Enhancement of Bone Tissue Regeneration with Multi-Functional Nanoparticles by Coordination of Immune, Osteogenic, and Angiogenic Responses.

Inorganic nanoparticles are promising materials for bone tissue engineering due to their chemical resemblance to the native bone structure. However, most studies are unable to capture the entirety of the defective environment, providing limited bone regenerative abilities. Hence, this study aims to develop a multifunctional nanoparticle to collectively control the defective bone niche, including immune, angiogenic, and osteogenic systems. The nanoparticles, self-assembled by biomimetic mineralization and tannic acid (TA)-mediated metal-polyphenol network (MPN), are released sustainably after the incorporation within a gelatin cryogel. The released nanoparticles display a reduction in M1 macrophages by means of reactive oxygen species (ROS) elimination. Consequently, osteoclast maturation is also reduced, which is observed by the minimal formation of multinucleated cells (0.4%). Furthermore, the proportion of M2 macrophages, osteogenic differentiation, and angiogenic potential are consistently increased by the effects of magnesium from the nanoparticles. This orchestrated control of multiple systems influences the in vivo vascularized bone regeneration in which 80% of the critical-sized bone defect is regenerated with new bones with mature lamellar structure and arteriole-scale micro-vessels. Altogether, this study emphasizes the importance of the coordinated modulation of immune, osteogenic, and angiogenic systems at the bone defect site for robust bone regeneration.

Cell-homing and immunomodulatory composite hydrogels for effective wound healing with neovascularization.

Wound healing in cases of excessive inflammation poses a significant challenge due to compromised neovascularization. Here, we propose a multi-functional composite hydrogel engineered to overcome such conditions through recruitment and activation of macrophages with adapted degradation of the hydrogel. The composite hydrogel (G-TSrP) is created by combining gelatin methacryloyl (GelMA) and nanoparticles (TSrP) composed of tannic acid (TA) and Sr2+. These nanoparticles are prepared using a one-step mineralization process assisted by metal-phenolic network formation. G-TSrP exhibits the ability to eliminate reactive oxygen species and direct polarization of macrophages toward M2 phenotype. It has been observed that the liberation of TA and Sr2+ from G-TSrP actively facilitate the recruitment and up-regulation of the expression of extracellular matrix remodeling genes of macrophages, and thereby, coordinate in vivo adapted degradation of the G-TSrP. Most significantly, G-TSrP accelerates angiogenesis despite the TA's inhibitory properties, which are counteracted by the released Sr2+. Moreover, G-TSrP enhances wound closure under inflammation and promotes normal tissue formation with strong vessel growth. Genetic analysis confirms macrophage-mediated wound healing by the composite hydrogel. Collectively, these findings pave the way for the development of biomaterials that promote wound healing by creating regenerative environment.

Engineering pre-vascularized 3D tissue and rapid vascular integration with host blood vessels via co-cultured spheroids-laden hydrogel.

Recent advances in regenerative medicine and tissue engineering have enabled the biofabrication of three-dimensional (3D) tissue analogues with the potential for use in transplants and disease modeling. However, the practical use of these biomimetic tissues has been hindered by the challenge posed by reconstructing anatomical-scale micro-vasculature tissues. In this study, we suggest that co-cultured spheroids within hydrogels hold promise for regenerating highly vascularized and innervated tissues, bothin vitroandin vivo. Human adipose-derived stem cells (hADSCs) and human umbilical vein cells (HUVECs) were prepared as spheroids, which were encapsulated in gelatin methacryloyl hydrogels to fabricate a 3D pre-vascularized tissue. The vasculogenic responses, extracellular matrix production, and remodeling depending on parameters like co-culture ratio, hydrogel strength, and pre-vascularization time forin vivointegration with native vessels were then delicately characterized. The co-cultured spheroids with 3:1 ratio (hADSCs/HUVECs) within the hydrogel and with a pliable storage modulus showed the greatest vasculogenic potential, and ultimately formedin vitroarteriole-scale vasculature with a longitudinal lumen structure and a complex vascular network after long-term culturing. Importantly, the pre-vascularized tissue also showed anastomotic vascular integration with host blood vessels after transplantation, and successful vascularization that was positive for both CD31 and alpha-smooth muscle actin covering 18.6 ± 3.6µm2of the luminal area. The described co-cultured spheroids-laden hydrogel can therefore serve as effective platform for engineering 3D vascularized complex tissues.

Bioassembly of multicellular spheroids to mimic complex tissue structure using surface-modified magnetized nanofibers.

Advancements in biofabrication have led to major strides toward creating authentic organ models; however, replicating intricate organ structures without scaffolds remains challenging. In this study, we introduce a method utilizing surface-modifiable magnetic nanofibers to achieve precise control over spheroid functions and geometrical features, allowing the creation of multiple functional domains within a single microtissue. We generated magnetized nanofibers by electrospinning magnetic nanoparticles dispersed in poly-L-lactic acid solution. These fibers were then coated with polydopamine (PD) to enhance their biological functions, particularly reactive oxygen species (ROS) scavenging. These PD-coated magnetic fibers (PMFs) had magnetic-responsive properties when incorporated into human dermal fibroblast spheroids (0.019 ± 0.001 emu g-1). Furthermore, PMFs within the spheroids effectively regulated ROS levels by upregulating the expression of key anti-oxidative genes such assuperoxide dismutase-1(2.2 ± 0.1) andglutathione peroxidase-1(2.6 ± 0.1). By exploiting the magnetic responsiveness of spheroids, we were able to assemble them into various structures such as linear, triangular, and square structures using remotely applied magnetic forces. Within the assembled three-dimensional constructs, the cells in spheroids incorporating PMFs demonstrated resistance to ROS regulatory activity in the presence of hydrogen peroxide, while spheroids composed of bare fibers exhibited high ROS levels. Furthermore, we assembled spheroids containing fibroblasts and endothelial cells into complex tissue structures resembling vessels under magnetic manipulation. This innovative method holds tremendous promise for organ modeling and regenerative medicine due to the unprecedented control it allows in developing microtissues that closely emulate real organs.

Composite Spheroid-Laden Bilayer Hydrogel for Engineering Three-Dimensional Osteochondral Tissue.

A combination of hydrogels and stem cell spheroids has been used to engineer three-dimensional (3D) osteochondral tissue, but precise zonal control directing cell fate within the hydrogel remains a challenge. In this study, we developed a composite spheroid-laden bilayer hydrogel to imitate osteochondral tissue by spatially controlled differentiation of human adipose-derived stem cells. Meticulous optimization of the spheroid-size and mechanical strength of gelatin methacryloyl (GelMA) hydrogel enables the cells to homogeneously sprout within the hydrogel. Moreover, fibers immobilizing transforming growth factor beta-1 (TGF-ß1) or bone morphogenetic protein-2 (BMP-2) were incorporated within the spheroids, which induced chondrogenic or osteogenic differentiation of cells in general media, respectively. The spheroids-filled GelMA solution was crosslinked to create the bilayer hydrogel, which demonstrated a strong interfacial adhesion between the two layers. The cell sprouting enhanced the adhesion of each hydrogel, demonstrated by increase in tensile strength from 4.8 ± 0.4 to 6.9 ± 1.2 MPa after 14 days of culture. Importantly, the spatially confined delivery of BMP-2 within the spheroids increased mineral deposition and more than threefold enhanced osteogenic genes of cells in the bone layer while the cells induced by TGF-ß1 signals were apparently differentiated into chondrocytes within the cartilage layer. The results suggest that our composite spheroid-laden hydrogel could be used for the biofabrication of osteochondral tissue, which can be applied to engineer other complex tissues by delivery of appropriate biomolecules.

Development of a composite hydrogel incorporating anti-inflammatory and osteoinductive nanoparticles for effective bone regeneration.

BACKGROUND: Bone tissue regeneration is regulated by complex events, including inflammation, osteoinduction, and remodeling. Therefore, to induce the complete restoration of defective bone tissue, biomaterials with the ability to regulate the collective bone regenerative system are beneficial. Although some studies conclude that reducing reactive oxygen species created a favorable environment for bone regeneration by controlling inflammation, biomaterials that can simultaneously promote osteogenesis and regulate inflammation have not been developed. Herein, we describe the development of a multi-functional nanoparticle and its hydrogel composite with osteoinductive, anti-inflammatory, and osteoclast-maturation regulatory functions for enhanced bone regeneration. METHODS: Tannic acid-mineral nanoparticles (TMP) were prepared by self-assembly of tannic acid in an ion-rich simulated body fluid containing Ca2+ and PO43-. Particles with a diameter of 443 ± 91 nm were selected for their stable spherical morphology and minimal tendency to aggregate. The particles were homogeneously embedded within a gelatin-based cryogel (TMP/Gel) to be used in further experiments. The osteoinductive properties, anti-inflammatory and osteoclast-maturation regulatory functions in vitro were tested by culturing corresponding cells on either TMP/Gel or a gelatin-based cryogel without the particles (Gel). For in vivo analyses, a murine calvarial defect model was used. Statistical analyses were carried out using a Graphpad Prism 7 software (San Diego, CA, USA) to perform one-way analysis of variance ANOVA with Tukey's honest significant difference test and a Student's t-test (for two variables) (P < 0.05). RESULTS: Excellent biocompatibility and radical scavenging abilities were exhibited by the TMP/Gel. The expression of osteogenic mRNA is significantly increased in human adipose-derived stem cells seeded on the TMP/Gel compared to those without the particles. Furthermore, RAW264.7 cells seeded on the TMP/Gel displayed significantly lower-than-normal levels of pro-inflammatory and osteoclastogenic genes. Finally, the in vivo results indicated that, compared with the cryogel with no anti-inflammatory effect, the TMP/Gel significantly enhanced both the quality and quantity of newly formed bone, demonstrating the importance of combining anti-inflammation with osteoinduction. CONCLUSION: Collectively, these findings suggest our nanoparticle-hydrogel composite could be an effective tool to regulate complex events within the bone healing process.

Harvest of Cell-Only Muscle Fibers Using Thermally Expandable Hydrogels with Adhesive Patterns.

Muscle tissue engineering has been the focus of extensive research due to its potential for numerous medical applications, including ex vivo actuator development and clinical treatments. In this study, we developed a method for harvesting muscle fiber in a floatable and translocatable manner utilizing thermally expandable hydrogels with a chemically patterned polydopamine (PD) layer generated by microcontact printing (µCP). The µCP of PD on the hydrogel facilitated the formation of stripe patterns with varying widths of printed/nonprinted area (50/50, 100/100, and 200/200 µm). The spatially controlled adhesion of C2C12 myoblasts on the PD patterns produced clearly distinguishable muscle fibers, and translocated muscle fibers exhibited preserved extracellular matrix and junction proteins. Furthermore, the development of anisotropic arrangements and mature myotubes within the fibers suggests the potential for functional control of engineered muscle tissues. Overall, the muscle fiber harvesting method developed herein is suitable for both translocation and floating and is a promising technique for muscle tissue engineering as it mimics the structure-function relationship of natural tissue.

Biomimetic anti-inflammatory and osteogenic nanoparticles self-assembled with mineral ions and tannic acid for tissue engineering.

Bone healing involves complex processes including inflammation, induction, and remodeling. In this context, anti-inflammatory and osteoconductive multi-functional nanoparticles have attracted considerable attention for application in improved bone tissue regeneration. In particular, nanoparticles that promote suppression of inflammatory response after injury and direction of desirable tissue regeneration events are of immense interest to researchers. We herein report a one-step method to prepare multi-functional nanoparticles using tannic acid (TA) and simulated body fluid (SBF) containing multiple mineral ions. Mineral-tannic acid nanoparticles (mTNs) were rapidly fabricated in 10 min, and their size (around 250-350 nm) and chemical composition were controlled through the TA concentration. In vitro analysis using human adipose derived stem cells (hADSCs) showed that mTNs effectively scavenged reactive oxygen species (ROS) and enhanced osteogenesis of hADSCs by inducing secretion of alkaline phosphatase. mTNs also increased osteogenic marker gene expression even in the presence of ROS, which can generally arrest osteogenesis (OPN: 1.74, RUNX2: 1.90, OCN: 1.47-fold changes relative to cells not treated with mTNs). In vivo analysis using a mouse peritonitis model revealed that mTNs showed anti-inflammatory effects by decreasing levels of pro-inflammatory cytokines in blood (IL-6: 73 ± 4, TNF-α: 42 ± 2%) and peritoneal fluid (IL-6: 78 ± 2, TNF-α: 21 ± 6%). We believe that this one-step method for fabrication of multi-functional nanoparticles has considerable potential in tissue engineering approaches that require control of complex microenvironments, as required for tissue regeneration.

Composite Multicellular Spheroids Containing Fibers with Pores and Epigallocatechin Gallate (EGCG) Coating on the Surface for Enhanced Proliferation of Stem Cells.

Multicellular spheroids are formed by strong cell-cell and cell-extracellular matrix interactions and are widely utilized in tissue engineering for therapeutic treatments or ex vivo tissue modeling. However, diffusion of oxygen into the spheroid gradually decreases, forming a necrotic core. In this study, polycaprolactone (PCL) fibers with pores and epigallocatechin gallate (EGCG) coating on their surface to provide a structural framework within the spheroids and investigated their ability to mitigate diffusional limitation and control over the proliferation of human adipose-derived stem cells (hADSCs) is engineered. The DNA content of composite spheroids prepared from fibers and hADSCs decreased in unadjusted cells (1224 ± 134 ng), in those with fibers with a smooth surface (SF) (1447 ± 331 ng), and in those EGCG-coated with SF (E-SF) (1437 ± 289 ng). Cells with fibers with pores on the surface (PF) (2020 ± 32 ng) and those with EGCG-coated PF (E-PF) (1911 ± 80 ng) increased after 7 days of culture, with a significantly greater number of proliferating cells (29 ± 8% and 30 ± 8%, respectively). These results indicate that physical modification through the formation of pores on the fiber surface alleviates diffusion limitation of composite spheroids, playing a dominant role over chemical modification.

Spatially arranged encapsulation of stem cell spheroids within hydrogels for the regulation of spheroid fusion and cell migration.

Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. STATEMENT OF SIGNIFICANCE: Recently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.

Surface engineering of 3D-printed scaffolds with minerals and a pro-angiogenic factor for vascularized bone regeneration.

Scaffolds functionalized with biomolecules have been developed for bone regeneration but inducing the regeneration of complex structured bone with neovessels remains a challenge. For this study, we developed three-dimensional printed scaffolds with bioactive surfaces coated with minerals and platelet-derived growth factor. The minerals were homogeneously deposited on the surface of the scaffold using 0.01 M NaHCO3 with epigallocatechin gallate in simulated body fluid solution (M2). The M2 scaffold demonstrated enhanced mineral coating amount per scaffold with a greater compressive modulus than the others which used different concentration of NaHCO3. Then, we immobilized PDGF on the mineralized scaffold (M2/P), which enhanced the osteogenic differentiation of human adipose derived stem cells in vitro and promoted the secretion of pro-angiogenic factors. Cells cultured in M2/P showed remarkable ratio of osteocalcin- and osteopontin-positive nuclei, and M2/P-derived medium induced endothelial cells to form tubule structures. Finally, the implanted M2/P scaffolds onto mouse calvarial defects had regenerated bone in 80.8 ± 9.8% of the defect area with the arterioles were formed, after 8 weeks. In summary, our scaffold, which composed of minerals and pro-angiogenic growth factor, could be used therapeutically to improve the regeneration of bone with a highly vascularized structure. STATEMENT OF SIGNIFICANCE: Surface engineered scaffolds have been developed for bone regeneration but inducing the volumetric regeneration of bone with neovessels remains a challenge. In here, we developed 3D printed scaffolds with bioactive surfaces coated with bio-minerals and platelet-derived growth factors. We proved that the 0.01 M NaHCO3 with polyphenol in simulated body fluid solution enhanced the deposition of bio-minerals and even distribution on the surface of scaffold. The in vitro studies demonstrated that the attached cells on the bioactive surface showed the enhanced osteogenic differentiation and secretion of pro-angiogenic factors. Finally, the scaffold with bioactive surface not only improved the regenerated volume of bone tissues but also increased neovessel formation after in vivo implantation onto mouse calvarial defect.

Magnetism-controlled assembly of composite stem cell spheroids for the biofabrication of contraction-modulatory 3D tissue.

Biofabrication of organ-like engineered 3D tissue through the assembly of magnetized 3D multi-cellular spheroids has been recently investigated in tissue engineering. However, the cytotoxicity of magnetic nanoparticles (MNPs) and contraction-induced structural deformation of the constructs have been major limitations. In this study, we developed a method to fabricate composite stem cell spheroids using MNP-coated fibers, alleviating MNP-mediated toxicity and controlling structural assembly under external magnetic stimuli. The MNP-coated synthetic fibers (MSFs) were prepared by coating various amounts of MNPs on the fibers via electrostatic interactions. The MSFs showed magnetic hysteresis and no cytotoxicity on 2D-cultured adipose-derived stem cells (ADSCs). The composite spheroids containing MSFs and ADSCs were rapidly formed in which the amount of impregnated MSFs modulated the spheroid size. The fusion ofin vitrocomposite spheroids was then monitored at the contacting interface; the fused spheroids with over 10µg of MSF showed minimal contraction after 7 d, retaining around 90% of total area ratio regardless of the number of cells, indicating that the presence of fibers within the composite spheroid supported its structural maintenance. The fusion of MSF spheroids was modulated by external magnetic stimulation, and the effect of magnetic force on the movement and fusion of the spheroids was investigated using COMSOL simulation. Finally, ring and lamellar structures were successfully assembled using remote-controlled MSF spheroids, showing limited deformation and high viability up to 50 d duringin vitroculture. In addition, the MSFs demonstrated no adverse effects on ADSC osteochondral differentiation. Altogether, we envision that our magnetic assembly system would be a promising method for the tissue engineering of structurally controlled organ-like constructs.

Evaluation of the anti-oxidative and ROS scavenging properties of biomaterials coated with epigallocatechin gallate for tissue engineering.

In tissue engineering, excessively generated reactive oxygen species (ROS) during biomaterial implantation or cell transplantation is a one of major causes of diminishing therapeutic effects. In this study, we prepared biomaterial surfaces coated with antioxidant epigallocatechin gallate (EGCG) and metal ions, and evaluated their anti-oxidative and ROS scavenging properties. We revealed that EGCG-coating on polycaprolactone (PCL) film surface increased hydrophilicity and anti-oxidative properties as a function of total phenol content (TPC) potentially due to the increase in phenolic -OH and π-electrons from structural maintenance and directly removed the hydrogen peroxide (H2O2) by resonance-stabilization. Furthermore, EGCG-coated PCL film increased attachment, spreading area, and viability of human adipose-derived stem cells (hADSCs) against H2O2 treatment while stimulated the cellular signaling to reduce apoptotic gene and enhance anti-oxidative enzyme expression. Further, we applied EGCG coating on the surface of poly-L-lactic acid (PLLA) fibers. Spheroids incorporating EGCG-coated PLLA fibers were able to maintain their shape and showed improved viability and anti-oxidative activities in response to H2O2-induced oxidative stress than control spheroids. Therefore, metal-phenolic network (MPN) coating of EGCG is a suitable method to impart the anti-oxidative properties to biomaterials by evaluating the structural properties and biological effects.

Stem cell spheroid engineering with osteoinductive and ROS scavenging nanofibers for bone regeneration.

Stem cell spheroids have been widely investigated to accelerate bone tissue regeneration. However, the directed differentiation of stem cells into osteoblastic lineage and the prevention of cells from damage by reactive oxygen species (ROS) remain challenge. Here, we developed osteoinductive and ROS scavenging extracellular matrix-mimicking synthetic fibers based on epigallocatechin gallate (EGCG) coating. They were then utilized to fabricate engineered spheroids with human adipose-derived stem cells (hADSCs) for bone tissue regeneration. The EGCG-mineral fibers (EMF) effectively conferred osteoinductive and ROS scavenging signals on the hADSCs within spheroids, demonstrating relative upregulation of antioxidant genes (SOD-1(25.8 ± 2.1) andGPX-1(3.3 ± 0.1) and greater level of expression of osteogenic markers,runt-related transcription factor(5.8 ± 0.1) andosteopontin(5.9 ± 0.1), compared to hADSCs in the spheroids without EMF. Thein vitrooverexpression of osteogenic genes from hADSCs was achieved from absence of osteogenic supplements. Furthermore,in vivotransplantation of hADSCs spheroids with the EMF significantly promoted calvarial bone regeneration (48.39 ± 9.24%) compared to that from defect only (17.38 ± 6.63%), suggesting that the stem cell spheroid biofabrication system with our novel mineralization method described here is a promising tool for bone tissue regeneration.

3D printed micro-chambers carrying stem cell spheroids and pro-proliferative growth factors for bone tissue regeneration.

Three-dimensional (3D)-printed scaffolds have proved to be effective tools for delivering growth factors and cells in bone-tissue engineering. However, delivering spheroids that enhance cellular function remains challenging because the spheroids tend to suffer from low viability, which limits bone regenerationin vivo. Here, we describe a 3D-printed polycaprolactone micro-chamber that can deliver human adipose-derived stem cell spheroids. Anin vitroculture of cells from spheroids in the micro-chamber exhibited greater viability and proliferation compared with cells cultured without the chamber. We coated the surface of the chamber with 500 ng of platelet-derived growth factors (PDGFs), and immobilized 50 ng of bone morphogenetic protein 2 (BMP-2) on fragmented fibers, which were incorporated within the spheroids as a new platform for a dual-growth-factor delivery system. The PDGF detached from the chamber within 8 h and the remains were retained on the surface of chamber while the BMP-2 was entrapped by the spheroid.In vitroosteogenic differentiation of the cells from the spheroids in the micro-chamber with dual growth factors enhanced alkaline phosphatase and collagen type 1A expression by factors of 126.7 ± 19.6 and 89.7 ± 0.3, respectively, compared with expression in a micro-chamber with no growth factors.In vivotransplantation of the chambers with dual growth factors into mouse calvarial defects resulted in a 77.0 ± 15.9% of regenerated bone area, while the chamber without growth factors and a defect-only group achieved 7.6 ± 3.9% and 5.0 ± 1.9% of regenerated bone areas, respectively. These findings indicate that a spheroid-loaded micro-chamber supplied with dual growth factors can serve as an effective protein-delivery platform that increases stem-cell functioning and bone regeneration.

Polydopamine-assisted one-step modification of nanofiber surfaces with adenosine to tune the osteogenic differentiation of mesenchymal stem cells and the maturation of osteoclasts.

Adenosine and its receptors have emerged as alternative targets to control cellular functions for bone healing. However, the soluble delivery of adenosine has not proven effective because of its fast degradation in vivo. We therefore designed a stable coating of adenosine for biomaterial surfaces through polydopamine chemistry to control osteogenesis and osteoclastogenesis via A2bR signaling. First, we prepared electrospun poly (ι-lactic acid) (PLLA) nanofiber sheets, which were modified through a one-step adenosine polydopamine coating process. Scanning electron microscopy (SEM) revealed deposition of particles on the adenosine polydopamine-coated PLLA (AP-PL) sheets compared to the polydopamine-only sheets. Moreover, X-ray photoelectron spectroscopy analysis confirmed an increase in nitrogen signals due to adenosine. Furthermore, adenosine loading efficiency and retention were significantly enhanced in AP-PL sheets compared to polydopamine-only sheets. Human adipose-derived stem cells (hADSCs) cultured on AP-PL expressed A2bR (1.30 ± 0.19 fold) at significantly higher levels than those cultured on polydopamine-only sheets. This in turn significantly elevated the expression of Runx2 (16.94 ± 1.68 and 51.69 ± 0.07 fold), OPN (1.63 ± 0.16 and 30.56 ± 0.25 fold), OCN (1.16 ± 0.13 and 5.23 ± 0.16 fold), and OSX (10.01 ± 0.81 and 62.48 ± 0.25 fold) in cells grown in growth media on days 14 and 21, respectively. Similarly, mineral deposition was enhanced to a greater extent in the AP-PL group than the polydopamine group, while blocking of A2bR significantly downregulated osteogenesis. Finally, osteoclast differentiation of RAW 264.7 cells was significantly inhibited by growth on AP-PL sheets. However, osteoclast differentiation was significantly stimulated after A2bR was blocked. Taken together, we propose that polydopamine-assisted one-step coating of adenosine is a viable method for surface modification of biomaterials to control osteogenic differentiation of stem cells and bone healing.

Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering.

Although stem cell spheroids offer great potential as functional building blocks for bottom-up bone tissue engineering, delivery of bioactive signals remain challenging. Here, we engineered adenosine-ligand-modified fiber fragments to create a 3D cell-instructive microenvironment for bone. Briefly, the Poly(ι-lactic acid) (PLLA) nanofiber sheet was partially degraded into fragmented fibers (FFs) through aminolysis and adenosine was stably incorporated via one-step polydopamine coating. The SEM and XPS analysis demonstrated that polydopamine assisted adenosine coating efficiency was significantly increased, which led to high coating efficiency of adenosine and its significant retention. The engineered fibers were then assembled into stable spheroids with human-adipose-derived stem cells (hADSCs). The adenosine in the spheroids effectively stimulated A2bR (1.768 ± 0.08) signaling, which further significantly induced the expression of osteogenic markers such as Runx2 (3.216 ± 0.25), OPN (4.136 ± 0.14), OCN (10.16 ± 0.34), and OSX (2.27 ± 0.11) with improved mineral deposition (1.375 ± 0.05 µg per spheroid). In contrast, the adipogenic differentiation of hADSCs was significantly suppressed within the engineered spheroids. Transplantation of engineered spheroids strongly induced osteogenic differentiation of hADSCs in ectopic subcutaneous tissue. Finally, the bone regeneration was significantly enhanced by implanting AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). We propose that stem cell spheroids impregnated with engineered fibers enabling adenosine delivery could be promising building blocks for a bottom-up approach to create large tissues for regeneration of damaged bone.

Fabrication of size-controllable human mesenchymal stromal cell spheroids from micro-scaled cell sheets.

Recently, stromal cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stromal cell spheroids in different sizes from micro-scaled cell sheets (µCS) using thermosensitive hydrogels and investigated their effects on stromal cell function. Mesenchymal stromal cells isolated from different tissues such as human turbinate tissue, bone marrow, and adipose tissue were adhered selectively to each micro-pattern (squares with widths of 100 and 400 µm) on the surface of the hydrogel and formed µCS. The diameters of the spheroids were modulated by the size of the patterns (45 ± 5 and 129 ± 4 µm in diameter for the 100 and 400 µm micro-patterns, respectively) and the seeding density (129 ± 4, 149 ± 6, and 163 ± 6 µm for 5.0, 10.0, and 15.0 × 104 cells cm-2, respectively, on 400 µm micro-pattern). In addition, the spheroids were successfully fabricated regardless of stromal cell origin, and the diameter of the spheroids was also affected by cell spreading area on a cell culture dish. Stemness markers were highly expressed in the spheroids regardless of the spheroid size. Furthermore, an increase in E-cadherin and decrease in N-cadherin gene expression showed the stable formation of spheroids of different sizes. Gene expression levels of hypoxia inducible factors and secretion of vascular endothelial growth factor were increased (13.2 ± 1.4, 325 ± 83.4 and 534.3 ± 121.5 pg ng-1 DNA in a monolayer, and 100 and 400 µm micro-patterned spheroids, respectively) proportional to the diameters of the spheroids. The size of spheroids were maintained even after injection, cryopreservation and 7 d of suspension culture with high viability (∼90%). In conclusion, this novel technique to fabricate spheroids with controlled size could be widely applied in various applications that require a controlled size in regenerative medicine.

Current Advances in Immunomodulatory Biomaterials for Bone Regeneration.

Biomaterials with suitable surface modification strategies are contributing significantly to the rapid development of the field of bone tissue engineering. Despite these encouraging results, utilization of biomaterials is poorly translated to human clinical trials potentially due to lack of knowledge about the interaction between biomaterials and the body defense mechanism, the "immune system". The highly complex immune system involves the coordinated action of many immune cells that can produce various inflammatory and anti-inflammatory cytokines. Besides, bone fracture healing initiates with acute inflammation and may later transform to a regenerative or degenerative phase mainly due to the cross-talk between immune cells and other cells in the bone regeneration process. Among various immune cells, macrophages possess a significant role in the immune defense, where their polarization state plays a key role in the wound healing process. Growing evidence shows that the macrophage polarization state is highly sensitive to the biomaterial's physiochemical properties, and advances in biomaterial research now allow well controlled surface properties. This review provides an overview of biomaterial-mediated modulation of the immune response for regulating key bone regeneration events, such as osteogenesis, osteoclastogenesis, and inflammation, and it discusses how these strategies can be utilized for future bone tissue engineering applications.

Hydrogels with an embossed surface: An all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids.

Stem cell spheroids have been studied extensively in organoid culture and therapeutic transplantation. Herein, hydrogels with an embossed surface (HES) were developed as an all-in-one platform that can enable the rapid formation and culture of a large quantity of size-controllable stem cell spheroids. The embossed structure on the hydrogel was adjustable according to the grit designation of the sandpaper. Human adipose-derived stem cells (hADSCs) were rapidly assembled into spheroids on the hydrogel, with their size distribution precisely controlled from 95 ±â€¯6 µm to 181 ±â€¯15 µm depending on surface roughness. The hADSC spheroids prepared from the HES demonstrated expression of stemness markers and differentiation capacity. In addition, HES-based spheroids showed significantly greater VEGF secretion than spheroids grown on a commercially available low-attachment culture plate. Exploiting those advantages, the HES-based spheroids were used for 3D bioprinting, and the spheroids within the 3D-printed construct showed improved retention and VEGF secretion compared to the same 3D structure containing single cell suspension. Collectively, HES would offer a useful platform for mass fabrication and culture of stem cell spheroids with controlled sizes for a variety of biomedical applications.