A review on gold nanoparticles as an innovative therapeutic cue in bone tissue engineering: Prospects and future clinical applications.

Bone damage is a complex orthopedic problem primarily caused by trauma, cancer, or bacterial infection of bone tissue. Clinical care management for bone damage remains a significant clinical challenge and there is a growing need for more advanced bone therapy options. Nanotechnology has been widely explored in the field of orthopedic therapy for the treatment of a severe bone disease. Among nanomaterials, gold nanoparticles (GNPs) along with other biomaterials are emerging as a new paradigm for treatment with excellent potential for bone tissue engineering and regenerative medicine applications. In recent years, a great deal of research has focused on demonstrating the potential for GNPs to provide for enhancement of osteogenesis, reduction of osteoclastogenesis/osteomyelitis, and treatment of bone cancer. This review details the latest understandings in regards to GNPs based therapeutic systems, mechanisms, and the applications of GNPs against various bone disorders. The present review aims to summarize i) the mechanisms of GNPs in bone tissue remodeling, ii) preparation methods of GNPs, and iii) functionalization of GNPs and its decoration on biomaterials as a delivery vehicle in a specific bone tissue engineering for future clinical application.

Dual-channel fluorescent probe for discriminative detection of H2S and N2H4: Exploring sensing mechanism and real-time applications.

A highly efficient system incorporates the real-time visualization of the two toxic molecules (H2S and N2H4) and the recognition of corresponding transforms using a fluorescent sensor. In this paper, a dual-responsive probe (QS-DNP) based on methylquinolinium-salicyaldehyde-2,4-dinitrophenyl was developed that can simultaneously detect H2S and N2H4 at two independent fluorescent channels without signal crosstalk. QS-DNP showed excellent anti-interference, high selectivity, outstanding water solubility, low LOD values (H2S: 51 nM; N2H4: 40 nM), low cytotoxicity, and mitochondrial localization properties. The 2,4-dinitrophenyl site was sensitive to H2S, and the CC bridge was reactive to N2H4, with strong fluorescence at 680 and 488 nm, respectively. The wavelength gap between these two channels is 192 nm; verify that there is no signal crosstalk throughout detection. By this means, the probe was used to simultaneously detect H2S and N2H4 in real soil samples, food samples, and living cells. The endogenous H2S and N2H4 were monitored in HeLa cells and investigated the mitochondria organelle of living cells with a positive charge on QS-DNP. Overall, all results emphasize that the QS-DNP probe is a powerful tool for the simultaneous detection of H2S and N2H4 and presents a potential new sensing approach.

Development of a Temperature-Responsive Hydrogel Incorporating PVA into NIPAAm for Controllable Drug Release in Skin Regeneration.

Melanoma, a highly malignant and aggressive form of skin cancer, poses a significant global health threat, with limited treatment options and potential side effects. In this study, we developed a temperature-responsive hydrogel for skin regeneration with a controllable drug release. The hydrogel was fabricated using an interpenetrating polymer network (IPN) of N-isopropylacrylamide (NIPAAm) and poly(vinyl alcohol) (PVA). PVA was chosen for its adhesive properties, biocompatibility, and ability to address hydrophobicity issues associated with NIPAAm. The hydrogel was loaded with doxorubicin (DOX), an anticancer drug, for the treatment of melanoma. The NIPAAm-PVA (N-P) hydrogel demonstrated temperature-responsive behavior with a lower critical solution temperature (LCST) around 34 °C. The addition of PVA led to increased porosity and faster drug release. In vitro biocompatibility tests showed nontoxicity and supported cell proliferation. The N-P hydrogel exhibited effective anticancer effects on melanoma cells due to its rapid drug release behavior. This N-P hydrogel system shows great promise for controlled drug delivery and potential applications in skin regeneration and cancer treatment. Further research, including in vivo studies, will be essential to advance this hydrogel system toward clinical translation and impactful advancements in regenerative medicine and cancer therapeutics.

Development of a plasma-based 3D printing system for enhancing the biocompatibility of 3D scaffold.

Fused deposition modeling (FDM) is a three-dimensional (3D) printing technology typically used in tissue engineering. However, 3D-printed row scaffolds manufactured using material extrusion techniques have low cell affinity on the surface and an insufficient biocompatible environment for desirable tissue regeneration. Thus, in this study, plasma treatment was used to render surface modification for enhancing the biocompatibility of 3D-printed scaffolds. We designed a plasma-based 3D printing system with dual heads comprising a plasma device and a regular 3D FDM printer head for a layer-by-layer nitrogen plasma treatment. Accordingly, the wettability, roughness, and protein adsorption capability of the 3D-printed scaffold significantly increased with the plasma treatment time. Hence, the layer-by-layer plasma-treated (LBLT) scaffold exhibited significantly enhanced cell adhesion and proliferation in anin vitroassay. Furthermore, the LBLT scaffold demonstrated a higher tissue infiltration and lower collagen encapsulation than those demonstrated by a non-plasma-treated scaffold in anin vivoassay. Our approach has great potential for various tissue-engineering applications via the adjustment of gas or precursor levels. In particular, this system can fabricate scaffolds capable of holding a biocompatible surface on an entire 3D-printed strut. Thus, our one-step 3D printing approach is a promising platform to overcome the limitations of current biocompatible 3D scaffold engineering.

Ginseng-derived exosome-like nanovesicles extracted by sucrose gradient ultracentrifugation to inhibit osteoclast differentiation.

Plant-derived extracellular nanovesicles contain RNA and proteins with unique and diverse pharmacological mechanisms. The extracellular nanovesicles encapsulating plant extracts resemble exosomes as they have a round, lipid bilayer morphology. Ginseng is anti-inflammatory, anti-cancer, immunostimulant, and osteogenic/anti-osteoporotic. Here, we confirmed that ginseng-derived extracellular nanovesicles (GDNs) inhibit osteoclast differentiation and elucidated the associated molecular mechanisms. We isolated GDNs by centrifugation with a sucrose gradient. We measured their dynamic light scattering and zeta potentials and examined their morphology by transmission electron microscopy. We used bone marrow-derived macrophages (BMMs) to determine the potential cytotoxicity of GDNs and establish their ability to inhibit osteoclast differentiation. The GDNs treatment maintained high BMM viability and proliferation whilst impeding osteoclastogenesis. Tartrate-resistant acid phosphatase and F-actin staining revealed that GDNs at concentrations >1 µg mL-1 strongly hindered osteoclast differentiation. Moreover, they substantially suppressed the RANKL-induced IκBα, c-JUN n-terminal kinase, and extracellular signal-regulated kinase signaling pathways and the genes regulating osteoclast maturation. The GDNs contained elevated proportions of Rb1 and Rg1 ginsenosides and were more effective than either of them alone or in combination at inhibiting osteoclast differentiation. In vivo bone analysis via microcomputerized tomography, bone volume/total volume ratios, and bone mineral density and bone cavity measurements demonstrated the inhibitory effect of GDNs against osteoclast differentiation in lipopolysaccharide-induced bone resorption mouse models. The results of this work suggest that GDNs are anti-osteoporotic by inhibiting osteoclast differentiation and are, therefore, promising for use in the clinical prevention and treatment of bone loss diseases.

Dual-sensitive drug-loaded hydrogel system for local inhibition of post-surgical glioma recurrence.

Local treatment after resection to inhibit glioma recurrence is thought to able to meet the real medical needs. However, the only clinically approved local glioma treatment-wafer containing bis(2-chloroethyl) nitrosourea (BCNU) showed very limited effects. Herein, in order to inhibit tumor recurrence with prolonged and synergistic therapeutic effect of drugs after tumor resection, an in situ dual-sensitive hydrogel drug delivery system loaded with two synergistic chemo-drugs BCNU and temozolomide (TMZ) was developed. The thermosensitive hydrogel was loaded with reactive oxygen species (ROS)-sensitive poly (lactic-co-glycolic) acid nanoparticles (NPs) encapsulating both BCNU and TMZ and also free BCNU and TMZ. The in vitro synergistic effect of BCNU and TMZ and in vivo presence of ROS at the residual tumor site were confirmed. The prepared ROS-sensitive NPs and thermosensitive hydrogel, as well as the long-term release behavior of drugs and NPs, were fully characterized both in vitro and in vivo. After >90% glioblastoma resection, the dual-sensitive hydrogel drug delivery system was injected into the resection cavity. The median survival time of the experimental group reached 65 days which was twice as long as the Resection only group, implying that this in situ drug delivery system effectively inhibited tumor recurrence. Overall, this study provides new ideas and strategies for the inhibition of postoperative glioma recurrence.

The Effectiveness of Compartmentalized Bone Graft Sponges Made Using Complementary Bone Graft Materials and Succinylated Chitosan Hydrogels.

Bone defects can occur from many causes, including disease or trauma. Bone graft materials (BGMs) have been used to fill damaged areas for the reconstruction of diseased bone tissues since they are cost effective and readily available. However, BGMs quickly disperse around the tissue area, which ultimately leads to it migrating away from the defect after transplantation. We tested chitosan hydrogels as a useful carrier to hold BGMs in the transplantation area. In this study, we synthesized succinylated chitosan (SCS)-based hydrogels with a high decomposition rate and excellent biocompatibility. We confirmed that BGMs were well distributed inside the SCS hydrogel. The SCS-B hydrogel showed a decrease in mechanical properties, such as compressive strength and Young's modulus, as the succinylation rate increased. SCS-B hydrogels also exhibited a high cell growth rate and bone differentiation rate. Moreover, the in vivo results showed that the SCS hydrogel resorbed into the surrounding tissues while maintaining the BGMs in the transplantation area for up to 6 weeks. These data support the idea that SCS hydrogel can be useful as a bioactive drug carrier for a broad range of biomedical applications.

Facile Preparation of ß-Cyclodextrin-grafted Chitosan Electrospun Nanofibrous Scaffolds as a Hydrophobic Drug Delivery Vehicle for Tissue Engineering Applications.

Despite advances in the bio-tissue engineering area, the technical basis to directly load hydrophobic drugs on chitosan (CTS) electrospun nanofibers (ENs) has not yet been fully established. In this study, we fabricated CTS ENs by using an electrospinning (ELSP) system, followed by surface modification using succinyl-beta-cyclodextrin (ß-CD) under mild conditions. The ß-CD-modified CTS (ßCTS) ENs had slightly increased hydrophobicity compared to pristine CTS ENs as well as decreased residual amine content on the surface. Through FTIR spectroscopy and thermogravimetric analysis (TGA), we characterized the surface treatment physiochemically. In the drug release test, we demonstrated the stable and sustained release of a hydrophobic drug (e.g., dexamethasone) loaded on ß-CD ENs. During in vitro biocompatibility assessments, the grafting of ß-CD was shown to not reduce cell viability compared to pristine CTS ENs. Additionally, cells proliferated well on ß-CD ENs, and this was confirmed by F-actin fluorescence staining. Overall, the material and strategies developed in this study have the potential to load a wide array of hydrophobic drugs. This could be applied as a drug carrier for a broad range of tissue engineering applications.

Development of photo-crosslinkable platelet lysate-based hydrogels for 3D printing and tissue engineering.

Three-dimensional (3D) printing shows potential for use as an advanced technology for forming biomimetic tissue and other complex structures. However, there are limits and restrictions on selection of conventional bioinks. Here we report the first 3D-printable platelet lysate (PLMA)-based hydrogel, which consists of platelet lysate from whole blood of humans that can simulate the 3D structure of tissues and can be formed into a crosslinked hydrogel layer-by-layer to build cell-laden hydrogel constructs through methacrylated photo-polymerization. Furthermore, it can be customized for use with various tissues by controlling the physical properties according to irradiation time and concentration. In particular, different cells can be mixed and printed, and the integrity of the 3D printed structure can maintain its shape after crosslinking. The bio-ink exhibits excellent cell diffusion and proliferation at low concentrations, which improves moldability and biocompatibility. The 3D-printable PLMA bioinks may constitute a new strategy to create customized microenvironments for the repair of various tissuesin vivousing materials derived from the human body.

Aspiration-assisted bioprinting of co-cultured osteogenic spheroids for bone tissue engineering.

Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 d, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

3D Bioprinting of Carbohydrazide-Modified Gelatin into Microparticle-Suspended Oxidized Alginate for the Fabrication of Complex-Shaped Tissue Constructs.

Extrusion-based bioprinting of hydrogels in a granular secondary gel enables the fabrication of cell-laden three-dimensional (3D) constructs in an anatomically accurate manner, which is challenging using conventional extrusion-based bioprinting processes. In this study, carbohydrazide-modified gelatin (Gel-CDH) was synthesized and deposited into a new multifunctional support bath consisting of gelatin microparticles suspended in an oxidized alginate (OAlg) solution. During extrusion, Gel-CDH and OAlg were rapidly cross-linked because of the Schiff base formation between aldehyde groups of OAlg and amino groups of Gel-CDH, which has not been demonstrated in the domain of 3D bioprinting before. Rheological results indicated that hydrogels with lower OAlg to Gel-CDH ratios possessed superior mechanical rigidity. Different 3D geometrically intricate constructs were successfully created upon the determination of optimal bioprinting parameters. Human mesenchymal stem cells and human umbilical vein endothelial cells were also bioprinted at physiologically relevant cell densities. The presented study has offered a novel strategy for bioprinting of natural polymer-based hydrogels into 3D complex-shaped biomimetic constructs, which eliminated the need for cytotoxic supplements as external cross-linkers or additional cross-linking processes, therefore expanding the availability of bioinks.

Aspiration-assisted bioprinting for precise positioning of biologics.

Three-dimensional (3D) bioprinting is an appealing approach for building tissues; however, bioprinting of mini-tissue blocks (i.e., spheroids) with precise control on their positioning in 3D space has been a major obstacle. Here, we unveil "aspiration-assisted bioprinting (AAB)," which enables picking and bioprinting biologics in 3D through harnessing the power of aspiration forces, and when coupled with microvalve bioprinting, it facilitated different biofabrication schemes including scaffold-based or scaffold-free bioprinting at an unprecedented placement precision, ~11% with respect to the spheroid size. We studied the underlying physical mechanism of AAB to understand interactions between aspirated viscoelastic spheroids and physical governing forces during aspiration and bioprinting. We bioprinted a wide range of biologics with dimensions in an order-of-magnitude range including tissue spheroids (80 to 600 µm), tissue strands (~800 µm), or single cells (electrocytes, ~400 µm), and as applications, we illustrated the patterning of angiogenic sprouting spheroids and self-assembly of osteogenic spheroids.

Emerging Potential of Exosomes in Regenerative Medicine for Temporomandibular Joint Osteoarthritis.

Exosomes are nanosized vesicles (30-140 nm) of endocytic origin that play important roles in regenerative medicine. They are derived from cell membranes during endocytic internalization and stabilize in biological fluids such as blood and synovia. Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, which, in addition to chronic pain, is characterized by progressive cartilage breakdown, condylar bone remodeling, and synovitis. However, traditional clinical treatments have limited symptom- and structure-modifying effects to restore damaged cartilage and other TMJ tissues. This is due to the limited self-healing capacity of condylar cartilage. Recently, stem-cell-derived exosomes have been studied as an alternative therapeutic approach to tissue repair and regeneration. It is known that trophic regulation of mesenchymal stem cells (MSCs) has anti-inflammatory and immunomodulatory effects under pathological conditions, and research on MSC-derived exosomes is rapidly accumulating. MSC-derived exosomes mimic the major therapeutic effects of MSCs. They affect the activity of immune effector cells and possess multilineage differentiation potential, including chondrogenic and osteogenic differentiation. Furthermore, exosomes are capable of regenerating cartilage or osseous compartments and restoring injured tissues and can treat dysfunction and pain caused by TMJ OA. In this review, we looked at the uniqueness of TMJ, the pathogenesis of TMJ OA, and the potential role of MSC-derived exosomes for TMJ cartilage and bone regeneration.

Double layers of gold nanoparticles immobilized titanium implants improve the osseointegration in rabbit models.

Osseointegration is important in osteopenia and osteoporosis patients due to their low bone densities. Gold nanoparticles (GNPs) are greatly beneficial materials as osteogenic agents. The aim of this study is to investigate osseointegration between bones and double layers of GNP-immobilized titanium (Ti) implants. The physicochemical properties of the Ti surface were evaluated by scanning electron microscopy, by atomic force microscopy, by means of the contact angle using water drops, and by x-ray photoelectron spectroscopy. Osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells was analyzed and showed the higher values in double layers of GNP (GNP2) groups. In addition, we performed an in vivo study using hydroxyapatite (HA) and GNP2 spine pedicle screws in ovariectomized (OVX) and SHAM rabbits. Osseointegration parameters also showed higher values in GNP2 than in HA groups. These findings suggest that implants with double layers of GNPs can be a useful alternative in osteoporotic patients.

Comparison of polysaccharides in articular cartilage regeneration associated with chondrogenic and autophagy-related gene expression.

Articular cartilage exhibits reduced self-healing following degeneration. This research evaluated the effects of hydrogels derived from various polysaccharides-gellan gum (GG), alginate, and agarose-on cartilage regeneration compared with that of hyaluronic acid (HA), which is commonly used in cartilage tissue engineering. Chondrocytes were isolated from the articular cartilage of New Zealand White (NZW) rabbits and stimulated with IL-1ß followed by incubation with polysaccharides. The expressions of NF-κB and Cox-2 were decreased and those of IκBα, Sox-9, aggrecan, and type II collagen were increased in HA, GG, and Alginate groups. Osteochondral defects in NZW rabbits were treated with intra-articular polysaccharide injections; all except alginate resulted in tissue regeneration. Significant improvements were observed in cartilage regeneration in the GG and agarose groups. These results show that GG and agarose improve cartilage regeneration by suppressing inflammatory mediators and inducing cartilage formation and autophagy-related gene expression, indicating their potential for cartilage tissue engineering.

Vitamin D-conjugated gold nanoparticles as functional carriers to enhancing osteogenic differentiation.

In an aging society, bone disorders such as osteopenia, osteoporosis, and degenerative arthritis cause serious public health problems. In order to solve these problems, researchers continue to develop therapeutic agents, increase the efficacy of developed therapeutic agents, and reduce side effects. Gold nanoparticles (GNPs) are widely used in tissue engineering applications as biosensors, drug delivery carriers, and bioactive materials. Their special surface property enables easy conjugation with ligands including functional groups such as thiols, phosphines, and amines. This creates an attractive advantage to GNPs for use in the bone tissue engineering field. However, GNPs alone are limited in their biological effects. In this study, we used thiol-PEG-vitamin D (SPVD) to conjugate vitamin D, an essential nutrient critical for maintaining normal skeletal homeostasis, to GNPs. To characterize vitamin D-conjugated GNPs (VGNPs), field emission transmission electron microscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, and ultraviolet/visible absorption analysis were carried out. The developed VGNPs were well bound through the thiol groups between GNPs and vitamin D, and were fabricated in size of 60 nm. Moreover, to demonstrate VGNPs osteogenic differentiation effect, various assays were carried out through cell viability test, alkaline phosphatase assay, calcium deposition assay, real-time polymerase chain reaction, and immunofluorescence staining. As a result, the fabricated VGNPs were found to effectively enhance osteogenic differentiation of human adipose-derived stem cells (hADSCs) in vitro. Based on these results, VGNPs can be utilized as functional nanomaterials for bone regeneration in the tissue engineering field.

Simple and facile preparation of recombinant human bone morphogenetic protein-2 immobilized titanium implant via initiated chemical vapor deposition technique to promote osteogenesis for bone tissue engineering application.

Over the past few decades, titanium (Ti) implants have been widely used to repair fractured bones. To promote osteogenesis, immobilization of osteoinductive agents, such as recombinant human bone morphogenic protein-2 (rhBMP2), onto the Ti surface is required. In this study, we prepared rhBMP2 immobilized on glycidyl methacrylate (GMA) deposited Ti surface through initiated chemical vapor deposition (iCVD) technique. After preparation, the bio-functionalized Ti surface was characterized by physicochemical analysis. For in vitro analysis, the developed Ti was evaluated by cell proliferation, alkaline phosphatase activity, calcium deposition, and real-time polymerase chain reaction to verify their osteogenic activity against human adipose-derived stem cells (hASCs). The GMA deposited Ti surface was found to effectively immobilize a large dose of rhBMP2 as compared to untreated Ti. Additionally, rhBMP2 immobilized on Ti showed significantly enhanced osteogenic differentiation and increased calcium deposition with nontoxic cell viability. These results clearly confirm that our strategy may provide a simple, solvent-free strategy to prepare an osteoinductive Ti surface for bone tissue engineering applications.

Development of 3D printable conductive hydrogel with crystallized PEDOT:PSS for neural tissue engineering.

Bioelectronic devices enable efficient and effective communication between medical devices and human tissue in order to directly treat patients with various neurological disorders. Due to the mechanical similarity to human tissue, hydrogel-based electronic devices are considered to be promising for biological signal recording and stimulation of living tissues. Here, we report the first three-dimensionally (3D) printable conductive hydrogel that can be photocrosslinked while retaining high electrical conductivity. In addition, we prepared dorsal root ganglion (DRG) cell-encapsulated gelatin methacryloyl (GelMA) hydrogels which were integrated with the 3D printed conductive structure and evaluated for efficiency neural differentiation under electrical stimulation (ES). For enhanced electrical conductivity, a poly(3,4-ethylenedioxythiophene) (PEDOT): polystyrene sulfonate (PSS) aqueous solution was freeze-dried and mixed with polyethylene glycol diacrylate (PEGDA) as the photocurable polymer base. Next, the conductive hydrogel was patterned on the substrate by using a table-top stereolithography (SLA) 3D printer. The fabricated hydrogel was characterized for electrochemical conductivity. After printing with the PEDOT:PSS conductive solution, the patterned hydrogel exhibited decreased printing diameters with increasing of PEDOT:PSS concentration. Also, the resultant conductive hydrogel had significantly increased electrochemical properties with increasing PEDOT:PSS concentration. The 3D printed conductive hydrogel provides excellent structural support to systematically transfer the ES toward encapsulated DRG cells for enhanced neuronal differentiation. The results from this study indicate that the conductive hydrogel can be useful as a 3D printing material for electrical applications.

Synergistic interplay between human MSCs and HUVECs in 3D spheroids laden in collagen/fibrin hydrogels for bone tissue engineering.

Stem cell encapsulation in hydrogels has been widely employed in tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such a strategy has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. In particular, scalable bone tissue constructs require vascular network to provide enough oxygen and nutrient supplies to encapsulated cells. To enhance stem cell function and generate pre-vascularized network, we here employed collagen/fibrin hydrogel as an encapsulation matrix for the incorporation of human mesenchymal stem cell/human umbilical vein endothelial cell (MSC/HUVEC) spheroids, and investigated their cellular behavior (including cell viability, morphology, proliferation, and gene expression profile) and compared to that of cell suspension- or MSC spheroids-laden hydrogels. MSC/HUVEC spheroids encapsulated in collagen/fibrin hydrogel showed better cell spreading and proliferation, and up-regulated osteogenic differentiation, and demonstrated pre-vascular network formation. Overall, MSC/HUVEC spheroids-laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering, disease modeling and drug screening. STATEMENT OF SIGNIFICANCE: Stem cell encapsulation in hydrogels has been widely used in various areas such as tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such an approach has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. Here in this work, we have encapsulated spheroids of human mesenchymal stems cells (MSCs) in collagen/fibrin hydrogel and evaluated their viability, proliferation, osteogenic differentiation, and bone formation potential in vitro with respect to cell suspension-laden hydrogel samples. We have further incorporated human umbilical vein endothelial cells (HUVECs) into MSC spheroids and demonstrated that the presence of HUVECs in 3D spheroid culture in collagen/fibrin gel induced the formation of pre-vascular network, improved cell viability and proliferation, enhanced the osteogenic differentiation of spheroids, and increased their bone mineral deposition. In sum, MSC/HUVEC spheroids laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering and regenerative medicine, disease modeling and drug screening.

Injectable hydrogel composite containing modified gold nanoparticles: implication in bone tissue regeneration.

BACKGROUND: For effective bone regeneration, it is necessary to implant a biocompatible scaffold that is capable of inducing cell growth and continuous osteogenic stimulation at the defected site. Here, we suggest an injectable hydrogel system using enzymatic cross-linkable gelatin (Gel) and functionalized gold nanoparticles (GNPs). METHODS: In this work, tyramine (Ty) was synthesized on the gelatin backbone (Gel-Ty) to enable a phenol crosslinking reaction with horseradish peroxidase (HRP). N-acetyl cysteine (NAC) was attached to the GNPs surface (G-NAC) for promoting osteodifferentiation. RESULTS: The Gel-Ty hydrogels containing G-NAC (Gel-Ty/G-NAC) had suitable mechanical strength and biocompatibility to embed and support the growth of human adipose derived stem cells (hASCs) during a proliferation test for three days. In addition, G-NAC promoted osteodifferentiation both when it was included in Gel-Ty and when it was used directly in hASCs. The osteogenic effects were demonstrated by the alkaline phosphatase (ALP) activity test. CONCLUSION: These findings indicate that the phenol crosslinking reaction is suitable for injectable hydrogels for tissue regeneration and G-NAC stimulate bone regeneration. Based on our results, we suggest that Gel-Ty/G-NAC hydrogels can serve both as a biodegradable graft material for bone defect treatment and as a good template for tissue engineering applications such as drug delivery, cell delivery, and various tissue regeneration uses.