results were compared to those of the commercial hemostatic product Celox™, a chitosan-based product. Adhesion of h9e peptide was evaluated using the platelet adhesion test. Biocompatibility of h9e peptide was studied in vivo using a mouse model. Results: After h9e peptide solution was mixed with blood, gelation started immediately, increased rapidly with time, and reached more than 100 Pa within 3 s. Blood coagulation strength increased as h9e peptide wt% concentration increased. In the rat model, h9e peptide solution at 5% weight concentration significantly reduced both bleeding time and blood loss, outperforming Celox. Preliminary pathological studies indicate that h9e peptide solution is biocompatible and did not have negative effects when injected subcutaneously in a mouse model. Innovation: For the first time, h9e peptide was found to have highly efficient hemostatic effects by forming nanoweb-like structures, which act as a preliminary thrombus and a surface to arrest bleeding 82% faster compared to the commercial hemostatic agent Celox. Conclusion: This study demonstrates that h9e peptide is a promising hemostatic biomaterial, not only because of its greater hemostatic effect than commercial product Celox but also because of its excellent biocompatibility based on the in vivo mouse model study.

Romosozumab in Postmenopausal Korean Women with Osteoporosis: A Randomized, Double-Blind, Placebo-Controlled Efficacy and Safety Study.

BACKGROUND: This phase 3 study evaluated the efficacy and safety of 6-month treatment with romosozumab in Korean postmenopausal women with osteoporosis. METHODS: Sixty-seven postmenopausal women with osteoporosis (bone mineral density [BMD] T-scores ≤-2.5 at the lumbar spine, total hip, or femoral neck) were randomized (1:1) to receive monthly subcutaneous injections of romosozumab (210 mg; n=34) or placebo (n=33) for 6 months. RESULTS: At month 6, the difference in the least square (LS) mean percent change from baseline in lumbar spine BMD (primary efficacy endpoint) between the romosozumab (9.5%) and placebo (-0.1%) groups was significant (9.6%; 95% confidence interval, 7.6 to 11.5; P<0.001). The difference in the LS mean percent change from baseline was also significant for total hip and femoral neck BMD (secondary efficacy endpoints). After treatment with romosozumab, the percent change from baseline in procollagen type 1 N-terminal propeptide transiently increased at months 1 and 3, while that in C-terminal telopeptide of type 1 collagen showed a sustained decrease. No events of cancer, hypocalcemia, injection site reaction, positively adjudicated atypical femoral fracture or osteonecrosis of the jaw, or positively adjudicated serious cardiovascular adverse events were observed. At month 9, 17.6% and 2.9% of patients in the romosozumab group developed binding and neutralizing antibodies, respectively. CONCLUSION: Treatment with romosozumab for 6 months was well tolerated and significantly increased lumbar spine, total hip, and femoral neck BMD compared with placebo in Korean postmenopausal women with osteoporosis (ClinicalTrials.gov identifier NCT02791516).

3D Printing and NIR Fluorescence Imaging Techniques for the Fabrication of Implants.

Three-dimensional (3D) printing technology holds great potential to fabricate complex constructs in the field of regenerative medicine. Researchers in the surgical fields have used 3D printing techniques and their associated biomaterials for education, training, consultation, organ transplantation, plastic surgery, surgical planning, dentures, and more. In addition, the universal utilization of 3D printing techniques enables researchers to exploit different types of hardware and software in, for example, the surgical fields. To realize the 3D-printed structures to implant them in the body and tissue regeneration, it is important to understand 3D printing technology and its enabling technologies. This paper concisely reviews 3D printing techniques in terms of hardware, software, and materials with a focus on surgery. In addition, it reviews bioprinting technology and a non-invasive monitoring method using near-infrared (NIR) fluorescence, with special attention to the 3D-bioprinted tissue constructs. NIR fluorescence imaging applied to 3D printing technology can play a significant role in monitoring the therapeutic efficacy of 3D structures for clinical implants. Consequently, these techniques can provide individually customized products and improve the treatment outcome of surgeries.

The effect of BMP-mimetic peptide tethering bioinks on the differentiation of dental pulp stem cells (DPSCs) in 3D bioprinted dental constructs.

The goal of this study was to use 3D bioprinting technology to create a bioengineered dental construct containing human dental pulp stem cells (hDPSCs). To accomplish this, we first developed a novel bone morphogenetic protein (BMP) peptide-tethering bioink formulation and examined its rheological properties, its printability, and the structural stability of the bioprinted construct. Second, we evaluated the survival and differentiation of hDPSCs in the bioprinted dental construct by measuring cell viability, proliferation, and gene expression, as well as histological and immunofluorescent analyses. Our results showed that the peptide conjugation into the gelatin methacrylate-based bioink formulation was successfully performed. We determined that greater than 50% of the peptides remained in the bioprinted construct after three weeks in vitro cell culture. Human DPSC viability was >90% in the bioprinted constructs immediately after the printing process. Alizarin Red staining showed that the BMP peptide construct group exhibited the highest calcification as compared to the growth medium, osteogenic medium, and non-BMP peptide construct groups. In addition, immunofluorescent and quantitative reverse transcription-polymerase chain reaction analyses showed robust expression of dentin sialophosphoprotein and osteocalcin in the BMP peptide dental constructs. Together, these results strongly suggested that BMP peptide-tethering bioink could accelerate the differentiation of hDPSCs in 3D bioprinted dental constructs.

A 3D-printed polycaprolactone/ß-tricalcium phosphate mandibular prosthesis: A pilot animal study.

OBJECTIVE: In this study, we assessed the effectiveness of a tonsil-derived mesenchymal stem cell (TMSC)-transplanted polycaprolactone/beta-tricalcium phosphate prosthesis (specifically designed for easier fixing and grafting with a single scaffold) on rabbit mandible osteogenesis. METHODS: The mandibles of 18 rabbits were exposed, and 10 × 8-mm bone defects were made. Two rabbits did not receive implants; four were reconstructed with the scaffold control (SC) (SC group); four were reconstructed with scaffolds soaked in peripheral blood (PB) (PB group); four were reconstructed with TMSC-transplanted scaffolds (TMSC group); and four were reconstructed with differentiated osteocyte-transplanted scaffolds (DOC) (DOC group). Each rabbit was sacrificed 12 weeks after surgery, and the area of new bone formation was investigated by mechanical testing, histology, and micro-computed tomography. RESULTS: More extended and denser new bone masses were observed in the TMSC and DOC groups, although fibrosis and vascular formation levels were similar in all groups, suggesting that the dual-structured scaffold alone provides a good environment for bone attachment and regeneration. The bone volumes of representative scaffolds from the SC, PB, TMSC, and DOC groups were 43.12, 48.35, 53.10, and 57.44% of the total volumes, respectively. CONCLUSION: The design of the scaffold resulted in effective osteogenesis, and TMSCs showed osteogenic potency, indicating that their combination could enable effective bone regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 130:358-366, 2020.