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![Characterization of methacrylate-modified hyaluronic acid. A) Schematic of transesterification reaction between methacrylic anhydride (MA) and hyaluronic acid (HA) (see van Dijk-Wolthuis et al. [45], Smeds et al. [39], and Derouet et al. [51] for details on reaction mechanism). B) Proton NMR spectrum of purified product of HA reacted with a 20-fold molar ratio of MA. Degree of modification was determined by comparing the ratio of the areas under the proton peaks at 5.6 and 6.1 ppm (methacrylate protons, indicated by bracket and arrow) to the peak at 1.9 ppm (N-acetyl glucosamine of HA, indicated by arrow) after performing a standard deconvolution algorithm to separate closely spaced peaks. C) Enzymatic degradation (50 U mL À1 hyaluronidase, 37 C, pH 7.4) of hydrogels photocrosslinked from methacrylate-modified HA with varying degrees of modification. Hydrogels were photocrosslinked using the UV-activated photoinitiator Irgacure 2959. Data shown here represent a single experiment (n ¼ 5 for each group), which was repeated two additional times to confirm results. Error bars represent standard deviation.](profile/Zin-Khaing-2/publication/41508490/figure/fig1/AS:1002172384833537@1615947924921/Characterization-of-methacrylate-modified-hyaluronic-acid-A-Schematic-of.png)
Characterization of methacrylate-modified hyaluronic acid. A) Schematic of transesterification reaction between methacrylic anhydride (MA) and hyaluronic acid (HA) (see van Dijk-Wolthuis et al. [45], Smeds et al. [39], and Derouet et al. [51] for details on reaction mechanism). B) Proton NMR spectrum of purified product of HA reacted with a 20-fold molar ratio of MA. Degree of modification was determined by comparing the ratio of the areas under the proton peaks at 5.6 and 6.1 ppm (methacrylate protons, indicated by bracket and arrow) to the peak at 1.9 ppm (N-acetyl glucosamine of HA, indicated by arrow) after performing a standard deconvolution algorithm to separate closely spaced peaks. C) Enzymatic degradation (50 U mL À1 hyaluronidase, 37 C, pH 7.4) of hydrogels photocrosslinked from methacrylate-modified HA with varying degrees of modification. Hydrogels were photocrosslinked using the UV-activated photoinitiator Irgacure 2959. Data shown here represent a single experiment (n ¼ 5 for each group), which was repeated two additional times to confirm results. Error bars represent standard deviation.
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We report the ability to direct the differentiation pathway of neural progenitor cells (NPCs) within hydrogels having tunable mechanical properties. By modifying the polymeric sugar hyaluronic acid (HA), a major extracellular matrix component in the fetal mammalian brain, with varying numbers of photocrosslinkable methacrylate groups, hydrogels cou...
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... was modified using methacrylate groups with a predictable degree of substitution (defined as the percentage of methacrylates per disaccharide) over a fivefold range by varying the molar ratio of MA to HA during transesterification (Fig. 1a). Ratios of 5-, 10-and 20-fold MA to HA were used, yielding MA-modified HA (MAHA) with 37 AE 18% (n ¼ 13), 87 AE 22% (n ¼ 12), and 160 AE 32% (n ¼ 9) degrees of substitution (mean AE standard deviation), respectively, as determined from H þ NMR (Table 1). Because there are four hydroxyls available for modification per HA disaccharide, ...
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... HA (MAHA) with 37 AE 18% (n ¼ 13), 87 AE 22% (n ¼ 12), and 160 AE 32% (n ¼ 9) degrees of substitution (mean AE standard deviation), respectively, as determined from H þ NMR (Table 1). Because there are four hydroxyls available for modification per HA disaccharide, substitution degrees up to 400% are theoretically possible. The schematic in Fig. 1a depicts transesterification at the site most readily available for modification. Hereafter, hydrogels produced using MAHA with different degrees of substitution will be referred to as 5Â, 10Â and 20Â MAHA hydrogels, to define the molar ratio of MA to HA used for HA ...
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... methods have reported only a single purification step, either precipitation in ethanol [24,39,46] or dialysis against water [40]. When only one of these purification methods was performed, we observed impurities on H þ NMR spectra that appeared as additional splitting of the methacrylate peaks ( Supplementary Fig. S1a), indicating the presence of unreacted MA or methacrylic acid contaminants in the product. Therefore, we devised a purification scheme that included precipitating twice in cold ethanol, then dialyzing against water for at least 72 h. ...
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... MA or methacrylic acid contaminants in the product. Therefore, we devised a purification scheme that included precipitating twice in cold ethanol, then dialyzing against water for at least 72 h. After completion of both purification steps, extraneous proton peaks were not observed on H þ NMR spectra of the product, indicating a purified product ( Fig. 1b; Supplementary Fig. S1b, c). We assume that unreacted MA and methacrylic acid contaminants were removed during ethanol precipitation; however, some MA and methacrylic acid likely became entrapped in the large HA chains even after extensive rinsing in ethanol. Thus, these molecules were removed by molecular weight separation during ...
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... were crosslinked from MAHA (5Â, 10Â, 20Â) using I2959 as a radical photoinitiator and were degraded in vitro by hyaluronidase (Fig. 1c). In general, degradation rates correlated with degree of methacrylation, with the most highly substituted MAHA hydrogels degrading slowest. Significant differences in weight loss between different hydrogels were first observed at the 4 h time point, when 50-60% weight loss had already occurred for all three hydrogel types. By 6 h, 5Â ...
Citations
... Hydrogel solutions were prepared by mixing 0.75 mg/mL laminin I (R&D Systems, 34446-005-01), 1.25 mg/ mL MAHA (Sigma-Aldrich, 53747-10G) and 4.5 mg/mL sodium hydroxide neutralized collagen type I (IBIDI, 50205) in 1X PBS (pH 7.3-7.5). MAHA was prepared via methacrylation using methacrylic anhydride as previously described by Seidlits et al. [91] and used previously by our lab [89,90]. The degree of MAHA methacrylation used in this study ranged between 85 to 115%. ...
Low back pain, knee osteoarthritis, and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Axonal dieback induced by capsaicin (Caps) denervation has been FDA-approved to treat painful neuropathies and knee osteoarthritis but with short-term efficacy and discomfort. Herein, we propose to evaluate pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as axonal dieback compounds for denervation with potential to alleviate pain. Previous literature suggests Pyr, Vcr, and Imy can cause undesired axonal degeneration, but no previous work has evaluated axonal dieback and cytotoxicity on adult rat dorsal root ganglia (DRG) explants. Thus, we performed axonal dieback screening using adult rat DRG explants in vitro with Caps as a positive control and assessed cytotoxicity. Imy inhibited axonal outgrowth and slowed axonal dieback, while Pyr and Vcr at high concentrations produced significant reduction in axon length and robust axonal dieback within three days. DRGs treated with Caps, Vcr, or Imy had increased DRG cytotoxicity compared to matched controls, but overall cytotoxicity was minimal and at least 88% lower compared to lysed DRGs. Pyr did not lead to any DRG cytotoxicity. Further, neither Pyr nor Vcr triggered intervertebral disc cell death or affected cellular metabolic activity after three days of incubation in vitro. Overall, our findings suggest Pyr and Vcr are not toxic to DRGs and intervertebral disc cells, and there is potential for repurposing these compounds for axonal dieback compounds to cause local denervation and alleviate pain.
... The bioprinting process is controlled and can produce constructs previously determined in size, thickness and shape [199,200,202]. Accordingly, 3D culturing and bioprinting increase the dimension of exploratory evaluations of cell-cell [189,190,203,204], cell-hydrogel [205] and cell-ECM interactions [206] in processes of cell migration [207], shape remodeling [122], differentiation [208,209], neuropathological paradigms [210,211] and so on, with greater chance of compatibility with in vivo conditions. A 3D collagen hydrogel-based culture was successfully employed with astrocytes, thus generating a culture system that consistently generates less reactive state, resembling undamaged brain phenotypes. ...
Astrocytes are the predominant glial cells that provide essential support to neurons and promote microenvironment changes in neuropathological states. Astrocyte and astrocytic-like cell culture have substantially contributed to elucidating the molecular pathways involved in key glial roles, including those relevant to neurodevelopment, brain physiology and metabolism, which are not readily accessible with traditional approaches. The in vitro methodology has also been applied to neuroinflammatory and neurodegeneration contexts, revealing cellular changes involved in brain dysfunction. Astrocytes studies in culture started with primary cell approaches using embryonic and postmortem tissue. Further developments included newborn rodent primary cells, cell lines and immortalized astrocytes, which resulted in homogeneous cell-type preparations grown on flat surfaces. To overcome some in vitro shortcomings, tridimensional bioprinted models and organoid culture enabled the mimicking of tissue cellular arrangements and, above these achievements, complex astrocyte cell culture can be generated from induced pluripotent stem cells (iPSCs) to model diseases. These unprecedented breakthroughs allowed the development of platforms to test new therapies in brain cells derived from human material noninvasively obtained from live patients. In this work, we reviewed the most studied astrocytic cell models for discussing limitations, advantages and reliable experimental readouts for neuroinflammation in neurodegeneration research.
... Hydrogels offer intricate biological environments by modulating their physicochemical and mechanical characteristics through using various materials and crosslinking methods [4,5]. Among the various characteristics of hydrogels, the mechanical properties play a crucial role in cellular functions, including proliferation through mechanotransduction associated with cell-extracellular matrix (ECM) interactions [6][7][8][9][10][11][12]. In vitro models that control the mechanical properties of hydrogels can constitute a more advanced microenvironment, providing valuable insights for performing more accurate drug evaluations [13,14]. ...
Versatile and efficient regulation of the mechanical properties of the extracellular matrix (ECM) is crucial not only for understanding the dynamic changes in biological systems, but also for obtaining precise and effective cellular responses in drug testing. In this study, we developed a well plate-based hydrogel photo-crosslinking system to effectively control the mechanical properties of hydrogels and perform high-throughput assays. We improved cell biocompatibility by using gelatin methacryloyl (GelMA) with a visible light photo-crosslinking method. Multiple cell-laden GelMA hydrogels were simultaneously and uniformly created using multi-arrayed 520 nm light-emitting diodes (LEDs) in a well plate format. The elastic modulus of the hydrogels can be widely adjusted (0.5–30 kPa) using a photo-crosslinking system capable of independently controlling the light intensity or exposure time for multiple samples. We demonstrate the feasibility of our system by observing enhanced bone differentiation of human mesenchymal stem cells (hMSCs) cultured on stiffer hydrogels. Additionally, we observed that the osteogenic fate of hMSCs, affected by the different mechanical properties of the gel, was regulated by parathyroid hormone (PTH). Notably, in response to PTH, hMSCs in a high-stiffness microenvironment upregulate osteogenic differentiation while exhibiting increased proliferation in a low-stiffness microenvironment. Overall, the developed system enables the generation of multiple cell-laden 3D cell culture models with diverse mechanical properties and holds significant potential for expansion into drug testing.
... (2010) 48 showed that astrocytes grown on hydrogels expressed lower levels of GFAP compared to those grown on glass coverslips, which exhibit a reactive phenotype. This suggests that astrocytes cultured in a 3D environment may be in a more quiescent state. ...
... This suggests that astrocytes cultured in a 3D environment may be in a more quiescent state. 48 ...
Combinations of different biomaterials with their own advantages as well as functionalization with other components have long been implemented in tissue engineering to improve the performance of the overall material. Biomaterials, particularly hydrogel platforms, have shown great potential for delivering compounds such as drugs, growth factors, and neurotrophic factors, as well as cells, in neural tissue engineering applications. In central the nervous system, astrocyte reactivity and glial scar formation are significant and complex challenges to tackle for neural and functional recovery. GelMA hydrogel‐based tissue constructs have been developed in this study and combined with two different formulations of phosphate glass fibers (PGFs) (with Fe ³⁺ or Ti ²⁺ oxide) to impose physical and mechanical cues for modulating astrocyte cell behavior. This study was also aimed at investigating the effects of lithium‐loaded GelMA‐PGFs hydrogels in alleviating astrocyte reactivity and glial scar formation offering novel perspectives for neural tissue engineering applications. The rationale behind introducing lithium is driven by its long‐proven therapeutic benefits in mental disorders, and neuroprotective and pronounced anti‐inflammatory properties. The optimal concentrations of lithium and LPS were determined in vitro on primary rat astrocytes. Furthermore, qPCR was conducted for gene expression analysis of GFAP and IL‐6 markers on primary astrocytes cultured 3D into GelMA and GelMA‐PGFs hydrogels with and without lithium and in vitro stimulated with LPS for astrocyte reactivity. The results suggest that the combination of bioactive phosphate‐based glass fibers and lithium loading into GelMA structures may impact GFAP expression and early IL‐6 expression. Furthermore, GelMA‐PGFs (Fe) constructs have shown improved performance in modulating glial scarring over GFAP regulation.
... It has been discovered that when NPCs are enclosed in HA hydrogels, they develop into astrocytes or neurons, depending on the mechanical characteristics of the hydrogel. Because of its capacity to influence the fate of NPCs, HA is a significant tool in the treatment of neurodegenerative illnesses [41][42][43][44][45][46][47]. ...
... Due to the need for a coupling agent or a photo/radical initiator in existing crosslinking chemistries, the potential presence of free radicals may be damaging to the adjacent tissues. [14] HA hydrogels overcome intrinsic limitations of the most commonly used solid type-I collagen scaffolds such as to instruct chondrogenesis and challenging in situ fixation. However, the effect of photocrosslinkable acellular HA-TEG-coumarin hydrogels on chondrogenesis following MFX remains to be determined. ...
Articular cartilage defects represent an unsolved clinical challenge. Photopolymerizable hydrogels are attractive candidates supporting repair. This study investigates the short‐term safety and efficacy of two novel hyaluronic acid (HA)‐triethylene glycol (TEG)‐coumarin hydrogels photocrosslinked in situ in a clinically relevant large animal model. It is hypothesized that HA‐hydrogel‐augmented microfracture (MFX) is superior to MFX in enhancing early cartilage repair, and that the molar degree of substitution and concentration of HA affects repair. Chondral full‐thickness defects in the knees of adult minipigs are treated with either 1) debridement (No MFX), 2) debridement and MFX, 3) debridement, MFX, and HA hydrogel (30% molar derivatization, 30 mg mL⁻¹ HA; F3) (MFX+F3), and 4) debridement, MFX, and HA hydrogel (40% molar derivatization, 20 mg mL⁻¹ HA; F4) (MFX+F4). After 8 weeks postoperatively, MFX+F3 significantly improves total macroscopic and histological scores compared with all other groups without negative effects, besides significantly enhancing the individual repair parameters “defect architecture,” “repair tissue surface” (compared with No MFX, MFX), and “subchondral bone” (compared with MFX). These data indicate that photopolymerizable HA hydrogels enable a favorable metastable microenvironment promoting early chondrogenesis in vivo. This work also uncovers a mechanism for effective HA‐augmented cartilage repair by combining lower molar derivatization with higher concentrations.
... [13] Besides, an easy-and fine-tuning of the mechanical properties is possible thanks to this control of the photopolymerization conditions, which has a large impact in its biological performance. [10,[14][15][16] In particular, stiffness is a crucial material parameter that influences cellular behavior. According to Daniele et al., a higher spreading of adherent cells was observed with increasing stiffness of GelMA-based hydrogels. ...
From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization have evolved extensively. A number of properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, we describe the influence of the reaction media and buffer media –phosphate buffer saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations– in GelMA hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco‐2 and HCT‐116). All scaffolds were photocrosslinked using UV light under identical conditions and evaluated for mass swelling ratio and stiffness. Our results indicate that stiffness is highly susceptible to the reaction media, but not to the swelling media. In addition, PBS‐prepared hydrogels exhibited a higher photopolymerization degree as confirmed by HR‐MAS NMR. These findings correlate with the biological response of Caco‐2 and HCT‐116 cells seeded on the substrates, which demonstrated flatter morphologies on stiffer hydrogels. Overall, cell viability and proliferation were excellent for both cell lines, and Caco‐2 cells displayed a characteristic apical‐basal polarization as observed in F‐actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture.
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... To overcome these challenges, we took advantage of a microneedle (MN) array, which has facilitated painless localized delivery of drugs or therapeutic biomolecules with good tolerability in clinical trials [24][25][26] . We then fabricated a patch with an MN array with appropriate mechanical strength matching soft spinal tissues and with a suitable pore size for MSC-EV delivery when it was mounted onto a spinal cord lesion beneath the dura 27,28 . To sustainably deliver MSC-EVs, the MN arrays were additionally mounted with a gelatin methacryloyl (GelMA) hydrogel block embedded with MSCs during surgery (Fig. 1). ...
... Quantitative analysis with the CCK-8 test indicated that MSCs in the GelMA hydrogel of the MN-MSC patch survived and proliferated well (Fig. 2d). The needle with a porous structure exhibited comparatively lower mechanical strength than the conventional needle (porous 10.77 kPa vs traditional 30.92 kPa, Fig. S3e), and matched that of the soft rat's spinal cord tissues (around 8.1 kPa) 27,28 . We next assessed the MSC-EV release capacity of the MN patch with this porous structure. ...
The transplantation of mesenchymal stem cells-derived secretome, particularly extracellular vesicles is a promising therapy to suppress spinal cord injury-triggered neuroinflammation. However, efficient delivery of extracellular vesicles to the injured spinal cord, with minimal damage, remains a challenge. Here we present a device for the delivery of extracellular vesicles to treat spinal cord injury. We show that the device incorporating mesenchymal stem cells and porous microneedles enables the delivery of extracellular vesicles. We demonstrate that topical application to the spinal cord lesion beneath the spinal dura, does not damage the lesion. We evaluate the efficacy of our device in a contusive spinal cord injury model and find that it reduces the cavity and scar tissue formation, promotes angiogenesis, and improves survival of nearby tissues and axons. Importantly, the sustained delivery of extracellular vesicles for at least 7 days results in significant functional recovery. Thus, our device provides an efficient and sustained extracellular vesicles delivery platform for spinal cord injury treatment.
... As shown in Fig. 2G, the average compression modulus of GM was higher than that of MNEVs@GM (GM: 628.6 ± 10.2 Pa; MNEVs@GM: 590.4 ± 10.4 Pa). According to a previous study, the compressive moduli of native neural tissue were in the range of 300 to 1000 Pa [41]. The mechanical properties of GM and MNEVs@GM both matched neural tissue mechanics, which was beneficial for their in vivo application. ...
... Drug stock solutions were prepared in either 1X PBS or EtOH solvent then diluted in incomplete DRG media until the desired nal concentrations in gels are achieved. To prepare the drug stock solutions, 2122.6 mg/mL Capsaicin MAHA was prepared via methacrylation using methacrylic anhydride as previously described by Seidlits et al. 102 and used previously by our lab 90,91 . The degree of MAHA methacrylation used in this study ranged between 85-115%. ...
Low back pain, knee osteoarthritis and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Retraction of nerve fibers via capsaicin (Caps) or resiniferatoxin denervation have been used to treat painful neuropathies and knee osteoarthritis but with short-term efficacy and discomfort. Herein, we propose to repurpose pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as potential axonal retraction compounds for denervation. Previous literature suggests Pyr, Vcr, and Imy each have undesired axonal degeneration as an off-target effect. Thus, we performed axonal retraction phenotype screening using adult rat dorsal root ganglia explants in vitro and assessed neurotoxicity. Imy did not induce axonal retraction, while Pyr and Vcr produced robust axonal retraction within three days. All DRGs treated with Caps, Pyr, Vcr, Imy or solvent had minimal neurotoxicity. Further, neither Pyr nor Vcr triggered nucleus pulposus cell death or affected cellular metabolic activity after three days of incubation. Overall, our findings suggest Pyr and Vcr are non-cytotoxic to dorsal root ganglia and nucleus pulposus cells, and there is potential for repurposing as axonal retraction compounds for local denervation.
