Punicalagin attenuates TNF-α-induced oxidative damage and promotes osteogenic differentiation of bone mesenchymal stem cells by activating the Nrf2/HO-1 pathway.

Oxidative stress is one of the most important factors in changing bone homeostasis. Redox homeostasis plays a key role in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and the angiogenesis ability of human umbilical vein endothelial cells (HUVECs) for bone regeneration. Currently, this study assessed the effects of punicalagin (PUN) on BMSCs and HUVECs. Cell viability was determined by CCK-8 assay. A flow cytometry analysis was adopted to detect macrophage polarization. The production of reactive oxygen stress (ROS), glutathione (GSH), malonaldehyde (MDA) and superoxide dismutase (SOD) activities were evaluated by using commercially-available kits. Osteogenic capacity of BMSCs was evaluated by ALP activity, ALP staining and ARS staining. The expression of osteogenic-related proteins (OCN, Runx-2, OPN) and Nrf/HO-1 levles were evaluated by Western blotting. Osteogenic-related genes (Osterix, COL-1, BMP-4, ALP) were evaluated by RT-PCR. Migration ability and invasion ability of HUVECs were evaluated by wound healing assay and Transwell assay. Angiogenic ability was detected by tube formation assay and the expression of angiogenic-related genes (VEGF, vWF, CD31) were evaluated by RT-PCR. Results showed that PUN alleviated oxidative stress by TNF-α, enhanced osteogenic differentiation in BMSCs and angiogenesis in HUVECs. Moreover, PUN regulate immune microenvironment by promoting the polarization of M2 macrophages and reduce the oxidative stress related products by activating Nrf2/HO-1 pathway. Altogether, these results suggested that PUN can promote osteogenic capacity of BMSCs, angiogenesis of HUVECs, alleviate oxidative stress via Nrf2/HO-1 pathway, offering PUN as a novel antioxidant agent for treating bone loss diseases.

Repair of Sciatic Nerve Defect in Rats With Acellular Nerve Allograft Carrying Vascular Endothelial Cells.

BACKGROUND: Acellular nerve allografts (ANAs) were developed to replace the autologous nerve grafts (ANGs) to fill the peripheral nerve defects. Poor vascularization relative to ANGs has been a limitation of application of ANAs. METHODS: A total of 60 female Sprague-Dawley rats were assigned 3 groups. The rats in A group received ANGs, the rats in B group received ANAs, and the rats in C group were transplanted with ANA carrying endothelial cells (ANA + ECs). In the 1st, 2nd, 4th, and 12th postoperative weeks, 5 rats were selected from each group for evaluating sciatic function index (SFI), electrophysiology, maximum tetanic force recovery rate, tibialis anterior muscle weights recovery rate, and microvessel density. In the 12th postoperative week, the nerves were harvested and stained with toluidine blue and observed under an electron microscope to compare nerve fibers, myelin width, and G-ratio. RESULTS: All the rats survived. In the first and second postoperative weeks, more microvessels were found in the ANA + EC group. In the 12th postoperative week, the nerve fibers were more numerous, and G-ratio was smaller in the C group compared with the B group. The compound muscle action potential and maximum tetanic force recovery rate in the tibialis anterior muscle in the C group were better than those in the B group in the 12th postoperative week. The A group showed better performances in electrophysiology, maximum tetanic force, muscle wet weight, and nerve regeneration. CONCLUSION: ANA + ECs can promote early angiogenesis, promoting nerve regeneration and neurological function recovery.

Efficacy and Safety of the Babysitter Procedure With Different Percentages of Partial Neurectomy.

BACKGROUND: After 2 months of denervation, the number of motor units in the muscle decreases; after 6 months of denervation, muscle atrophy and weakness are irreversible and successful nerve reconstruction does not generally restore function. The babysitter procedure was reported to successfully avoid muscle atrophy. One study found that the babysitter procedure with a 40% neurectomy was most suitable; however, the amount of donor nerve that can be borrowed for the babysitter procedure in peripheral nerve injury is unknown. METHOD: One hundred adult female Sprague-Dawley rats were used in this study. The rats were randomly allocated to 5 groups (groups A-E; n = 20 each). The rats underwent different surgeries based on their grouping. At 6, 12, 18, and 24 weeks after surgery, 5 rats in each group were selected for electrophysiology and muscle force tests. These rats were then killed, and the gastrocnemius and tibialis anterior muscles were harvested for weight measurement and cross-sectional muscle measurement. RESULT: The results of the effects on the peroneal nerves and tibialis anterior muscles after the babysitter procedure with 40% and 80% neurectomies showed that the functional ability of the recipient nerves was maintained and the muscle was effectively prevented from atrophy, whereas the 20% neurectomy and end-to-side procedures showed relatively poor performance. The results of the effects on the tibial nerve and gastrocnemius muscles after the babysitter procedure with 20% and 40% neurectomies showed that there was little effect on the donor nerve. By contrast, 80% neurectomy strongly and negatively affected the donor nerve. CONCLUSIONS: Our results indicate that the babysitter procedure using a donor nerve with a partial neurectomy of 40% was the best choice for effectively treating peripheral (peroneal) nerve injury in rats.

Adipose-derived stem cells induce autophagic activation and inhibit catabolic response to pro-inflammatory cytokines in rat chondrocytes.

OBJECTIVE: Adipose-derived stem cells (ADSCs) have been demonstrated to have an anti-apoptosis effect on chondrocytes; However, their effect on autophagic activation remains unclear. We sought to explore whether ADSCs can activate autophagy and inhibit IL-1ß- and lipopolysaccharide (LPS)-induced catabolism in chondrocytes. METHODS: ADSCs and chondrocytes were collected from SD rats. The biologic characteristics of ADSCs were analyzed by flow cytometric analysis, Oil red O and Alizarin Red staining. Autophagic level and autophagic flux were revealed by Western blotting for LC3-II and SQSTM1/P62, MDC (monodansylcadaverine) staining and mRFP-GFP-LC3 analysis. The mTOR pathway was investigated by Western blotting for p-mTOR. The mRNA level of matrix metalloproteinases (MMPs) and thrombospondin motifs (ADAMTSs) was detected by real-time PCR. RESULTS: The typical surface markers and differentiation potentials of ADSCs were proved. ADSCs enhanced the expression of LC3-II/LC3-I and reduced SQSTM1 levels in IL-1ß-induced chondrocytes after 24 and 48 h co-culturing and in LPS-induced chondrocytes after 48 h co-culturing respectively. mRFP-GFP-LC3 analysis suggested that autophagosomes and autolysosomes were formed earlier in IL-1ß-treated chondrocytes than in LPS-treated chondrocytes. Bafilomycin A1 treatment further increased the LC3-II/LC3-I level in chondrocytes in co-culture with ADSCs. The mTOR pathway was inhibited in the chondrocytes in co-culture with ADSCs. Finally, ADSCs inhibited the increase of MMPs and ADAMTSs in chondrocytes induced by IL-1ß and LPS. CONCLUSIONS: ADSCs seem able to activate autophagy and inhibit catabolism in chondrocytes in an inflammation environment, and the mTOR pathway might be involved in the autophagy activation.

[Nerve classification with hyperspectral imaging technology].

In surgical nerve repair surgery, the identification of nerve fascicles is a key to a good repair of their broken end. Some of the existing nerve fascicles identification method are not ideal. Hyperspectral imaging (HSI) technology provides information of images and spectra of biological tissue at the same time. It can supply a qualitative, quantitative and positioning description of the test objectives, and identify different biological tissues by biochemical characteristic difference, and classify and position these tissues in the image. Compared to other medical imaging technology, this techriology has unique advantages. In this study, the hyperspectral imaging technology is used in the identification and classification of the nerve fascicles by the spectral characteristics of different nerve fascicles, and in determining the orientation of the nerve fascicles in the image by the image spectral information in order to better help surgical personnel to carry out the nerve repair surgery. The significance of this paper is: the first to propose a new method of identification and location of the nerve fascicles and assist surgical staff to improve the efficacy of nerve repair; the second to reserve hyperspectral imaging techniques used in qualitative and quantitative and orientation research combined with biological organization, and speed up the molecular hyperspectral imaging technology to the practical stage.

AOTF based molecular hyperspectral imaging system and its applications on nerve morphometry.

The neuroanatomical morphology of nerve fibers is an important description for understanding the pathological aspects of nerves. Different from the traditional automatic nerve morphometry methods, a molecular hyperspectral imaging system based on an acousto-optic tunable filter (AOTF) was developed and used to identify unstained nerve histological sections. The hardware, software, and system performance of the imaging system are presented and discussed. The gray correction coefficient was used to calibrate the system's spectral response and to remove the effects of noises and artifacts. A spatial-spectral kernel-based approach through the support vector machine formulation was proposed to identify nerve fibers. This algorithm can jointly use both the spatial and spectral information of molecular hyperspectral images for segmentation. Then, the morphological parameters such as fiber diameter, axon diameter, myelin sheath thickness, fiber area, and g-ratio were calculated and evaluated. Experimental results show that the hyperspectral-based method has the potential to recognize and measure the nerve fiber more accurately than traditional methods.

Spine Surgery with Electronic Conductivity Device: A Prospectively Multicenter Randomized Clinical Trial and Literature Review.

OBJECTIVE: Improving accuracy and safety of pedicle screw placement is of great clinical importance. Electronic conductivity device (ECD) can be a promising technique with features of affordability, portability, and real-time detection capabilities. This study aimed to validate the safety and effectiveness of a modified ECD. METHODS: The ECD underwent a modification where six lamps of various colors, and it was utilized in a prospectively multicenter randomized controlled clinical trial involving 96 patients across three hospitals from June 2018 to December 2018. The trial incorporated a self-control randomization with an equal distribution of left or right side of vertebral pedicle among two groups: the free-hand group and the ECD group. A total of 496 pedicle screws were inserted, with 248 inserted in each group. The primary outcomes focused on the accuracy of pedicle screw placement and the frequency of intraoperative X-rays. Meanwhile, the secondary indicator measured the time required for pedicle screw placement. Results were presented as means ± SD. Paired samples t-test and χ2 -test were used for comparison. Furthermore, an updated review was conducted, which included studies published from 2006 onwards. RESULTS: Baseline patient characteristics were recorded. The primary accuracy outcome revealed a 96.77% accuracy rate in the ECD group, compared to a 95.16% accuracy rate in the free-hand group, with no significant differences noted. In contrast, ECD demonstrated a significant reduction in radiation exposure frequency when compared to the free-hand group (1.11 ± 0.32 vs. 1.30 ± 0.53; p < 0.001), resulting in a 14.6% reduction. Moreover, ECD displayed a decrease of 30.38% in insertion time (70.88 ± 30.51 vs. 101.82 ± 54.00 s; p < 0.001). According to the results of the 21 studies, ECD has been utilized in various areas of the spine such as the atlas, thoracic and lumbar spine, as well as sacral 2-alar-iliac. The accuracy of ECD ranged from 85% to 100%. CONCLUSION: The prospectively randomized trial and the review indicate that the use of ECD presents a secure and precise approach to the placement of pedicle screws, with the added benefit of reducing both procedure time and radiation exposure.

Endothelial cells promote the proliferation and migration of Schwann cells.

Background: After peripheral nerve injury, Schwann cells proliferate and migrate to the injured site, thereby promoting peripheral nerve regeneration. The process is regulated by various factors. Endothelial cells participate in the process via angiogenesis. However, the effects of endothelial cells on Schwann cells are not yet known. The present study sought to evaluate whether endothelial cells accelerate Schwann cell proliferation and migration. Methods: We established a co-culture model of rat Schwann cells (RSC96s) and rat aortic endothelial cells (RAOECs), and studied the effects of endothelial cells on Schwann cells by evaluating changes in Schwann cell proliferation and migration and related multiple genes and their protein expressions in the co-culture model. Results: The results showed that increasing the proportion of endothelial cells in the co-culture model enhanced the proliferation. At days 1 and 3 following the co-culturing, the relative growth rates of the co-cultured cells were 122.87% and 127.37%, respectively, which showed a significant increase in the viability compared to that of the RSC96s (P<0.05). In this process, the expression of Ki67 increased. The migration ability of Schwann cells was also enhanced. The migration capacity of Schwann cells was detected by wound-healing and Transwell assays. The results of the group with 15% of endothelial cells was significantly higher than the results of the other groups (P<0.0001 and P<0.05, respectively). Further, neuregulin 1 and glial fibrillary acidic protein increased the process of Schwann cell migration. Conclusions: The results showed that endothelial cells can promote the proliferation and migration of Schwann cells and participate in peripheral nerve regeneration.

Anterior cervical discectomy and fusion with recombinant human bone morphogenetic protein-2-adsorbed ß-tricalcium phosphate granules: a preliminary report.

BACKGROUND: Anterior cervical discectomy and fusion (ACDF) is an alternative to conservative therapy in the treatment of cervical spondylopathy. This study evaluated the clinical outcome of ACDF with BMP-2-adsorbed ß-tricalcium phosphate granules. METHODS: Thirty-two patients with cervical spondylopathy received treatment of ACDF with BMP-2-adsorbed ß-tricalcium phosphate granules. The clinical outcomes were evaluated with the Japanese Orthopedic Association (JOA) scores and Neck Disability Index (NDI). Meanwhile, the cervical curvature and intervertebral heights were obtained through lateral cervical X-ray films pre- and postoperatively at each interval, and the precision of cervical fusion was assessed by three-dimensional computed tomography scan. RESULTS: The follow-up averaged 15.2 months (range 13-18). Average JOA scores significantly increased from a preoperative point (7.4 ± 1.2) to each interval after surgery (P < 0.05). NDI decreased from preoperative point (43.1 ± 9.0) to each interval after surgery (P < 0.05). The angles of cervical curvature and intervertebral heights were improved postoperatively and kept throughout the follow-up period. CT scan demonstrated a fusion rate of 82.9% at 6 months postoperatively and was improved to 100% at 12 months postoperatively. In all cases, no complications appeared and reported due to any lapse in surgical procedure skills throughout the follow-up period. CONCLUSIONS: Our preliminary findings suggest that BMP-2-adsorbed ß-tricalcium phosphate granules will be an effective alternative to autogenous bone grafting for cervical fusion in treating cervical spondylopathy. Our surgical procedure usingß-tricalcium phosphate granules could improve neurological function, recover intervertebral height and cervical curvature, and could be potentially exploitable in the clinical setting.

Dihydroartemisinin inhibits catabolism in rat chondrocytes by activating autophagy via inhibition of the NF-κB pathway.

Osteoarthritis is a disease with inflammatory and catabolic imbalance in cartilage. Dihydroartemisinin (DHA), a natural and safe anti-malarial agent, has been reported to inhibit inflammation, but its effects on chondrocytes have yet to be elucidated. We investigated the effects of DHA on catabolism in chondrocytes. Viability of SD rats chondrocytes was analyzed. Autophagy levels were determined via expression of autophagic markers LC3 and ATG5, GFP-LC3 analysis, acridine orange staining, and electron microscopy. ATG5 siRNA induced autophagic inhibition. Catabolic gene and chemokine expression was evaluated using qPCR. The NF-κB inhibitor SM7368 and p65 over-expression were used to analyze the role of NF-κB pathway in autophagic activation. A concentration of 1 µM DHA without cytotoxicity increased LC3-II and ATG5 levels as well as autophagosomal numbers in chondrocytes. DHA inhibited TNF-α-induced expression of MMP-3 and -9, ADAMTS5, CCL-2 and -5, and CXCL1, which was reversed by autophagic inhibition. TNF-α-stimulated nuclear translocation and degradation of the p65 and IκBα proteins, respectively, were attenuated in DHA-treated chondrocytes. NF-κB inhibition activated autophagy in TNF-α-treated chondrocytes, but p65 over-expression reduced the autophagic response to DHA. These results indicate that DHA might suppress the levels of catabolic and inflammatory factors in chondrocytes by promoting autophagy via NF-κB pathway inhibition.

Evaluation of quality of life and risk factors affecting quality of life in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common disease leading to spinal deformity in children ages 10 and over. With advances in the study of health-related quality of life (HRQoL), greater attention has been given to the quality of life (QoL) of patients with AIS and their perception of deformity instead of just focusing on improving the rate of surgical correction. This article provides an overview of the methods of evaluating HRQoL and it analyzes several main factors affecting QoL, such as severity of disease, method of treatment, gender, and social environment, based on previous studies of patients with AIS. The authors believe that radiological studies should no longer be taken as the only indicator of postoperative therapeutic evaluation and hope to build a new evaluation system with assessment of QoL for patients with AIS.

Microsurgical principles related to excision of intraneural ganglion at the elbow.

We describe the treatment of a ganglion within the ulnar nerve at the elbow and apply the concept that an intraneural ganglion arises from the joint adjacent to the nerve in which the ganglion is located. Successful treatment of nerve compression and prevention of recurrence of the ganglion require disconnection of the nerve from the joint and deflation, not excision, of the ganglion.

Repeated doses of intravenous tranexamic acid are effective and safe at reducing perioperative blood loss in total knee arthroplasty.

Fibrin sealant (FS) and tranexamic acid (TXA) have been used in total knee arthroplasty (TKA) to minimize perioperative blood loss. The efficacy of FS has been debated, and few studies have looked into the effects of FS and TXA on perioperative coagulability. The current study retrospectively reviewed 100 cases of unilateral primary TKA. Twenty-five cases served as blank controls, FS was used without TXA in 23, TXA was used without FS in 20, and both FS and TXA (FS + TXA) were used in 32. FS was sprayed before wound closure whereas 1 g of TXA was intravenously administered before incision and 1 g was administered 15 min before tourniquet release. Hematocrit and hemoglobin levels and thromboelastography (TEG) parameters were assessed pre-operatively and on day 1, 4, and 9 post-operatively. Blood transfusions were noted and the incidence of symptomatic DVT/PE was determined. Hematocrit and hemoglobin levels were significantly higher in the TXA and FS + TXA groups compared to the control and FS groups on day 1, 4, and 9 post-operatively. Hematocrit and hemoglobin levels in the control group were similar to those in the FS group and hematocrit and hemoglobin levels in the TXA group were similar to those in the FS + TXA group. TEG parameters (R, K, α, MA, and CI) remained within normal ranges. Mean CI was less than +3 in all four groups, suggesting that hypercoagulation was not promoted. One patient in the FS group received an allogeneic transfusion. Incidence of symptomatic DVT/PE was not noted. Intravenous TXA significantly reduced perioperative blood loss in patients undergoing a TKA but FS did not. Administration of FS in addition to TXA was not superior to TXA alone. FS and/or TXA did not increase the risk of hypercoagulation according to TEG parameters. Intravenous administration of 1 g of TXA pre-operatively and administration of 1 g before tourniquet release is an effective and safe method of reducing blood loss in TKA.

Differentiation of peripheral nerve functions and properties with spectral analysis and Karnovsky-Roots staining: a preliminary study.

OBJECTIVE: The purpose of this study was to explore the possibility for analyzing and differentiating between motor and sensory functions of peripheral nerve axons using spectral technology. METHODS: 10 µm slide section of S1 anterior and posterior rabbit spinal nerve roots were made and then stained with Karnovsky-Roots method for molecular hyperspectral imaging microscopy analysis. In addition, Raman spectra data of nerve axons on each slide was collected after Karnovsky-Roots staining for 30 minutes. RESULTS: Motor axons were differentiated from sensory axons in a nerve axon section hyperspectral image via Spectral angle mapper algorithm. Raman scatterings could be detected near 2110 cm(-1), and 2155 cm(-1) in motor axons after Karnvosky-Roots staining. The value of I2100/I1440 in motor axons are significantly different (P0.001) than in sensory axons after staining for 30 minutes. CONCLUSIONS: Motor and sensory nerve axons can be differentiated from their counterparts in 30 minutes by using Raman micro-spectroscopy analysis assisted with Karnovsky-Roots staining.

Automatic identification and quantitative morphometry of unstained spinal nerve using molecular hyperspectral imaging technology.

Quantitative observation of nerve fiber sections is often complemented by morphological analysis in both research and clinical condition. However, existing manual or semi-automated methods are tedious and labour intensive, fully automated morphometry methods are complicated as the information of color or gray images captured by traditional microscopy is limited. Moreover, most of the methods are time-consuming as the nerve sections need to be stained with some reagents before observation. To overcome these shortcomings, a molecular hyperspectral imaging system is developed and used to observe the spinal nerve sections. The molecular hyperspectral images contain both the structural and biochemical information of spinal nerve sections which is very useful for automatic identification and quantitative morphological analysis of nerve fibers. This characteristic makes it possible for researchers to observe the unstained spinal nerve and live cells in their native environment. To evaluate the performance of the new method, the molecular hyperspectral images were captured and the improved spectral angle mapper algorithm was proposed and used to segment the myelin contours. Then the morphological parameters such as myelin thickness and myelin area were calculated and evaluated. With these morphological parameters, the three dimension surface view images were drawn to help the investigators observe spinal nerve at different angles. The experiment results show that the hyperspectral based method has the potential to identify the spinal nerve more accurate than the traditional method as the new method contains both the spectral and spatial information of nerve sections.