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.

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.

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.

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.