Methacrylated Chondroitin Sulfate Research Articles

Local Sustained Dinutuximab Delivery and Release From Methacrylated Chondroitin Sulfate.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. High-risk NB is a subset of the disease that has poor prognosis and requires multimodal treatment regimens, with a 50% rate of recurrence despite intervention. There is a need for improved treatment strategies to reduce high-risk patient mortality. Dinutuximab is an anti-GD2 antibody ideal for targeting GD2 expressing NB cells, but binding of the antibody to peripheral nerve fibers leads to severe pain during systemic administration. Intratumoral delivery of the anti-GD2 antibody would allow for increased local antibody concentration, without increasing systemic toxicity. Chondroitin Sulfate (CS) is a biocompatible glycosaminoglycan that can be methacrylated to form CSMA, a photocrosslinkable hydrogel that can be loaded with therapeutic agents. The methacrylation reaction time can be varied to achieve different degrees of substitution, resulting in different release and degradation profiles. In this work, 4 and 24 h reacted CSMA was used to create hydrogels at 10% and 20% CSMA. Sustained in vitro release of dinutuximab from these formulations was observed over a 24-day period, and 4 h reacted 10% CSMA hydrogels had the highest overall dinutuximab release over time. An orthotropic mouse model was used to evaluate in vivo response to dinutuximab loaded 4 h methacrylated 10% CSMA hydrogels as compared to bolus tail vein injections. Tumor growth was monitored, and there was a statistically significant increase in the days to reach specific tumor size for tumors treated with intratumoral dinutuximab-loaded hydrogel compared to those treated with dinutuximab solution through tail vein injection. This supports the concept that locally delivering dinutuximab within the hydrogel formulation slowed tumor growth. The CSMA hydrogel-only treatment slowed tumor growth as well, an interesting effect that may indicate interactions between the CSMA and cell adhesion molecules in the tumor microenvironment. These findings demonstrate a potential avenue for local sustained delivery of dinutuximab for improved anti-tumoral response in high-risk NB.

GAPDH-Silence Microsphere via Reprogramming Macrophage Metabolism and eradicating Bacteria for Diabetic infection bone regeneration

Macrophage metabolism dysregulation, which is exacerbated by persistent stimulation in infectious and inflammatory diseases, such as diabetic infectious bone defects (DIBD), eventually leads to the failure of bone repair. Here, we have developed an injectable, macrophage-modulated GAPDH-Silence drug delivery system. This microsphere comprises chondroitin sulfate methacrylate (CM) and methacrylated gelatin (GM), while the dimethyl fumarate (DMF)-loaded liposome (D-lip) is encapsulated within the microsphere (CM@GM), named D-lip/CM@GM. Triggered by the over-expressed collagenase in DIBD, the microspheres degrade and release the encapsulated D-lip. D-lip could modulate metabolism by inhibiting GAPDH, which suppresses the over-activation of glycolysis, thus preventing the inflammatory response of macrophages in vitro. While beneficial for macrophages, D-lip/CM@GM is harmful to bacteria. GAPDH, while crucial for glycolysis of staphylococcal species (S. aureus), can be effectively countered by D-lip/CM@GM. We are utilizing existing drugs in innovative ways to target central metabolism for effective eradication of bacteria. In the DIBD model, our results confirmed that the D-lip/CM@GM enhanced bacteria clearance and reprogrammed dysregulated metabolism, thereby significantly improving bone regeneration. In conclusion, this GAPDH-Silence microsphere system may provide a viable strategy to promote diabetic infection bone regeneration.

Chondroitin Sulfate and Hyaluronic Acid-Based PolyHIPE Scaffolds for Improved Osteogenesis and Chondrogenesis In Vitro.

Osteochondral damage, affecting the articular cartilage and the underlying subchondral bone, presents significant challenges in clinical treatment. Such defects, commonly seen in knee and ankle joints, vary from small localized lesions to larger defects. Current medical therapies encounter several challenges, such as donor shortages, drug side effects, high costs, and rejection problems, often resulting in only temporary relief. Highly porous emulsion-templated polymers (polyHIPEs) offer numerous potential benefits in the fabrication of scaffolds for tissue engineering and regenerative medicine. Polymeric scaffolds synthesized using a high internal phase emulsion (HIPE) technique, called PolyHIPEs, involve polymerizing a continuous phase surrounding a dispersed internal phase to form a solid, foam-like structure. A dense, porous design encourages cell ingrowth, nutrient delivery, and waste disposal from the scaffold, mimicking the cells' natural microenvironment. This study used hydroxyethyl methacrylate (HEMA) and acrylamide (AAM) polyHIPE scaffolds combined with extracellular matrix (ECM) components of the tissue, such as methacrylated hyaluronic acid (MHA) and methacrylated chondroitin sulfate (MCS), to prepare polyHIPE scaffolds. The mouse preosteoblast MC3T3-E1 cells and primary rat chondrocytes (harvested from male Wistar rats) were seeded on the scaffolds and cultured for 21 days to assess the osteogenesis and chondrogenesis in vitro. When compared to the AAM-MHA and AAM-MCS groups at day 21, scaffold groups HEMA-MHA and HEMA-MCS showed a significant rise in alkaline phosphatase (ALP) and calcium content. Chondrogenic markers such as glycosaminoglycan (GAG) and hydroxyproline were also assessed over a 21-day time point. On day 21, it was found that GAG and hydroxyproline production were considerably higher in the HEMA-MHA and HEMA-MCS scaffolds than in the AAM-MHA and AAM-MCS scaffolds. The overall studies showed that polyHIPE monolith scaffolds could favor cell adherence, survival ability, proliferation, differentiation, and ECM formation over 21 days. Thus, incorporating ECM components enhanced osteogenesis and chondrogenesis in vitro and can be further used as tissue repair models.

Construction of a dual-component hydrogel matrix for 3D biomimetic skin based on photo-crosslinked chondroitin sulfate/collagen

The main challenge in the field of 3D biomimetic skin is to search for a suitable hydrogel matrix with good biocompatibility, appropriate mechanical property and inner porosity that can support the adhesion and proliferation of skin cells. In this study, photocurable chondroitin sulfate methacrylate (CSMA) and collagen methacrylate (CoLMA) synthesized from chondroitin sulfate (CS) and type I collagen I (CoL) in the dermal matrix were used to construct a photo-crosslinked dual-component CSMA-CoLMA hydrogel matrix. Due to the toughening effect of the dual-component, the CSMA-CoLMA hydrogel improved the intrinsic brittleness of the single-component CSMA hydrogel, presented good mechanical tunability. The average storage and elasticity modulus could reach 3.3 KPa and 30.3 KPa, respectively, which were close to those of natural skin. The CSMA-CoLMA hydrogel with a ratio of 8/6 showed suitable porous structure and good biocompatibility, supporting the adhesion and proliferation of skin cells. Furthermore, the expression of characteristic marker proteins was detected in the epidermal and dermal bi-layered models constructed with the hydrogel containing keratinocytes and fibroblasts. These results suggest that the dual-component CSMA-CoLMA hydrogel has promising potential as a matrix to construct 3D biomimetic skin.

Reprogramming macrophages via immune cell mobilized hydrogel microspheres for osteoarthritis treatments

Regulating macrophage activation precisely is crucial in treating chronic inflammation in osteoarthritis (OA). However, the stable pro-inflammatory state and deep distribution of macrophages in vivo pose a great challenge to treatment. In this study, inspired by the innate immune, immune cell mobilized hydrogel microspheres were constructed by microfluidic methods and load chemokines, macrophage antibodies and engineered cell membrane vesicles (sEVs) via covalent and non-covalent junctions. The immune cell mobilized hydrogel microspheres, based on a mixture of streptavidin grafted hyaluronic acid methacrylate (HAMA-SA) and Chondroitin sulfate methacrylate (ChSMA) microspheres (HCM), can recruit, capture and reprogram proinflammatory macrophages in the joint cavity to improve the joint inflammatory microenvironment. In vitro experiments demonstrated that immune cell mobilized hydrogel microspheres had excellent macrophage recruitment, capture, and reprogramming abilities. Pro-inflammatory macrophages can be transformed into anti-inflammatory macrophages with an efficiency of 88.5 %. Animal experiments also revealed significant reduction in synovial inflammation and cartilage matrix degradation of OA. Therefore, the immune cell mobilized hydrogel microspheres may be an effective treatment of OA inflammation for the future.

Eggshell integrated GelMA/CSMA/HyMA hybrid hydrogels for cell therapy/tissue engineering

AbstractThe use of biocompatible materials with improved behaviors for potential applications in the area of tissue engineering has increased as the developments in biomaterials have increased in recent years. Current approaches concentrate on hybrid combinations of synthetic and natural polymers. In this context, obtaining porous scaffolds with good mechanical properties is still challenging. Herein, a hybrid tissue scaffold consisting of methacrylated gelatin (GelMA), methacrylated chondroitin sulfate (CSMA), methacrylated hyaluronic acid (HyMA) and eggshell particles with enhanced pore morphology and mechanical property was fabricated by photo‐polymerization. The effect of different amounts of eggshell particles on the behaviors of hybrid hydrogel scaffolds was investigated. It was determined by scanning electron microscope analysis (SEM) that the particles were well‐dispersed and triggered the formation of an interconnected porous structure in the hybrid tissue scaffold containing 10% by weight of eggshell particles. It was concluded that the mechanical behaviors of the hybrid tissue scaffold containing 10% by weight of eggshell particles improved by 47% compared to the neat hydrogel scaffold. There were no toxic effects of eggshell particles‐integrated scaffolds against osteoblast cells. In addition, cells successfully adhered onto the scaffolds and proliferated. Consequently, GelMA/CSMA/HyMA scaffolds containing 10% eggshell particles were found to be biocompatible and suitable for cell therapy and tissue engineering applications.

3D printing a universal knee meniscus using a custom collagen ink

Tears of the meniscus are among the most commonly diagnosed knee injuries. Because most of the meniscus lacks the ability to self-heal due to its low vascularity, surgical intervention is needed in more than 85% of cases. Tissue-engineered meniscal implants may provide a treatment strategy that better supports healing and long-term health and mobility benefits. We used three-dimensional printing to develop a “universal” human meniscal tissue repair device that can be trimmed to match the corresponding area of damage debrided during the patient's surgical repair. Computer aided design software was used to design an adult meniscus of average shape based on published physical dimensions. To reproduce the natural fiber arrangement found in the meniscus, the tool path for 3D bioprinting was structured to use alternating layers of circumferential and radial extrusions. We also developed extrudable, shear thinning bioinks based on meniscus biochemical components, including collagen I methacrylate, collagen II, and chondroitin sulfate methacrylate. The combination of this tissue-specific bioink and the deposition pattern to build the meniscus are novel. Ink formulations were evaluated with rheology to assess the viscosity and post-gelling stiffness. Inks retained shape fidelity when thermally gelled after printing into a support bath, and the fabricated menisci maintained stable dimensions for up to 4 weeks post printing. Bioprinted menisci containing human mesenchymal stem cells were also dimensionally stable, and viable cells were present up to 4 weeks post printing. Increased glycosaminoglycan deposition was noted in the bioprinted meniscus over 21 days, and decorin and collagen type I gene expression increased. Compression testing demonstrated that Young's modulus approaches 100 kPa when molded as a solid object and 45 kPa when extruded into the meniscus shape. This 3D printed, anisotropic meniscus emulates the natural architecture and biochemical composition of the natural human meniscus and has potential to be developed into a device for use in treatment of meniscal injuries.

3D Bioprinting Using Synovium-Derived MSC-Laden Photo-Cross-Linked ECM Bioink for Cartilage Regeneration.

In this study, inspired by the components of cartilage matrix, a photo-cross-linked extracellular matrix (ECM) bioink composed of modified proteins and polysaccharides was presented, including gelatin methacrylate, hyaluronic acid methacrylate, and chondroitin sulfate methacrylate. The systematic experiments were performed, including morphology, swelling, degradation, mechanical and rheological tests, printability analysis, biocompatibility and chondrogenic differentiation characterization, and RNA sequencing (RNA-seq). The results indicated that the photo-cross-linked ECM hydrogels possessed suitable degradation rate and excellent mechanical properties, and the three-dimensional (3D) bioprinted ECM scaffolds obtained favorable shape fidelity and improved the basic properties, biological properties, and chondrogenesis of synovium-derived MSCs (SMSCs). The strong stimulation of transforming growth factor-beta 1 (TGF-β1) enhanced the aggregation, proliferation, and differentiation of SMSCs, thereby enhancing chondrogenic ECM deposition. In vivo animal experiments and gait analysis further confirmed that the ECM scaffold combined with TGF-β1 could effectively promote cartilage regeneration and functional recovery of injured joints. To sum up, the photo-cross-linked ECM bioink for 3D printing of functional cartilage tissue may become an attractive strategy for cartilage regeneration.

Baicalin Nanocomplexes with an In Situ-Forming Biomimetic Gel Implant for Repair of Calvarial Bone Defects via Localized Sclerostin Inhibition.

In situ-forming hydrogels are highly effective in covering complex and irregular tissue defects. Herein, a biomimetic gel implant (CS-GEL) consisting of methacrylated chondroitin sulfate and gelatin is obtained via visible light irradiation, which displays rapid gelation (∼30 s), suitable mechanical properties, and biological features to support osteoblast attachment and proliferation. Sclerostin is proven to be a viable target to promote osteogenesis. Hence, baicalin, a natural flavonoid with a high affinity to sclerostin, is selected as the therapeutic compound to achieve localized neutralization of sclerostin. To overcome its poor solubility and permeability, a baicalin nanocomplex (BNP) is synthesized using Solutol HS15, which is then dispersed in the CS-GEL to afford a nanocomposite delivery system, i.e., BNP-loaded gel (BNP@CS-GEL). In vitro, BNP significantly downregulated the level of sclerostin in MLO-Y4 osteocytes. In vivo, either CS-GEL or BNP@CS-GEL is proven to effectively promote osteogenesis and angiogenesis in a calvarial critical-sized bone defect rat model, with BNP@CS-GEL showing the best pro-healing effect. Specifically, the BNP@CS-GEL-treated group significantly downregulated the sclerostin level as compared to the sham group (p < 0.05). RANKL expression was also significantly suppressed by BNP in MLO-Y4 cells and BNP@CS-GEL in vivo. Collectively, our study offers a facile and viable gel platform in combination with nanoparticulated baicalin for the localized neutralization of sclerostin to promote bone regeneration and repair.

The effect of chondroitin sulfate concentration and matrix stiffness on chondrogenic differentiation of mesenchymal stem cells.

Chondroitin sulfate (CS), a glycosaminoglycan of native cartilage, has shown its potential in promoting chondrogenesis of mesenchymal stem cells (MSCs), whereas the effect of matrix stiffness in a CS-containing 3D environment on chondrogenesis is still poorly understood. Herein, this study aimed at assessing the effect of CS concentration and stiffness of CS-containing hydrogels on the chondrogenesis of MSCs. Hydrogels composed of 6% (w/v) gelatin methacryloyl (GelMA) and three concentrations, i.e., 4%, 6%, or 10% (w/v), of methacrylated chondroitin sulfate (CSMA) were prepared. The hydrogels of each composition were prepared with two stiffness values (33.36 ± 8.25 kPa vs. 8.42 ± 2.83 kPa). Physical characterization showed similar microporous structures among the six groups, higher swelling ratios and faster degradation in the soft hydrogel groups. MSCs were encapsulated in the six groups of hydrogels and they underwent 28-day chondrogenic differentiation. The cell viability in each group on day 1 was similar and most cells exhibited a round shape without spreading. Afterwards, cellular protrusions in soft hydrogels remained filopodium-like from day 14 to day 28, while most protrusions were lamellipodium-like in stiff hydrogels on day 14 and then transformed into a spherical shape on day 28. The expression of chondrogenic markers analysed by real-time qPCR and immunohistochemical staining demonstrated that the optimal CS concentration for chondrogenesis was 6% (w/v) regardless of the stiffness of hydrogels. In addition, with the same CSMA concentration, the trend was observed that the stiff hydrogels supported superior chondrogenesis of MSCs compared to the soft hydrogel. To summarize, this study presents an advancement in the optimization of CSMA concentration and stiffness of hydrogels for chondrogenesis. In the CSMA/GelMA hydrogel, 6% (w/v) CSMA with an initial Young's modulus around 33 kPa was recommended for cartilage tissue engineering.

The effect of HydroSpacer implant placement on the wear of opposing and adjacent cartilage.

A HydroSpacer implant, that is,a swelling hydrogel confined by a spacer fabric, was developed to repair focal cartilage defects and to prevent progression into osteoarthritis. The present study evaluated the effect of implant placement height in an osteochondral (OC) plug on wear of the opposing and adjacent cartilage. Three-dimensional warp-knitted spacer fabrics, polycaprolactone with poly(4-hydroxybutyrate) pile yarns, were filled with a hyaluronic acid methacrylate and chondroitin sulfate methacrylate hydrogel. After polymerization of the hydrogel, these HydroSpacers were implanted in OC defects (ø 6 mm) created in bovine OC plugs (ø 10 mm) and allowed to swell to equilibrium. A custom-made pin-on-plate wear apparatus was used to apply simultaneous compression and sliding against bovine cartilage. Cartilage damage, visualized with Indian ink, was only seen for the group in which the HydroSpacer was placed flush with the surrounding cartilage. A significant increase on average surface roughness of the sliding path compared to the adjacent cartilage confirmed surface damage for this group. When the implants were recessed (with and without extra hydrogel layer on top of the implant), this damage was not observed, but the cartilage surrounding the implants was compressed (without damage) indicating substantial load sharing with the implant. Furthermore, it was shown that all defects treated with a HydroSpacer implant resulted in shear forces comparable to intact cartilage.Clinical significance: The present study suggests that placing a HydroSpacer implant recessed into the surrounding cartilage would decrease wear of the opposing cartilage. Altogether, this study supports the development of textile-constraining hydrogels for cartilage replacement.

A semi-interpenetrating network-based microneedle for rapid local anesthesia

To improve the mechanical strength of a polyvinylpyrrolidone (PVP K29/32)-derived dissolvable microneedle, a semi-interpenetrating network is developed using covalently crosslinked methacrylated chondroitin sulfate (CS-MA) in combination with PVP K29/32, which is used to successfully load lidocaine hydrochloride (LIDH) and applied as a microneedle patch for rapid local anesthesia. Specifically, CS-MA when mixed with soluble PVP and photo initiators afforded a covalently cross-linked three-dimensional network (CS-MA/PVP MN) after irradiation at 405 nm for 1 min. CS-MA/PVP MNs demonstrated greatly enhanced mechanical strength and reduced moisture absorption rate as compared to PVP MN. The content of LIDH in each microneedle array was determined as 802.8 ± 22.8 μg. The obtained CS-MA/PVP MNs displayed sharp needle edges with a uniform appearance under scanning electron microscope. Penetration studies showed that the prepared microneedles were able to penetrate the skin of experimental animals causing minimum irritation. The biosafety study showed that the trauma to the skin was almost negligible after the application of microneedles. Furthermore, preliminary pharmacodynamic studies in guinea pigs revealed the prepared microneedles achieved a rapid onset of action (<1 min) compared to the commercially available lidocaine cream (about 1 h). Overall, LIDH-loaded semi-interpenetrating network-based microneedles serve as a promising transdermal delivery platform for achieving rapid and efficient local anesthesia.

Insights into the reaction of chondroitin sulfate with glycidyl methacrylate: 1D and 2D NMR investigation

Chondroitin sulfate methacrylate (CS-MA) is a semisynthetic biopolymer increasingly used for the fabrication of chemical hydrogels. In this study, the methacrylation reaction of native CS was carried out with glycidyl methacrylate in dimethyl sulfoxide and optimized to obtain tunable and reproducible methacrylation degrees in a short reaction time. The methacrylation reaction was deeply characterized by mono- and bi-dimensional (1D, 2D) NMR spectroscopy of CS-MA derivatives with different methacrylation degrees. In contrast to what previously reported in the literature, HSQC, HMBC and TOCSY analyses revealed that the methacrylation reaction proceeds via both epoxy ring-opening and transesterification, involving predominantly the primary hydroxyl groups of CS, while preserving sulfate and carboxyl groups of the biopolymer. These findings are of fundamental importance for appropriate and rational design of CS-MA-based biomaterials.

Hydrolytic (In)stability of Methacrylate Esters in Covalently Cross-Linked Hydrogels Based on Chondroitin Sulfate and Hyaluronic Acid Methacrylate.

Chondroitin sulfate (CS) and hyaluronic acid (HA) methacrylate (MA) hydrogels are under investigation for biomedical applications. Here, the hydrolytic (in)stability of the MA esters in these polysaccharides and hydrogels is investigated. Hydrogels made with glycidyl methacrylate-derivatized CS (CSGMA) or methacrylic anhydride (CSMA) degraded after 2–25 days in a cross-linking density-dependent manner (pH 7.4, 37 °C). HA methacrylate (HAMA) hydrogels were stable over 50 days under the same conditions. CS(G)MA hydrogel degradation rates increased with pH, due to hydroxide-driven ester hydrolysis. Desulfated chondroitin MA hydrogels also degrade, indicating that sulfate groups are not responsible for CS(G)MA’s hydrolytic sensitivity (pH 7.0–8.0, 37 °C). This sensitivity is likely because CS(G)MA’s N-acetyl-galactosamines do not form hydrogen bonds with adjacent glucuronic acid oxygens, whereas HAMA’s N-acetyl-glucosamines do. This bond absence allows CS(G)MA higher chain flexibility and hydration and could increase ester hydrolysis sensitivity in CS(G)MA networks. This report helps in biodegradable hydrogel development based on endogenous polysaccharides for clinical applications.

Enhanced Neovascularization Using Injectable and rhVEGF‐Releasing Cryogel Microparticles

Cryogels are gel networks or scaffolds with a large porous structure; they can be tailored for injectability and for possessing a shape-memory ability. Herein, a growth factor-releasing cryogel microparticle (CMP) system is fabricated, and the therapeutic efficacy of recombinant human vascular endothelial growth factor (rhVEGF)-loaded CMP (V-CMP) for neovascularization is investigated. To prepare the cryogels, both methacrylated chitosan (Chi-MA) and methacrylated chondroitin sulfate (CS-MA) are used, and crosslinking using a radical crosslinking reaction is established. The physical, mechanical, and biological properties of the cryogels are analyzed by varying the amount of CS-MA used. The cryogels are then pulverized, and microsized CMPs are fabricated. CMPs dispersed in saline demonstrate a shear-thinning property, and can thus be extruded through a 23G needle. Additionally, V-CMP exhibit a sustained release profile of rhVEGF and enhance the in vitro proliferation of endothelial cells. Finally, neovascularization and effective tissue necrosis prevention are observed when V-CMPs are injected into a hindlimb ischemia mouse model. Thus, the injectable V-CMP system developed herein demonstrates a high potential utility in various tissue regeneration applications based on cell or growth factor delivery.

Fabrication of 3D-Printed Interpenetrating Hydrogel Scaffolds for Promoting Chondrogenic Differentiation.

The limited self-healing ability of cartilage necessitates the application of alternative tissue engineering strategies for repairing the damaged tissue and restoring its normal function. Compared to conventional tissue engineering strategies, three-dimensional (3D) printing offers a greater potential for developing tissue-engineered scaffolds. Herein, we prepared a novel photocrosslinked printable cartilage ink comprising of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and chondroitin sulfate methacrylate (CSMA). The PEGDA-GelMA-CSMA scaffolds possessed favorable compressive elastic modulus and degradation rate. In vitro experiments showed good adhesion, proliferation, and F-actin and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. When the CSMA concentration was increased, the compressive elastic modulus, GAG production, and expression of F-actin and cartilage-specific genes (COL2, ACAN, SOX9, PRG4) were significantly improved while the osteogenic marker genes of COL1 and ALP were decreased. The findings of the study indicate that the 3D-printed PEGDA-GelMA-CSMA scaffolds possessed not only adequate mechanical strength but also maintained a suitable 3D microenvironment for differentiation, proliferation, and extracellular matrix production of BMSCs, which suggested this customizable 3D-printed PEGDA-GelMA-CSMA scaffold may have great potential for cartilage repair and regeneration in vivo.

Photocurable Biopolymers for Coaxial Bioprinting.

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication-in one step-of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition.To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers-namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen-that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications.

Investigating the Effects of Tissue-Specific Extracellular Matrix on the Adipogenic and Osteogenic Differentiation of Human Adipose-Derived Stromal Cells Within Composite Hydrogel Scaffolds.

While it has been postulated that tissue-specific bioscaffolds derived from the extracellular matrix (ECM) can direct stem cell differentiation, systematic comparisons of multiple ECM sources are needed to more fully assess the benefits of incorporating tissue-specific ECM in stem cell culture and delivery platforms. To probe the effects of ECM sourced from decellularized adipose tissue (DAT) or decellularized trabecular bone (DTB) on the adipogenic and osteogenic differentiation of human adipose-derived stem/stromal cells (ASCs), a novel detergent-free decellularization protocol was developed for bovine trabecular bone that complemented our established detergent-free decellularization protocol for human adipose tissue and did not require specialized equipment or prolonged incubation times. Immunohistochemical and biochemical characterization revealed enhanced sulphated glycosaminoglycan content in the DTB, while the DAT contained higher levels of collagen IV, collagen VI and laminin. To generate platforms with similar structural and biomechanical properties to enable assessment of the compositional effects of the ECM on ASC differentiation, micronized DAT and DTB were encapsulated with human ASCs within methacrylated chondroitin sulfate (MCS) hydrogels through UV-initiated crosslinking. High ASC viability (>90%) was observed over 14 days in culture. Adipogenic differentiation was enhanced in the MCS+DAT composites relative to the MCS+DTB composites and MCS controls after 14 days of culture in adipogenic medium. Osteogenic differentiation studies revealed a peak in alkaline phosphatase (ALP) enzyme activity at 7 days in the MCS+DTB group cultured in osteogenic medium, suggesting that the DTB had bioactive effects on osteogenic protein expression. Overall, the current study suggests that tissue-specific ECM sourced from DAT or DTB can act synergistically with soluble differentiation factors to enhance the lineage-specific differentiation of human ASCs within 3-D hydrogel systems.

Inhibition of astrocytic differentiation of transplanted neural stem cells by chondroitin sulfate methacrylate hydrogels for the repair of injured spinal cord.

Neural stem cell (NSC) transplantation exerts a therapeutic effect on spinal cord injury (SCI) but is limited to an unregulated differentiation pattern by which NSCs preferentially differentiate into astrocytes, with relatively few neurons. It is well established that the increased NSC-derived astrocytes exhibit aberrant axonal sprouting associated with allodynia-like symptoms of the forepaws. Some strategies have been used to overcome this issue, such as regulation of major pathways, ex vivo gene transfer, and genetic overexpression. However, lack of efficiency, viral vector safety issues and the risk of tumorigenesis have hindered the clinical application of these treatments. Here, we show that astrocytic differentiation of NSCs in vitro and in vivo can be inhibited by encapsulation of cells in a three-dimensional chondroitin sulfate methacrylate (CSMA) hydrogel. When CSMA hydrogels were used to transplant NSCs, the combinatory implant promoted functional recovery and attenuated the hypersensitivity responses of the forepaws. Further analysis showed that transplantation of NSCs within CSMA hydrogels reduced injured cavity areas and promoted neurogenesis rather than fibroglial formation after graft implantation. Furthermore, the treatment prevented allodynia-related CGRP/GAP43-positive nociception due to fibers sprouting into inappropriate lamina regions. Taken together, these findings show that CSMA/NSCs combined transplantation helps prevent adverse side effects of NSCs treatment and promotes recovery of SCI.

Optimizing Glutaraldehyde-Fixed Tissue Heart Valves with Chondroitin Sulfate Hydrogel for Endothelialization and Shielding against Deterioration.

Porcine glutaraldehyde-fixed pericardium is widely used to replace human heart valves. Despite the stabilizing effects of glutaraldehyde fixation, the lack of endothelialization and the occurrence of immune reactions contribute to calcification and structural valve deterioration, which is particularly significant in young patients, in whom valve longevity is crucial. This report shows an optimization system with which to enhance endothelialization of fixed pericardium to mimic the biological function of a native heart valve. The glutaraldehyde detoxification, together with the application of a biodegradable methacrylated chondroitin sulfate hydrogel, reduces aldehydes cytotoxicity, increases the migration and proliferation of endothelial cells and the recruitment of endothelial cell progenitors, and confers thromboresistance in fixed pericardium. The combination of glutaraldehyde detoxification and a coating with chondroitin sulfate hydrogel promotes in situ mechanisms of endothelialization in fixed pericardium. We offer a new solution for improving the long life of bioprosthetic valves and exploring the means of making valves suitable to endothelialization.