Three-dimensional Scaffolds Research Articles

Evaluation of innovative polyvinyl alcohol/ alginate/ green palladium nanoparticles composite scaffolds: Effect on differentiated human dental pulp stem cells into osteoblasts

Three-dimensional (3D) scaffolds are attracting great concern for bone tissue engineering applications. However, selecting an appropriate material with optimal physical, chemical, and mechanical properties is considered a great challenge. The green synthesis approach is essential to avoid the production of harmful by-products through textured construction, sustainable, and eco-friendly procedures. This work aimed at the implementation of natural green synthesized metallic nanoparticles for the development of composite scaffolds for dental applications. In this study, innovative hybrid scaffolds of polyvinyl alcohol/alginate (PVA/Alg) composite loaded with various concentrations of green palladium nanoparticles (Pd NPs) have been synthesized. Various characteristic analysis techniques were used to investigate the synthesized composite scaffold's properties. The SEM analysis revealed impressive microstructure of the synthesized scaffolds dependent on the Pd NPs concentration. The results confirmed the positive effect of Pd NPs doping on the sample stability over time. The synthesized scaffolds were characterized by the oriented lamellar porous structure. The results confirmed the shape stability, without pores breakdown during the drying process. The XRD analysis confirmed that doping with Pd NPs does not affect the crystallinity degree of the PVA/Alg hybrid scaffolds. The mechanical properties results (up to 50 MPa) confirmed the remarkable effect of Pd NPs doping and its concentration on the developed scaffolds. The MTT assay results showed that the incorporation of Pd NPs into the nanocomposite scaffolds is necessary for increasing cell viability. According to the SEM results, the scaffolds with Pd NPs provided the differentiated grown osteoblast cells with enough mechanical support and stability and the cells had a regular form and were highly dense. In conclusion, the synthesized composite scaffolds expressed suitable biodegradable, osteoconductive properties, and the ability to construct 3D structures for bone regeneration, making them a potential option for treating critical deficiencies of bone.

Microfluidic Fabrication of Natural Polymer-Based Scaffolds for Tissue Engineering Applications: A Review

Natural polymers, thanks to their intrinsic biocompatibility and biomimicry, have been largely investigated as scaffold materials for tissue engineering applications. Traditional scaffold fabrication methods present several limitations, such as the use of organic solvents, the obtainment of a non-homogeneous structure, the variability in pore size and the lack of pore interconnectivity. These drawbacks can be overcome using innovative and more advanced production techniques based on the use of microfluidic platforms. Droplet microfluidics and microfluidic spinning techniques have recently found applications in the field of tissue engineering to produce microparticles and microfibers that can be used as scaffolds or as building blocks for three-dimensional structures. Compared to standard fabrication technologies, microfluidics-based ones offer several advantages, such as the possibility of obtaining particles and fibers with uniform dimensions. Thus, scaffolds with extremely precise geometry, pore distribution, pore interconnectivity and a uniform pores size can be obtained. Microfluidics can also represent a cheaper manufacturing technique. In this review, the microfluidic fabrication of microparticles, microfibers and three-dimensional scaffolds based on natural polymers will be illustrated. An overview of their applications in different tissue engineering fields will also be provided.

Well-aligned three-dimensional silk fibroin protein scaffold for orientation regulation of cells

The similar characteristics of biomaterials to the extracellular matrix are essential for efficient tissue repair through dictating cell behaviors. But the scaffold fabrication with complex shapes and controlled alignment have proven to be a difficult task. Herein, a well-designed three-dimensional silk fibroin scaffold is fabricated through ice template technology. The effect of the silk fibroin protein concentration and the freezing temperature on the microstructure and mechanical properties of scaffolds are investigated systematically. Cells behavior mediated by the obtained silk fibroin scaffolds is detected. The results show that the protein concentration plays a vital role in microstructure and scaffold strength. A well-aligned scaffold can be obtained when silk fibroin solution is kept at 12 wt%, which holds the highest mechanical properties. The pore size can be further adjusted in the range of 5–80 µm by changing the freezing temperature from −60 to −196 °C. The well-oriented scaffold with the appropriate pore size of 10–20 µm has the best ability to guide cell alignment. The resulting scaffolds provide an excellent matrix to guide cells behaviors and have a potential application in tissue engineering.

Additive manufacturing and characterization of mathematically designed bone scaffolds based on triply periodic minimal surface lattices

Lattice structures are widely employed in bone scaffolds to obtain high porosity with interconnected pores and high surface area-to-volume and strength-to-weight ratios. Here, 21 lattice-based scaffold architectures were modeled using implicit functions and additively manufactured using epoxidized soybean oil-based resin. The porosity of the scaffolds ranged from 53.42% ± 1.16% to 98.28% ± 0.75%. Scaffolds with the lowest overall porosity (Group 1) exhibited the highest compressive strength. The compressive strength of eighteen different three-dimensional scaffold designs was compatible with that of human trabecular bone. The results and methodology presented here can facilitate the mathematical design of complex porous scaffolds for bone tissue engineering.

Preliminary study on a novel biological scaffold loaded with Apelin-13 sustained-release microcapsules for promoting fallopian tube recanalization in rabbits.

Tubal factor infertility severely impairs the natural fertility of women, and there is for genuine tubal recanalization, including restoration of both the anatomy and function of the diseased fallopian tubes. Currently, there is no effective treatment available. This study aims to explore methods for promoting the repair and recanalization of fallopian tubes from these 2 aspects. Apelin-13 sustained-release microspheres and poly (lactic-co-glycolic acid) (PLGA) three-dimensional (3D) biodegradable scaffolds were prepared. The basic characteristics and in vivo degradation (mass loss rate) of the biodegradable scaffolds were tested, along with the in vitro drug release (cumulative release rate), the in vivo drug release (Apelin-13 plasma concentration), and in vitro degradation (degradation rate) of the microspheres. The Apelin-13 microspheres (microsphere group)/PLGA 3D scaffolds loaded with Apelin-13 sustained-release microspheres (scaffold-microcapsule group) were injected/placed into the fallopian tubes of New Zealand rabbit of chronic salpingitis models. The patency, microscopic structure, and positive expression of estrogen receptor and progesterone receptor of the fallopian tubes in the control group, the model group, the microcapsule group, and the scaffold-microcapsule group was observed and compared. At the 4th week post-operation, the mass loss rate of the PLGA 3D scaffolds, the degradation rate of the microspheres, and the Apelin-13 sustained-release microspheres-generated cumulative release rate in vitro over 30 days were 98.66%, 70.58%, and 98.68% respectively. The plasma concentration of Apelin-13 reached its peak within 5 days and remained stable for 25 days. Compared with the model and microsphere groups, the scaffold-microsphere group showed a milder inflammatory reaction within the tubal lumen, a higher rate of fallopian tube patency, and higher expression levels of estrogen and progesterone receptors (all P<0.05). The indicators of the scaffold-microsphere group were close to those of the control group. The PLGA 3D scaffolds loaded with Apelin-13 sustained-release microspheres can comprehensively repair the anatomical structure and physiological function of the fallopian tubes and hold promise for truly effective tubal recanalization.

Porous surface with fusion peptides embedded in strontium titanate nanotubes elevates osteogenic and antibacterial activity of additively manufactured titanium alloy

It is still a big challenge in orthopedics to treat infected bone defects properly using medical metals. The use of three-dimensional (3D) scaffold materials that simultaneously mimic the skeletal hierarchy and induce sustainable osteogenic and antibacterial functions are a promising solution with an increasing appeal. In this study, we first designed a bifunctional fusion peptide (HHC36-RGD, HR) by linking antimicrobial peptide (HHC36) and arginine-glycine-aspartate (RGD) peptide via 6-aminohexanoic acid. Then the 3D scaffold was fabricated by additive manufacturing, and the strontium titanate nanotube structure (3D-STN) was constructed on its surface. Finally, the HR was anchored to the 3D-STN with the aid of polydopamine (PDA, P), forming the 3D-STN-P-HR scaffold. The results showed that the scaffold exhibited an ordered 3D porous structure, and that the surface was covered by a dense HHC36-RGD layer. Expectedly, the adsorption of PDA effectively slowed down the release of HR. Moreover, the functionalized scaffold had a significant inhibitory effect on Staphylococcus aureus and Escherichia coli, and its antibacterial rate could reach more than 95%. The results of in vitro cell culture experiments demonstrated that the 3D-STN-P-HR scaffold possessed excellent cytocompatibility and could promote the transcription of osteogenic differentiation-related genes and the expression of related proteins. In conclusion, the functionally modified 3D porous titanium alloy scaffold (3D-STN-P-HR) has a balanced antibacterial and osteogenic function, which bodes well for future potential in the customized functional reconstruction of complex-shaped infected bone defects.

Poly(lactic acid) and Nanocrystalline Cellulose Methacrylated Particles for Preparation of Cryogelated and 3D-Printed Scaffolds for Tissue Engineering.

Different parts of bones possess different properties, such as the capacity for remodeling cell content, porosity, and protein composition. For various traumatic or surgical tissue defects, the application of tissue-engineered constructs seems to be a promising strategy. Despite significant research efforts, such constructs are still rarely available in the clinic. One of the reasons is the lack of resorbable materials, whose properties can be adjusted according to the intended tissue or tissue contacts. Here, we present our first results on the development of a toolbox, by which the scaffolds with easily tunable mechanical and biological properties could be prepared. Biodegradable poly(lactic acid) and nanocrystalline cellulose methacrylated particles were obtained, characterized, and used for preparation of three-dimensional scaffolds via cryogelation and 3D printing approaches. The composition of particles-based ink for 3D printing was optimized in order to allow formation of stable materials. Both the modified-particle cytotoxicity and the matrix-supported cell adhesion were evaluated and visualized in order to confirm the perspectives of materials application.

Encapsulation of human endometrial stem cells in chitosan hydrogel containing titanium oxide nanoparticles for dental pulp repair and tissue regeneration in male Wistar rats

This study aimed to determine the impact of human endometrial stem cells (EnSCs) and titanium oxide nanoparticles (TiO2 NPs) on dental pulp repair and regeneration in an animal model through dentine development and tissue regeneration. The EnSCs were put on a three-dimensional (3D) chitosan scaffold containing TiO2 NPs after obtaining and purifying the collagenase enzyme. Pulps were exposed on the maxillary left first molar of all rats followed by direct pulp capping with the experimental scaffolds, as follows. Groups were: 1, control group without any treatment; 2, chitosan group (CS); 3, chitosan group with stem cells (CS/SCs); 4, chitosan group with stem cells and TiO2 NPs (CS/EnSCs/TiO2). Glass ionomer was used as a sealant in all groups. The teeth were extracted and histologically evaluated after 8 weeks. The quality and amount of dentine in the CS/EnSCs/TiO2 group were higher than in the other groups. The combination of EnSCs with TiO2 NPs and 3D chitosan scaffolds had a synergistic effect on each other, evidencing increased speed and quality of dentine formation. Using EnSCs with TiO2 NPs on a 3D chitosan scaffold can be a suitable combination for direct pulp capping and dentine regeneration in a rat molar tooth model.

Polymeric microcarriers for minimally-invasive cell delivery.

Tissue engineering (TE) aims at restoring tissue defects by applying the three-dimensional (3D) biomimetic pre-formed scaffolds to restore, maintain, and enhance tissue growth. Broadly speaking, this approach has created a potential impact in anticipating organ-building, which could reduce the need for organ replacement therapy. However, the implantation of such cell-laden biomimetic constructs based on substantial open surgeries often results in severe inflammatory reactions at the incision site, leading to the generation of a harsh adverse environment where cell survival is low. To overcome such limitations, micro-sized injectable modularized units based on various biofabrication approaches as ideal delivery vehicles for cells and various growth factors have garnered compelling interest owing to their minimally-invasive nature, ease of packing cells, and improved cell retention efficacy. Several advancements have been made in fabricating various 3D biomimetic microscale carriers for cell delivery applications. In this review, we explicitly discuss the progress of the microscale cell carriers that potentially pushed the borders of TE, highlighting their design, ability to deliver cells and substantial tissue growth in situ and in vivo from different viewpoints of materials chemistry and biology. Finally, we summarize the perspectives highlighting current challenges and expanding opportunities of these innovative carriers.

Remote control of muscle-driven miniature robots with battery-free wireless optoelectronics.

Bioengineering approaches that combine living cellular components with three-dimensional scaffolds to generate motion can be used to develop a new generation of miniature robots. Integrating on-board electronics and remote control in these biological machines will enable various applications across engineering, biology, and medicine. Here, we present hybrid bioelectronic robots equipped with battery-free and microinorganic light-emitting diodes for wireless control and real-time communication. Centimeter-scale walking robots were computationally designed and optimized to host on-board optoelectronics with independent stimulation of multiple optogenetic skeletal muscles, achieving remote command of walking, turning, plowing, and transport functions both at individual and collective levels. This work paves the way toward a class of biohybrid machines able to combine biological actuation and sensing with on-board computing.

Silk Fibroin Bioink for 3D Printing in Tissue Regeneration: Controlled Release of MSC extracellular Vesicles.

Sodium alginate (SA)-based hydrogels are often employed as bioink for three-dimensional (3D) scaffold bioprinting. They offer a suitable environment for cell proliferation and differentiation during tissue regeneration and also control the release of growth factors and mesenchymal stem cell secretome, which is useful for scaffold biointegration. However, such hydrogels show poor mechanical properties, fast-release kinetics, and low biological performance, hampering their successful clinical application. In this work, silk fibroin (SF), a protein with excellent biomechanical properties frequently used for controlled drug release, was blended with SA to obtain improved bioink and scaffold properties. Firstly, we produced a printable SA solution containing SF capable of the conformational change from Silk I (random coil) to Silk II (β-sheet): this transition is a fundamental condition to improve the scaffold's mechanical properties. Then, the SA-SF blends' printability and shape fidelity were demonstrated, and mechanical characterization of the printed hydrogels was performed: SF significantly increased compressive elastic modulus, while no influence on tensile response was detected. Finally, the release profile of Lyosecretome-a freeze-dried formulation of MSC-secretome containing extracellular vesicles (EV)-from scaffolds was determined: SF not only dramatically slowed the EV release rate, but also modified the kinetics and mechanism release with respect to the baseline of SA hydrogel. Overall, these results lay the foundation for the development of SA-SF bioinks with modulable mechanical and EV-release properties, and their application in 3D scaffold printing.

Advances in Recellularization of Decellularized Liver Grafts with Different Liver (Stem) Cells: Towards Clinical Applications.

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

Effect of increasing hyaluronic acid content in collagen scaffolds on the maintenance of chondrogenic phenotype in chondrocytes and mesenchymal stem cells

IntroductionIn articular cartilage, hyaluronic acid (HA) has been shown to increase adhesion, differentiation, and proliferation of cartilage cells, and its addition to three-dimensional (3D) scaffolds has been shown to improve chondrocyte phenotype in vitro. ObjectivesThe purpose of this study was to determine (1) whether increasing the HA levels in collagen scaffolds leads to better maintenance of the chondrogenic phenotype in chondrocytes in a dose-dependent manner and (2) whether collagen scaffolds infused with HA promote the differentiation of mesenchymal stem cells (MSCs) into chondrocytes within 3D collagen/HA scaffolds. MethodsBovine chondrocytes and rat MSCs were seeded onto 2% or 6% collagen/HA scaffolds, or no scaffold for controls. Cells were harvested at days 6, 12 and 18. Real-time polymerase chain reaction was used to measure expression of Type I and Type II collagen, SOX-9, and aggrecan. ResultsExpression of Type I and II collagen was increased at day 18 in the chondrocytes of both groups. For the MSCs, at day 18 there was increased expression of Type I collagen and increased expression of aggrecan in both the 2 and 6% scaffold groups, plus increased SOX-9 expression in the 2% HA group. ConclusionsThough there was no significant dose-dependent change between the HA groups in their effects on Type II collagen production, both HA scaffold groups showed significantly increased expression of Type II collagen as compared to monolayer controls at 18 days. Future experiments should extend timepoints to elucidate these effects and human chondrocytes and MSCs should be used to determine biological applicability.

Nano-hydroxyapatite/poly(L-lactic acid) nanofibrous composite scaffolds prepared by in-situ precipitation combined with frozen extraction

A series of nano-hydroxyapatite/polyl-lactic acid (n-HA/PLLA) three-dimensional nanofibrous composite scaffolds were synthesized by a new in-situ precipitation method combined with the frozen extraction using Ca(NO3)2·4H2O and (NH4)2HPO4 as the hydroxyapatite precursor. Combining SEM and XRD results showed that the HA nanoparticles were generated on the surface of the scaffolds and uniformly distributed throughout the material. The effects of the aging temperature and the amount of calcium phosphate solution on the morphology of resulting nanofiber composite scaffolds and fiber diameters were investigated. The results showed that uniform nanofibers were formed at low aging temperatures (−10 °C or less), while the fibers tended to flake and the support structure was more compact at high aging temperatures (5 °C or higher). The amount of added (NH4)2HPO4 had a similar effect on the nanofiber structure. The volume of the (NH4)2HPO4 solution and aging temperature used to prepare the composites also influenced the porosity, crystallinity, and mechanical properties of the final materials. In vitro bioactivity assays using simulated body fluid (SBF) showed that large amounts of low crystallinity carbonate hydroxyapatite (CHA) formed on the scaffolds after 14 days, indicating that the n-HA/PLLA nanofiber composite scaffolds have good bioactivity. This study shows that nHA effectively improved the biological activity of composite scaffolds.

Bioactive Glass-Ceramic Scaffolds Coated with Hyaluronic Acid-Fatty Acid Conjugates: A Feasibility Study.

Promoting bone healing is a key challenge in our society that can be tackled by developing new implantable biomaterials provided with regenerative properties. In this work, the coating of three-dimensional porous glass-derived scaffolds with hyaluronic acid (HA)-fatty acids was investigated for the first time. The starting scaffolds, based on bioactive silicate glass, were produced by foam replication followed by sintering; then, HA-palmitate and HA-oleate conjugate coatings were deposited on the scaffold struts through a dipping procedure. FT-IR analysis confirmed the successful deposition of the coatings on the surface and struts of the scaffolds, the foam-like architecture of which was maintained as assessed by SEM investigations. The in vitro bioactivity of the HA-fatty-acid-coated scaffolds was studied by immersion tests in simulated body fluid and the subsequent evaluation of hydroxyapatite formation. The deposition of the polymeric coating did not inhibit the apatite-forming ability of scaffolds, as revealed by the formation of nanostructured hydroxyapatite agglomerates 48 h from immersion. These promising results motivate further investigation of these novel bioactive systems, which are expected to combine the bone-bonding properties of the glass with the wound-healing promotion carried out by the polymeric conjugates.

Regenerated cellulose sponge as sacrificial template for the synthesis of three-dimensional porous alumina-silica scaffold for tissue engineering

Regenerated cellulose sponge as sacrificial template for the synthesis of three-dimensional porous alumina-silica scaffold for tissue engineering

Imitating the microenvironment of native biofilms using nanofibrous scaffolds to emulate chronic wound infections.

Three-dimensional scaffolds of electrospun fibers are widely investigated for in vitro human tissue engineering, but to date, their application in the cultivation of bacterial biofilms has been neglected. In contrast, in a clinical setting, biofilms have received increasing recognition as major determinants of severe and chronic tissue infections, illustrating their immense threat to global public health. Their complex three-dimensional structure enables their persistence in harsh infection environments, tight attachment to human tissue and reduced susceptibility to antimicrobials. For the investigation of biofilm formation and persistence and for the development of novel infection therapies, mimicking the complex biofilm architecture with adequate in vitro models is essential. In this study, electrospun nanofibers were designed to simulate the matrix of native biofilms to serve as scaffolds for a novel biofilm model, which provides an in vivo-like growth environment and comprises biofilm-tissue interfaces. The three-dimensional scaffolds closely imitate the composition and structure of the matrix, while providing high mechanical support. The specific material properties of the developed scaffolds promote bacterial adhesion, infiltration, and homogenous distribution throughout the fiber network. Furthermore, matrix production and increased tolerance against antibiotics were proven, verifying adequate biofilm formation and maturation. In combination with human ex vivo wound models, the chronic state of infected wounds could be emulated allowing for investigation of biofilm-tissue interfaces and biofilm-host interactions. The here-described biofilm model based on nanofibers represents a valuable tool for simulating biofilm-associated infections in a pathophysiologically relevant manner paving new grounds for a multitude of possible applications beyond infection research.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Ca2+/Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Enhances Chondrogenesis of Bone Marrow Mesenchymal Stem Cells and Delays Chondrogenic Hypertrophy.

Cartilage tissue engineering has been popularly applied in the treatment of articular cartilage defect because it is more effective in generating functional engineered cartilage than traditional methods. Although the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is well established, it is often accompanied by undesired hypertrophy. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator in the ion channel pathway which is known to be involved in chondrogenic hypertrophy. Therefore, this study aimed to reduce the hypertrophy of BM-MSCs by inhibiting CaMKII activation. BM-MSCs were cultured in three-dimensional (3D) scaffold under chondrogenic induction with and without CaMKII inhibitor, KN-93. After cultivation, markers of chondrogenesis and hypertrophy were investigated. KN-93 at a concentration of 2.0 μM had no effect on the viability of BM-MSCs, while the activation of CaMKII was suppressed. A long period of KN-93 treatment significantly up-regulated the expression of SRY-box transcription factor 9 and aggrecan on day 28 compared to untreated BM-MSCs. Furthermore, KN-93 treatment significantly down-regulated the expression of RUNX family transcription factor 2 and collagen type X alpha 1 chain on days 21 and 28. Immunohistochemistry showed increased expression of aggrecan and type II collagen while the expression of type X collagen was reduced. A CaMKII inhibitor, KN-93 is able to enhance chondrogenesis of BM-MSCs and suppress chondrogenic hypertrophy, suggesting its potential applicability in cartilage tissue engineering.