Nanosized Silk-Magnesium Complexes for Promotion of Angiogenic and Osteogenic Activities.

Injectable hydrogels with osteogenic and angiogenetic properties are of interest in bone tissue engineering. Since the bioactivity of ions is concentration-dependent, nanosized silk-magnesium (Mg) complexes were previously developed and assembled into hydrogels with angiogenic capabilities but failed to control both osteogenic and angiogenetic activities effectively. Here, nanosized silk particles with different sizes were obtained by using ultrasonic treatment to control silk-Mg coordination and particle formation, resulting in silk-Mg hydrogels with different types of bioactivity. Fourier transform infrared and X-ray diffraction results revealed that different coordination intensities were present in the different complexes as a basis for the differences in activities. Slow Mg ion release was controlled by these nanosized silk-Mg complexes through degradation. With the same amount of Mg ions, the different silk-Mg complexes exhibited different angiogenic and osteogenic capacities. Complexes with both angiogenic and osteogenic capacities were developed by optimizing the sizes of the silk particles, resulting in faster and improved quality of bone formed in vivo than complexes with the same composition of silk and Mg but only angiogenic or osteogenic capacities. The biological selectivity of silk-Mg complexes should facilitate applications in tissue regeneration.

Nanosized Silk-Magnesium Complexes for Tissue Regeneration.

Metal ions provide multifunctional signals for cell and tissue functions, including regeneration. Inspired by metal-organic frameworks (MOFs), nanosized silk protein aggregates with a high negative charge density are used to form stable silk-magnesium ion complexes. Magnesium ions (Mg ions) are added directly to silk nanoparticle solutions, inducing gelation through the formation of silk-Mg coordination complexes. The Mg ions are released slowly from the nanoparticles through diffusion, with sustained release via tuning the degradation or dissolution of the nanosized silk aggregates. Studies in vitro reveal a dose-dependent influence of Mg ions on angiogenic and anti-inflammatory functions. Silk-Mg ion complexes in the form of hydrogels also stimulate tissue regeneration with a reduced formation of scar tissue in vivo, suggesting potential utility in tissue regeneration.

Tourniquet Use Improves Intraoperative Parameters, Leading to Similar Postoperative Outcomes Compared With No Tourniquet Use in Anterior Cruciate Ligament Reconstruction: A Prospective, Double-Blind, Randomized Clinical Trial.

PURPOSE: To examine the effect of tourniquet use in arthroscopic anterior cruciate ligament reconstruction in terms of: (1) intraoperative visualization with operative time and consumption of sterile saline, and (2) intra- and postoperative blood loss, postoperative pain, opioid consumption, swelling, serum creatine phosphokinase (CPK) and hemoglobin (Hb) concentrations, clinical outcomes, and graft healing. METHODS: In this prospective randomized clinical trial, patients were assigned to tourniquet inflation (tourniquet-up) or tourniquet deflation (tourniquet-down) groups. Primary outcomes were intraoperative visualization with operative time and sterile saline consumption. Secondary outcomes were intra- and postoperative blood loss, postoperative pain, opioid consumption, swelling, serum CPK, Hb concentration, subjective and objective functional scores, and graft healing. RESULTS: Intraoperative visualization was satisfactory in 100 of 100 cases in the tourniquet-up group and 64 of 100 cases in the tourniquet-down group (P < .05). The mean operative time was 58.4 ± 5.7 minutes in the tourniquet-up group and 72.5 ± 8.6 minutes in the tourniquet-down group (P < .05). The mean sterile saline consumption was 6.4 ± 2.5 L in the tourniquet-up group and 8.7 ± 4.6 L in the tourniquet-down group (P < .05). The respective amounts of estimated intraoperative and postoperative blood loss were 95.3 ± 25.1 mL and 240.3 ± 44.5 mL in the tourniquet-up group and 230.2 ± 22.3 mL and 75.6 ± 15.3 mL in the tourniquet-down group (P < .05). Our results showed no significant difference in postoperative pain, opioid consumption, percentage of patients using opioids, swelling, mean serum CPK and Hb levels, subjective and objective functional scores, or graft healing (P > .05) between the 2 groups. CONCLUSIONS: Tourniquet use during anterior cruciate ligament reconstruction significantly improves intraoperative visualization, shortens operative time, and decreases intraoperative sterile saline consumption and blood loss without serious adverse events or greater complication rates based on early postoperative outcomes. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Macroporous Silk Nanofiber Cryogels with Tunable Properties.

Cryogels are widely used in tissue regeneration due to their porous structures and friendly hydrogel performance. Silk-based cryogels were developed but failed to exhibit desirable tunable properties to adapt various biomedical applications. Here, amorphous short silk nanofibers (SSFs) were introduced to fabricate silk cryogels with versatile cues. Compared to previous silk cryogels, the SSF cryogels prepared under same conditions showed significantly enhanced mechanical properties. The microporous cryogels were achieved under lower silk concentrations, confirming better tunability. Versatile cryogels with the modulus in the range of 0.5-283.7 kPa were developed through adjusting silk concentration and crosslinking conditions, superior to previous silk cryogel systems. Besides better cytocompatibility, the SSF cryogels were endowed with effective mechanical cues to control osteogenetic differentiation behaviors of BMSCs. The mechanical properties could be further regulated finely through the introduction of ß-sheet-rich silk nanofibers (SNFs), which suggested possible optimization of mechanical niches. Bioactive cargo-laden SNFs were introduced to the SSF cryogel systems, bringing biochemical signals without the compromise of mechanical properties. Versatile SNF-based cryogels with different physical and biological cues were developed here to facilitate the applications in various tissue engineering.

Nerve Growth Factor-Laden Anisotropic Silk Nanofiber Hydrogels to Regulate Neuronal/Astroglial Differentiation for Scarless Spinal Cord Repair.

Scarless spinal cord regeneration remains a challenge due to the complicated microenvironment at lesion sites. In this study, the nerve growth factor (NGF) was immobilized in silk protein nanofiber hydrogels with hierarchical anisotropic microstructures to fabricate bioactive systems that provide multiple physical and biological cues to address spinal cord injury (SCI). The NGF maintained bioactivity inside the hydrogels and regulated the neuronal/astroglial differentiation of neural stem cells. The aligned microstructures facilitated the migration and orientation of cells, which further stimulated angiogenesis and neuron extensions both in vitro and in vivo. In a severe rat long-span hemisection SCI model, these hydrogel matrices reduced scar formation and achieved the scarless repair of the spinal cord and effective recovery of motor functions. Histological analysis confirmed the directional regenerated neuronal tissues, with a similar morphology to that of the normal spinal cord. The in vitro and in vivo results showed promising utility for these NGF-laden silk hydrogels for spinal cord regeneration while also demonstrating the feasibility of cell-free bioactive matrices with multiple cues to regulate endogenous cell responses.

Silk Biomaterials for Bone Tissue Engineering.

Silk is a natural fibrous polymer with application potential in regenerative medicine. Increasing interest remains for silk materials in bone tissue engineering due to their characteristics in biocompatibility, biodegradability and mechanical properties. Plenty of the in vitro and in vivo studies confirmed the advantages of silk in accelerating bone regeneration. Silk is processed into scaffolds, hydrogels, and films to facilitate different bone regenerative applications. Bioactive factors such as growth factors and drugs, and stem cells are introduced to silk-based matrices to create friendly and osteogenic microenvironments, directing cell behaviors and bone regeneration. The recent progress in silk-based bone biomaterials is discussed and focused on different fabrication and functionalization methods related to osteogenesis. The challenges and potential targets of silk bone materials are highlighted to evaluate the future development of silk-based bone materials.

Nerve Guidance Conduits with Hierarchical Anisotropic Architecture for Peripheral Nerve Regeneration.

Nerve guidance conduits with multifunctional features could offer microenvironments for improved nerve regeneration and functional recovery. However, the challenge remains to optimize multiple cues in nerve conduit systems due to the interplay of these factors during fabrication. Here, a modular assembly for the fabrication of nerve conduits is utilized to address the goal of incorporating multifunctional guidance cues for nerve regeneration. Silk-based hollow conduits with suitable size and mechanical properties, along with silk nanofiber fillers with tunable hierarchical anisotropic architectures and microporous structures, are developed and assembled into conduits. These conduits supported improves nerve regeneration in terms of cell proliferation (Schwann and PC12 cells) and growth factor secretion (BDNF, brain-derived neurotrophic factor) in vitro, and the in vivo repair and functional recovery of rat sciatic nerve defects. Nerve regeneration using these new conduit designs is comparable to autografts, providing a path towards future clinical impact.

Injectable Desferrioxamine-Laden Silk Nanofiber Hydrogels for Accelerating Diabetic Wound Healing.

Dysangiogenesis and chronic inflammation are two critical reasons for diabetic foot ulcers. Desferrioxamine (DFO) was used clinically in the treatment of diabetic foot ulcers by repeated injections because of its capacity to induce vascularization. Biocompatible carriers that release DFO slowly and facilitate healing simultaneously are preferable options to accelerate the healing of diabetic wounds. Here, DFO-laden silk nanofiber hydrogels that provided a sustained release of DFO for more than 40 days were used to treat diabetic wounds. The DFO-laden hydrogels stimulated the healing of diabetic wounds. In vitro cell studies revealed that the DFO-laden hydrogels modulated the migration and gene expression of endothelial cells, and they also tuned the inflammation behavior of macrophages. These results were confirmed in an in vivo diabetic wound model. The DFO-laden hydrogels alleviated dysangiogenesis and chronic inflammation in the diabetic wounds, resulting in a more rapid wound healing and increased collagen deposition. Both in vitro and in vivo studies suggested potential clinical applications of these DFO-laden hydrogels in the treatment of diabetic ulcers.

LncRNA MM2P-induced, exosome-mediated transfer of Sox9 from monocyte-derived cells modulates primary chondrocytes.

Monocyte-derived cells were shown to promote cartilage repair in osteoarthritis. The role of the long non-coding RNA (lncRNA) MM2P in this function of monocyte-derived cells remained unexplored. Treatment of RAW264.7 murine macrophages and mouse bone marrow-derived macrophages with IL-4 or IL-13 upregulated MM2P expression, upstream of STAT3 and STAT6 phosphorylation. Specifically, MM2P blocked SHP2-mediated dephosphorylation of STAT3 at Try705 and interacted with the RNA-binding protein FUS. In turn, p-STAT3 increased the Sox9 gene expression. These cells released Sox9 mRNA and protein-containing exosomes, as demonstrated by a transmission electron microscope, nanoparticle tracking analysis, and detection of typical surface markers. Their culture supernatant promoted the differentiation of mouse primary chondrocytes, i.e., upregulated the expression of Col1a2 and Acan genes and promoted the secretion of extracellular matrix components proteoglycan and type II collagen. These effects were mediated by Sox9 mRNA and protein delivered to chondrocytes by exosomes. Together, ex vivo treatment of monocyte-derived cells with IL-4 or IL-13 promoted chondrocyte differentiation and functions through exosome-mediated delivery of Sox9 mRNA and protein.

p38γ overexpression promotes osteosarcoma cell progression.

Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. Recent studies demonstrate that p38 gamma (p38γ) phosphorylates retinoblastoma (Rb) to promote cyclin expression, cell-cycle entry and tumorigenesis. Studying the potential function of p38γ in human OS, we show that p38γ mRNA and protein expression are significantly elevated in OS tissues and OS cells, whereas its expression is relatively low in normal bone tissue and in human osteoblasts/osteoblastic cells. Knockdown of p38γ in established (U2OS) and primary human OS cells potently inhibited cell growth, proliferation, migration and invasion, while promoting cell apoptosis. Furthermore, CRISPR/Cas9-induced p38γ knockout inhibited human OS cell progression in vitro. Conversely, ectopic overexpression of p38γ in primary human OS cells augmented cell growth, proliferation and migration. Signaling studies show that retinoblastoma (Rb) phosphorylation and cyclin E1/cyclin A expression were decreased following p38γ shRNA knockdown and knockout, but increased after ectopic p38γ overexpression. Collectively, these results show that p38γ overexpression promotes human OS cell progression.

Microskin-Inspired Injectable MSC-Laden Hydrogels for Scarless Wound Healing with Hair Follicles.

Scarless skin regeneration with functional tissue remains a challenge for full-thickness wounds. Here, mesenchymal stem cell (MSC)-laden hydrogels are developed for scarless wound healing with hair follicles. Microgels composed of aligned silk nanofibers are used to load MSCs to modulate the paracrine. MSC-laden microgels are dispersed into injectable silk nanofiber hydrogels, forming composites biomaterials containing the cells. The injectable hydrogels protect and stabilize the MSCs in the wounds. The synergistic action of silk-based composite hydrogels and MSCs stimulated angiogenesis and M1-M2 phenotype switching of macrophages, provides a suitable niche for functional recovery of wounds. Compared to skin defects treated with MSC-free hydrogels, the defects treated with the MSC-laden composite hydrogels heal faster and form scarless tissues with hair follicles. Wound healing can be further improved by adjusting the ratio of silk nanofibers and particles and the loaded MSCs, suggesting tunability of the system. To the best of current knowledge, this is the first time scarless skin regeneration with hair follicles based on silk material systems is reported. The improved wound healing capacity of the systems suggests future in vivo studies to compare to other biomaterial systems related to clinical goals in skin regeneration in the absence of scarring.

Injectable hydrogel systems with multiple biophysical and biochemical cues for bone regeneration.

Bone regeneration is a complex process in which angiogenesis and osteogenesis are crucial. Introducing multiple angiogenic and osteogenic cues simultaneously into a single system and tuning these cues to optimize the niche remains a challenge for bone tissue engineering. Herein, based on our injectable biomimetic hydrogels composed of silk nanofibers (SNF) and hydroxyapatite nanoparticles (HA), deferoxamine (DFO) and bone morphogenetic protein-2 (BMP-2) were loaded on SNF and HA to introduce more angiogenic and osteogenic cues. The angiogenesis and osteogenesis capacity of injectable hydrogels could be regulated by tuning the delivery of DFO and BMP-2 independently, resulting in vascularization and bone regeneration in cranial defects. The angiogenesis and osteogenesis outcomes accelerated the regeneration of vascularized bones toward similar composition and structure to natural bones. Therefore, the multiple biophysical and chemical cues provided by the nanofibrous structures, organic-inorganic compositions, and chemical and biochemical angiogenic and osteogenic inducing cues suggest the potential for clinical applicability of these hydrogels in bone tissue engineering.

Keap1-targeting microRNA-941 protects endometrial cells from oxygen and glucose deprivation-re-oxygenation via activation of Nrf2 signaling.

BACKGROUND: Mimicking ischemia-reperfusion injury, oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) applied to endometrial cells produces significant oxidative stress and programmed necrosis, which can be inhibited by nuclear-factor-E2-related factor 2 (Nrf2) signaling. MicroRNA (miRNA)-induced repression of Keap1, a Nrf2 suppressor protein that facilitates Nrf2 degradation, is novel strategy to activate Nrf2 cascade. METHODS: MicroRNA-941 (miR-941) was exogenously expressed in HESC and primary human endometrial cells, and the Nrf2 pathway examined by Western blotting and real-time quantitative PCR analysis. The endometrial cells were treated with OGDR, cell programmed necrosis and apoptosis were tested. RESULTS: MiR-941 is a novel Keap1-targeting miRNA that regulates Nrf2 activity. In T-HESC cells and primary human endometrial cells, ectopic overexpression of miR-941 suppressed Keap1 3'-UTR (untranslated region) expression and downregulated its mRNA/protein expression, leading to activation of the Nrf2 cascade. Conversely, inhibition of miR-941 elevated Keap1 expression and activity in endometrial cells, resulting in suppression of Nrf2 activation. MiR-941 overexpression in endometrial cells attenuated OGDR-induced oxidative stress and programmed necrosis, whereas miR-941 inhibition enhanced oxidative stress and programmed necrosis. MiR-941 overexpression and inhibition were completely ineffective in Keap1-/Nrf2-KO T-HESC cells (using CRISPR/Cas9 strategy). Restoring Keap1 expression, using an UTR-depleted Keap1 construct, abolished miR-941-induced anti-OGDR activity in T-HESC cells. Thus Keap1-Nrf2 cascade activation is required for miR-941-induced endometrial cell protection. CONCLUSIONS: Targeting Keap1 by miR-941 activates Nrf2 cascade to protect human endometrial cells from OGDR-induced oxidative stress and programmed necrosis. Video Abstract.

Tough Anisotropic Silk Nanofiber Hydrogels with Osteoinductive Capacity.

Multiple physical cues such as hierarchical microstructures, topography, and stiffness influence cell fate during tissue regeneration. Yet, introducing multiple physical cues to the same biomaterial remains a challenge. Here, a synergistic cross-linking strategy was developed to fabricate protein hydrogels with multiple physical cues based on combinations of two types of silk nanofibers. ß-sheet-rich silk nanofibers (BSNFs) were blended with amorphous silk nanofibers (ASNFs) to form composite nanofiber systems. The composites were transformed into tough hydrogels through horseradish peroxidase (HRP) cross-linking in an electric field, where ASNFs were cross-linked with HRP, while BSNFs were aligned by the electrical field. Anisotropic morphologies and higher stiffness of 120 kPa were achieved. These anisotropic hydrogels induced osteogenic differentiation and the aligned aggregation of stem cells in vitro while also exhibiting osteoinductive capacity in vivo. Improved tissue outcomes with the hydrogels suggest promising applications in bone tissue engineering, as the processing strategy described here provides options to form hydrogels with multiple physical cues.

Reactive Oxygen Species-Scavenging Scaffold with Rapamycin for Treatment of Intervertebral Disk Degeneration.

The chronic inflammatory microenvironment is characterized by the elevated level of reactive oxygen species (ROS). Here, it is hypothesized that developing an ROS-scavenging scaffold loaded with rapamycin (Rapa@Gel) may offer a new strategy for modulating the local inflammatory microenvironment to improve intervertebral disk tissue regeneration. The therapeutic scaffold consisting of ROS-degradable hydrogel can be injected into the injured degeneration site of intervertebral disk (IVD) and can release therapeutics in a programmed manner. The ROS scavenged by scaffold reduces the inflammatory responses. It is found that when rats are treated with Rapa@Gel, this results in an increase in the percentage of M2-like macrophages and a decrease in M1-like macrophages in the inflammatory environment, respectively. Regeneration of IVD is achieved by Rapa@Gel local treatment, due to the increased M2 macrophages and reduced inflammation. This strategy may be extended to the treatment of many other inflammatory diseases.

Robust multi-responsive supramolecular hydrogel based on a mono-component host-guest gelator.

Supramolecular hydrogels have been widely investigated, but the construction of stimuli-responsive mono-component host-guest hydrogels remains a challenge in that it is still hard to balance the solubility and gelation ability of the gelator. In this work, three azobenzene-modified ß-cyclodextrin derivatives with different alkyl lengths (ß-CD-Azo-Cn) have been synthesized. The length of the alkyl chain dramatically influences the solubility and gelation ability of ß-CD derivatives in water. Among these derivatives, ß-CD-Azo-C8 possesses the lowest minimum gelation concentration (MGC). Based on the host-guest interaction between ß-CD and azobenzene units in aqueous solution, which is confirmed by UV-visible and ROESY NMR spectra, the gelators self-assemble and further interwine into networks through the hydrogen bonds on the surface of ß-CD cavities. Hydrogels formed by mono-component gelators can collapse under external stimuli such as heating, competition guests and hosts, and UV irradiation. When the concentration of the gelator is more than 8 wt%, the hydrogel exhibits good self-supporting ability with a storage modulus higher than 104 Pa. The gel-sol transition temperature of the hydrogel is near body temperature, indicating its potential applications in biological materials.

Precisely Tuning Helical Twisting Power via Photoisomerization Kinetics of Dopants in Chiral Nematic Liquid Crystals.

It has been paid much attention to improve the helical twisting power (ß) of dopants in chiral nematic liquid crystals (CLCs); however, the correlations between the ß value and the molecular structures as well as the interaction with nematic LCs are far from clear. In this work, a series of reversibly photo-switchable axially chiral dopants with different lengths of alkyl or alkoxyl substituent groups have been successfully synthesized through nucleophilic substitution and the thiol-ene click reaction. Then, the effect of miscibility between these dopants and nematic LCs on the ß values, as well as the time-dependent decay/growth of the ß values upon irradiations, has been investigated. The theoretical Teas solubility parameter shows that the miscibility between dopants and nematic LCs decreases with increasing of the length of substituent groups from dopant 1 to dopant 4. The ß value of chiral dopants in nematic LCs decreases from dopant 1 to dopant 4 both at the visible light photostationary state (PSS) and at the UV PSS after UV irradiation. With increasing of the length of substituent groups, the photoisomerization rate constant of dopants increases for trans-cis transformation upon UV irradiation and decreases for the reverse process upon visible light irradiation either in isotropic ethyl acetate or in anisotropic LCs, although the constant in ethyl acetate is several times larger than the corresponding value in LCs. Also, the color of the CLCs could be tuned upon light irradiations. These results enable the precise tuning of the pitch and selective reflection wavelength/color of CLCs, which paves the way to the applications in electro-optic devices, information storage, high-tech anticounterfeit, and so forth.

Rehmannia glutinosa (Gaertn.) DC. polysaccharide ameliorates hyperglycemia, hyperlipemia and vascular inflammation in streptozotocin-induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmannia glutinosa (Gaertn.) DC. (RG) has been widely used as traditional Chinese herbal medicine for treatment of diabetes and its complications. The polysaccharide fraction of RG has been proposed to possess hypoglycemic effect by intraperitoneal administration, however, the mechanisms responsible for the hypoglycemic effect of RG polysaccharide (RGP) remain poorly understood. Here we studied the anti-hyperglycemic and anti-hyperlipidemic effect of oral administration of a purified RGP and its underlying mechanisms in streptozotocin (STZ)-induced diabetic mice. MATERIALS AND METHODS: The preliminary structure of RGP was determined by GC and FT-IR. Mice were injected with STZ to induce type 1 diabetes. RGP at doses of 20, 40 and 80 mg/kg/day was orally administered to mice for 4 weeks, and metformin was used as positive control. After 4 weeks, the blood biochemical parameters, the pancreatic insulin contents, in vitro insulin secretion, the hepatic glycogen contents and mRNA expression of phosphoenolpyruvate carboxyl kinase (PEPCK) were assayed. RESULTS: RGP was composed of rhamnose, arabinose, mannose, glucose and galactose in the molar ratio of 1.00:1.26:0.73:16.45:30.40 with the average molecular weight of 63.5 kDa. RGP administration significantly decreased the blood levels of glucose, total cholesterol, triglycerides, low density lipoprotein-cholesterol, and increased the blood levels of high density lipoprotein-cholesterol and insulin in diabetic mice, concurrent with increases in body weights and pancreatic insulin contents. The in vitro study revealed that RGP significantly enhanced both basal and glucose-stimulated insulin secretions, as well as islet insulin contents in the pancreatic islets of diabetic mice. Moreover, RGP reversed the increased mRNA expression of PEPCK and the reduced glycogen contents in the liver of diabetic mice. Furthermore, RGP exhibited potent anti-inflammatory and anti-oxidative activities, as evidenced by the decreased blood levels of TNF-α, IL-6, monocyte chemoattractant protein-1, MDA, and also the elevated blood levels of SOD and GPx activities in diabetic mice. CONCLUSIONS: Taken together, RGP can effectively ameliorate hyperglycemia, hyperlipemia, vascular inflammation and oxidative stress in STZ-induced diabetic mice, and thus may be a potential therapeutic option for type 1 diabetes.