Concurrent Joint Contact in Anterior Cruciate Ligament Injury induces cartilage micro-injury and subchondral bone sclerosis, resulting in knee osteoarthritis.

Objective: Anterior Cruciate Ligament (ACL) injury develops the Osteoarthritis (OA) via two distinct processes: initial direct micro-injury of the cartilage surface by compressive force during ACL injury, and secondary joint instability due to the deficiency of the ACL. Using the conventional Compression-induced ACL-R and novel Non-Compression ACL-R models, we aimed to reveal the individual effects on OA progression after ACL injury. Methods: Twelve-week-old C57BL/6 male were randomly divided to three experimental groups: Compression ACL-R, Non-Compression ACL-R, and Intact. We performed the joint laxity test and microscope analysis at 0 days, in vivo imaging with matrix-metalloproteinases (MMPs) at 3 and 7 days, histological and micro-CT analysis at 0, 7, 14, and 28 days. Results: Although no differences in the joint laxity were observed between both ACL-R groups, the Compression ACL-R group exhibited a significant increase of cartilage roughness immediately after injury compared with the Non-Compression group. At 7 days, Compression group increased MMPs-induced fluorescence intensity slightly and MMP-13 positive cell ratio of chondrocytes significantly than that in the Non-Compression group. Moreover, histological cartilage degeneration initiated in the whole joint level of the Compression group at the same time point. Micro-CT analysis revealed that sclerosis of tibial Subchondral bone in the Compression group developed significantly more than in the Non-Compression group at 28 days, especially in the medial tibial compartment. Conclusions: Concurrent joint contact during ACL rupture caused initial micro-damage on the cartilage surface and early cartilage degeneration with MMP-13 production, leading to later bone formation in the subchondral bone.

The model for predicting the central lymph node metastasis in cN0 papillary thyroid microcarcinoma with Hashimoto's thyroiditis.

Background: The relationship between Hashimoto's thyroiditis (HT) and papillary thyroid microcarcinoma (PTMC) is controversial. These include central lymph node metastasis (CLNM), which affects the prognosis of PTMC patients. This study aimed to establish a predictive model combining ultrasonography and clinicopathological features to accurately evaluate latent CLNM in PTMC patients with HT at the clinical lymph node-negative (cN0) stage. Methods: In this study, 1102 PTMC patients who received thyroidectomy and central cervical lymph node dissection (CLND) from the First Affiliated Hospital of Shandong First Medical University from January 2021 to December 2022 and the 960th Hospital of PLA from January 2021 to December 2022 were jointly collected. The clinical differences between PTMCs with HT and those without HT were compared. A total of 373 PTMCs with HT in cN0 were randomly divided into a training cohort and a validation cohort. By analyzing and screening the risk factors of CLNM, a nomogram model was established and verified. The predictive performance was measured by the receiver operating characteristic (ROC) curve, calibration curve, and clinical decision curve analysis (DCA). Results: The ratio of central lymph node metastasis (CLNMR) in PTMCs with HT was 0.0% (0.0%, 15.0%) and 7.7% (0.0%, 40.0%) in the non-HT group (P<0.001). Multivariate logistic regression analysis showed that age, gender, calcification, adjacent to trachea or capsule, and TPOAB were predictors of CLNM in PTMCs with HT. The areas under the curve (AUC) of the prediction models in the training cohort and the validation cohort were 0.835 and 0.825, respectively, which showed good differentiation ability. DCA indicates that the prediction model also has high net benefit and clinical practical value. Conclusion: This study found that CLN involvement was significantly reduced in PTMC patients with HT, suggesting that different methods should be used to predict CLNM in PTMC patients with HT and without HT, to more accurately assist preoperative clinical evaluation. The actual CLNM situation of PTMCs with HT in cN0 can be accurately predicted by the combination of ultrasonography and clinicopathological features.

Surgical restabilization reduces the progression of post-traumatic osteoarthritis initiated by ACL rupture in mice.

OBJECTIVE: People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often develop post-traumatic osteoarthritis (PTOA). In human patients, ACL injuries are often treated with ACL reconstruction. However, it is still unclear how effective joint restabilization is for reducing the progression of PTOA. The goal of this study was to determine how surgical restabilization of a mouse knee joint following non-invasive ACL injury affects PTOA progression. DESIGN: In this study, 187 mice were subjected to non-invasive ACL injury or no injury. After injury, mice underwent restabilization surgery, sham surgery, or no surgery. Mice were then euthanized on day 14 or day 49 after injury/surgery. Functional analyses were performed at multiple time points to assess voluntary movement, gait, and pain. Knees were analyzed ex vivo with micro-computed tomography, RT-PCR, and whole-joint histology to assess articular cartilage degeneration, synovitis, and osteophyte formation. RESULTS: Both ACL injury and surgery resulted in loss of epiphyseal trabecular bone (-27-32%) and reduced voluntary movement at early time points. Joint restabilization successfully lowered OA score (-78% relative to injured at day 14, p < 0.0001), and synovitis scores (-37% relative to injured at day 14, p = 0.042), and diminished the formation of chondrophytes/osteophytes (-97% relative to injured at day 14, p < 0.001, -78% at day 49, p < 0.001). CONCLUSIONS: This study confirmed that surgical knee restabilization was effective at reducing articular cartilage degeneration and diminishing chondrophyte/osteophyte formation after ACL injury in mice, suggesting that these processes are largely driven by joint instability in this mouse model. However, restabilization was not able to mitigate the early inflammatory response and the loss of epiphyseal trabecular bone, indicating that these processes are independent of joint instability.

Combinatorial preparation and structural characterization of anthocyanins and aglycones from Purple-heart Radish for evaluation of physicochemical stability and pancreatic lipase inhibitory activity.

In this study, an efficient approach to preparation of different anthocyanins from Purple-heart Radish was developed by combining microwave-assisted extraction (MAE), macroporous resin purification (MRP) and ultrasound-assisted acid hydrolysis (UAAH) for evaluation of physicochemical stability and pancreatic lipase (PL) inhibitory activity. By optimization of MAE, MRP and UAAH processes, the anthocyanins reached the yield of 6.081 ± 0.106 mg/g, the purity of 78.54 ± 0.62 % (w/w) and the content of 76.29 ± 1.31 % (w/w), respectively. With high-resolution UHPLC-Q-Orbitrap/MS, 15 anthocyanins were identified as pelargonins with diverse glucosides and confirmed by pelargonidin standard. By glycosylation, pelargonins exhibited higher stability in different pH, temperature, light, metal ions environments than that of pelargonidin. However, PL inhibitory assay, kinetic analysis and molecular docking demonstrated that pelargonidin had higher PL inhibitory activity than pelargonins even though with similar binding sites and a dose-effect relationship. The above results revealed that the effect of glycosylation and deglycosylation on PL inhibitory activity and physicochemical stability.

Integration of ultrasound-based radiomics with clinical features for predicting cervical lymph node metastasis in postoperative patients with differentiated thyroid carcinoma.

OBJECTIVE: The primary objective was to establish a radiomics model utilizing longitudinal +cross-sectional ultrasound (US) images of lymph nodes (LNs) to predict cervical lymph node metastasis (CLNM) following differentiated thyroid carcinoma (DTC) surgery. METHODS: A retrospective collection of 211 LNs from 211 postoperative DTC patients who underwent neck US with suspicious LN fine needle aspiration cytopathology findings at our institution was conducted between June 2021 and April 2023. Conventional US and clinicopathological information of patients were gathered. Based on the pathological results, patients were categorized into CLNM and non-CLNM groups. The database was randomly divided into a training cohort (n = 147) and a test cohort (n = 64) at a 7:3 ratio. The least absolute shrinkage and selection operator algorithm was applied to screen the most relevant radiomic features from the longitudinal + cross-sectional US images, and a radiomics model was constructed. Univariate and multivariate analyses were used to assess US and clinicopathological significance features. Subsequently, a combined model for predicting CLNM was constructed by integrating radiomics, conventional US, and clinicopathological features and presented as a nomogram. RESULTS: The area under the curves (AUCs) of the longitudinal + cross-sectional radiomics models were 0.846 and 0.801 in the training and test sets, respectively, outperforming the single longitudinal and cross-sectional models (p < 0.05). In the testing cohort, the AUC of the combined model in predicting CLNM was 0.901, surpassing that of the single US model (AUC, 0.731) and radiomics model (AUC, 0.801). CONCLUSIONS: The US-based radiomics model exhibits the potential to accurately predict CLNM following DTC surgery, thereby enhancing diagnostic accuracy.

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Validation of a Low-Cost Portable Device for Inducing Noninvasive Anterior Cruciate Ligament Injury in Mice.

Noninvasive compression-induced anterior cruciate ligament rupture (ACL-R) is an easy and reproducible model for studying post-traumatic osteoarthritis (PTOA) in mice. However, equipment typically used for ACL-R is expensive, immobile, and not available to all researchers. In this study, we compared PTOA progression in mice injured with a low-cost custom ACL-rupture device (CARD) to mice injured with a standard system (ElectroForce 3200). We quantified anterior-posterior (AP) joint laxity immediately following injury, epiphyseal trabecular bone microstructure, and osteophyte volume at 2 and 6 weeks post injury using micro-computed tomography, and osteoarthritis progression and synovitis at 2 and 6 weeks post injury using whole-joint histology. We observed no significant differences in outcomes in mice injured with the CARD system compared to mice injured with the Electroforce (ELF) system. However, AP joint laxity data and week 2 micro-CT and histology outcomes suggested that injuries may have been slightly more severe and PTOA progressed slightly faster in mice injured with the CARD system compared to the ELF system. Altogether, these data confirm that ACL-R can be successfully and reproducibly performed with the CARD system and that osteoarthritis (OA) progression is mostly comparable to that of mice injured with the ELF system, though potentially slightly faster. The CARD system is low cost and portable, and we are making the plans and instructions freely available to all interested investigators in the hopes that they will find this system useful for their studies of OA in mice.

MSC-Exos: Important active factor of bone regeneration.

Bone defect and repair is a common but difficult problem in restorative and reconstructive surgery. Bone tissue defects of different sizes caused by different reasons bring functional limitations and cosmetic deformities to patients. Mesenchymal stem cells (MSC), a major hotspot in the field of regeneration in recent years, have been widely used in various studies on bone tissue regeneration. Numerous studies have shown that the bone regenerative effects of MSC can be achieved through exosome-delivered messages. Although its osteogenic mechanism is still unclear, it is clear that MSC-Exos can directly or indirectly support the action of bone regeneration. It can act directly on various cells associated with osteogenesis, or by carrying substances that affect cellular activators or the local internal environment in target cells, or it can achieve activation of the osteogenic framework by binding to materials. Therefore, this review aims to summarize the types and content of effective contents of MSC-Exos in bone regeneration, as well as recent advances in the currently commonly used methods to enable the binding of MSC-Exos to the framework and to conclude that MSC-Exos is effective in promoting osteogenesis.

Design and experimental performance of a novel piezoelectric inchworm actuator based on screw clamping principle.

The requirements of large thrust in bidirectional motion, high specific power (power/weight ratio), reliable self-locking at power off state, and compact size envelope for various applications of existing piezoelectric actuators are still urgent. To address these challenges, this study makes full use of the piezoelectric material's excellent dynamic performance, robust force output, and high energy density to propose a novel inchworm piezoelectric actuator, which relies on the alternate clamping of two nuts independently rotated by two ultrasonic motors on a feed-screw in accordance with a piezoelectric stack (piezo-stack) excitation to accumulate the piezo-stack's electric field-induced strain periodically and realize power output. The operation principle and design process of the presented actuator were elaborated, and a prototype was fabricated and experimented. Results indicate that the actuator has achieved an expected bidirectional motion for powering the tensile load and compressive load, attained a micrometer-sized step resolution, produced a 3.08 mm/s free speed, a 700 N stall load, and a 1.34 W/kg specific power, and shown a favorable prospect for further development.

Microbial response and adaption to thallium contamination in soil profiles.

Thallium (Tl) is a trace metal with high toxicity. Comprehensive investigation of spatial distribution of Tl and microorganism is still limited in soils from mining area. In this study, 16S rRNA sequencing and network analysis were used for deciphering the co-occurrence patterns of bacterial communities in two different types of soil profiles around a typical Tl-bearing pyrite mine. The results showed that geochemical parameters (such as pH, S, Tl, Fe and TOM) were the driving forces for shaping the vertical distribution of microbial community. According to network analysis, a wide diversity of microbial modules were present in both soil profiles and affected by depth, significantly associated with variations in Tl geochemical fractionation. Phylogenetic information further unveiled that the microbial modules were mainly dominated by Fe reducing bacteria (FeRB), Fe oxidizing bacteria (FeOB), S oxidizing bacteria and Mn reducing bacteria. The results of metagenome indicated that Fe, Mn and S cycle in soil are closely involved in the biogeochemical cycle of Tl. The findings of co-occurrence patterns in the bacterial network and correlation between microorganisms and different geochemical fractions of Tl may benefit the strategy of bioremediation of Tl-contaminated soils with indigenous microbes.

Stable isotope fractionation of thallium as novel evidence for its geochemical transfer during lead­zinc smelting activities.

Thallium (Tl) is a highly toxic trace metal. Lead (Pb)­zinc (Zn) smelting, which is a pillar industry in various countries, is regarded as one of the dominant anthropogenic sources of Tl contamination in the environment. In this study, thallium isotope data have been evaluated for raw material and a set of industrial wastes produced at different stages of Pb-Zn smelting in a representative large facility located by the North River, South China, in order to capture Tl isotope signatures of such typical anthropogenic origin for laying the foundation of tracking Tl pollution. Large variations in Tl isotopic compositions of raw Pb-Zn ores and solid smelting wastes produced along the process chain were observed. The ε205Tl values of raw Pb-Zn ores and return fines are -0.87 ± 0.26 and -1.0 ± 0.17, respectively, contrasted by increasingly more negative values for electrostatic precipitator dust (ε205Tl = -2.03 ± 0.14), lime neutralizing slag (ε205Tl = -2.36 ± 0.18), and acid sludge (ε205Tl = -4.62 ± 0.76). The heaviest ε205Tl (1.12 ± 0.51) was found in clinker. These results show that isotopic fractionation occurs during the smelting processes. Obviously, the lighter Tl isotope is enriched in the vapor phase (-3.75 ε205Tl units). Further XPS and STEM-EDS analyses show that Tl isotope fractionation conforms to the Rayleigh fractionation model, and adsorption of 205Tl onto hematite (Fe2O3) may play an important role in the enrichment of the heavier Tl isotope. The findings demonstrate that Tl isotope analysis is a robust tool to aid our understanding of Tl behavior in smelting processes and to provide a basis for source apportionment of Tl contaminations.

Enzyme and microwave co-assisted extraction, structural characterization and antioxidant activity of polysaccharides from Purple-heart Radish.

A novel method of simultaneous extraction and separation of diverse polysaccharides from Purple-heart Radish was developed by integrating EAE with MAATPE. The effects of different enzymes, the ATPS composition, extraction temperature, time etc. were investigated by single-factor experiments and RSM. Under the optimum conditions, the extraction yields of PTP, PBP and total polysaccharides were 9.107 ± 0.391%, 32.506 ± 0.046% and 41.613 ± 0.437%, respectively. By means of HPGPC and PMP-HPLC, Mw of PTP and Mw of PBP were 15935 Da and 27962 Da, respectively. PTP and PBP were mainly composed of mannose, glucuronic acid, aminogalactose, glucose, galactose and arabinose. Moreover, both polysaccharides exhibited stronger antioxidant activities for scavenging multiple radicals and anti-lipid peroxidation. Compared to the conventional extraction methods, EAE-MAATPE achieved higher extraction efficiency due to the synergistic effect between EAE and MAATPE leading to rupture and enzymolysis of cell. Thus, EAE-MAATPE provided an efficient alternative to simultaneous extraction of different polysaccharides from natural products.

Altered canalicular remodeling associated with femur fracture in mice.

We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post-fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar-canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high-resolution microcomputed tomography to quantify the morphology of the lacunar-canalicular network at the contralateral tibia, and we used quantitative real-time polymerase chain reaction (RT-PCR) and RNA-seq to measure the expression of PLR-associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post-fracture, we observed downregulation of PLR-associated genes; RNA-seq analysis at 3 days post-fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury.

Quantification of smelter-derived contributions to thallium contamination in river sediments: Novel insights from thallium isotope evidence.

Thallium(Tl), an extremely toxic metal, is posing great hazards to water safety through anthropogenic activities (e.g., Pb-Zn smelter) and natural weathering in riverine systems. However, the relative contribution from each source remains obscure. This study investigated enrichment pattern of Tl and its isotopic compositions in sediment profiles from a recipient river, which was continuously collecting various Tl-bearing wastes discharged from a large Pb-Zn smelter in South China. Results show that high Tl content and ultra-fine particles (~ µm) of Tl-bearing mineral assemblages, probably derived from Pb-Zn smelting wastes, were ubiquitously observed in both of the depth profiles. In addition, the sediments generally yielded intermediate ε205Tl values of -3.76 to 1.01, which resembled those found in smelting wastes. A ternary mixing model was for the first time proposed for quantifying relative Tl contributions from each possible source. The calculation suggests that the smelter wastes are the major contributors, contributing approximately 80% of Tl contamination. All these results indicate that Tl isotope can be used as powerful proxies for quantitatively identifying potential different contributors in the environment. This is of critical importance to further implementation of pollution control and remediation strategy for the riverine systems in the near future.

Accumulation and passive sampling of bisphenol analogues using triolein-embedded cellulose acetate membrane in waters.

Bisphenol analogues (BPs) are emerging contaminants that have been widely detected in water environments. The presence of substituted hydrophilic and hydrophobic groups in the molecule may lead to unclear performance in passive sampling. This study tested the accumulation capacity and passive sampling of fifteen BPs in a triolein-embedded cellulose acetate membrane (TECAM) passive sampler. In a dynamic accumulation experiment, twelve hydrophobic BPs accumulated in the TECAM with concentrations ranging from 251 ng g-1 to 6283 ng g-1, and three hydrophilic BPs did not accumulate during the 72 h exposure duration. BPs accumulations were determined by the hydrophilic and hydrophobic substituent groups in molecule structures. The estimated passive sampling parameters showed correlations to both the log Kow values and chemical structures, and compared to other contaminants, such as organophosphorus flame retardants. Environmental factors, including flow rate, temperature, salinity, and pH, that affect the accumulation of BPs in the TECAM were tested, and the flow rate was found to be an important factor affecting the uptake rate. The isotropic exchange kinetics for BPs in the TECAM were verified, and the results indicated that BPs can be calibrated with performance reference compounds (PRCs) in field applications. Finally, a field deployment of TECAM in river waters successfully estimated the time-weighted concentrations of two hydrophobic BPs. To address the inherent weaknesses of TECAM in sampling hydrophilic and moderately hydrophobic BPs, future studies should explore alternative passive samplers, such as hydrophilic-lipophilic balance sorbent-embedded cellulose acetate membranes, to sample BPs in surface waters.

Spatial pattern analysis reveals multiple sources of organophosphorus flame retardants in coastal waters.

Organophosphorus flame retardants (PFRs) are a group of emerging contaminants which have been detected in worldwide waters. However, source of various PFRs in the large-scale area like coastal water environment have not been clearly revealed. In this study, fifteen PFRs in coast of Guangdong-Hong Kong-Macao Greater Bay area (GBA), China were investigated, and a method of spatial pattern analysis was firstly used for pollution source identification. Seawater samples from different segments of GBA coast were analyzed and thirteen PFRs were quantified with total concentrations ranging from 32.7 to 1032.7 ng L-1. GBA coasts have been seriously polluted by PFRs. A hierarchical cluster analysis of the PFR concentrations in different GBA sites showed significant spatial distributions for different types of PFRs. A series of correlation analysis between PFRs distributions and spatial pattern of GBA socio-economic indicators were performed, and multiple sources including human settlement, wastewater, manufacture, construction industry, vehicles, and shipping transport were found to be correlated to PFRs pollutions in the coasts. This study indicates that spatial pattern analysis based on statistical analysis would be a promising method of analyzing environmental data and exploring pollution source in large-scale area.

Development and placement accuracy evaluation of an MR conditional robot for prostate intervention.

Robot-assisted prostate intervention under magnetic resonance imaging (MRI) guidance is a promising method to improve the clinical performance compared with the manual method. An MR conditional 6-DOF prostate intervention serial robot is developed and a binocular vision system (BVS) is established to evaluate the needle placement accuracy and located the penetration point precisely. The robot is designed by the MR conditional criteria. The serial configuration of the robot provides adequate flexibility and large workspace and excellent friendliness to the physicians. The kinematics are deduced and the needle placement control flow is proposed according to the configuration of the robot. The robot-assisted prostate intervention is divided into two phases including needle placement and needle penetration. A custom-made robust BVS is developed to obtain the needle tip position automatically in the needle placement phase where the needle cannot be detected by the MRI for lack of hydrogen atom. A simple and general algorithm used for needle tip camera coordinate estimation is proposed. Experiments on the BVS validation and robot accuracy evaluation are performed. The experiment results show that the errors of the BVS are under 0.3621 mm and the position error of the proposed robot is 2.815 mm which indicate the adequate accuracy for the prostate intervention. Graphical abstract.

Thallium geochemical fractionation and migration in Tl-As rich soils: The key controls.

Thallium (Tl) pollution caused by mining and processing of Tl-enriched ores has become an increasing concern. This study explored the geochemical fractionation and vertical transfer of Tl in a soil profile (200 cm) from a representative Tl-As mineralized area, Southwest China. The results showed that the soils were heavily enriched by Tl and As, with concentration ranging from 3.91-17.3 and 1830-8840 mg/kg (6.79 and 2973 mg/kg in average), respectively. Approximately 50% of Tl occurred in geochemically mobile fractions in the topsoil, wherein the reducible fraction was the most enriched fraction. Further characterization using LA-ICP-MS and TEM revealed that enriched Tl and As in soils were mainly inherited from the weathering of mine tailing piles upstream. XPS characterization indicated that Fe oxides herein may play a critical role in the oxidation of Tl(I) to Tl(III) which provoked further adsorption of Tl onto Fe oxides, thereby facilitating Tl enrichment in the reducible fraction. The findings highlight that the pivotal role of Fe oxides from mineralized area in the co-mobility and migration of Tl and As in the depth profile.

Gelatin-Poly (γ-Glutamic Acid) Hydrogel as a Potential Adhesive for Repair of Intervertebral Disc Annulus Fibrosus: Evaluation of Cytocompatibility and Degradability.

STUDY DESIGN: An in vitro experimental study testing a Gelatin-poly (γ-glutamic acid) hydrogel for disc repair. OBJECTIVE: To evaluate the cytocompatibility and degradability of the above mentioned hydrogel for intervertebral disc annular fibrosis (AF) repair. SUMMARY OF BACKGROUND DATA: No repair strategies for correcting annular defects in lumbar discectomy have been clinically well recognized. Exogenous supplementation of regenerative materials to fill defects is a minimally invasive way to restore compromised mechanical properties. The injected materials, most commonly gelatin-based materials with cross-linking agents, serve as sealants and as a scaffold for incorporating biomaterials for augmentation. However, cytotoxicity of hydrogel crosslinking agents is of concern in developing viable materials. METHODS: This in vitro experimental study evaluated a newly developed gelatin-based hydrogel for intervertebral disc AF repair. Mechanical strength was augmented by γ-PGA, and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) was used for material crosslinking. Isolated bovine tail intervertebral discs (IVDs) were used to test the hydrogel, and hydrogel surface monolayer AF cell culture was used to investigate efficacy in hydrogel constructs of different EDC concentrations. Cell metabolic activity was evaluated with Alamar blue assay, cell viability assay with live/dead stain, and sulfated glycosaminoglycan (GAG) and double strain DNA were quantified to evaluate proliferation of implanted cells and synthesis of extracellular matrix (ECM) proteins. RESULTS: EDC concentrations from 10 to 40 mM resulted in significant decreases in AF cell proliferation without obvious influence on cell viability. Higher EDC concentrations resulted in decreased percentage of Alamar blue reduction and GAG and DNA concentration, but did not affect GAG/DNA and live-dead ratios. Degradation tests revealed that higher EDC concentrations decreased the hydrogel degradation rate. CONCLUSION: The developed gelatin-poly (γ-PGA) hydrogel with 20 mM EDC concentration provides an effective gap-filling biomaterial with good cytocompatibility, suggesting substantial promise for use as a sealant for small AF defects.Level of Evidence: N/A.

Sequential extraction and enrichment of flavonoids from Euonymus alatus by ultrasonic-assisted polyethylene glycol-based extraction coupled to temperature-induced cloud point extraction.

A green method for simultaneous extraction and enrichment of flavonoids from Euonymus alatus was developed by ultrasonic-assisted extraction (UAE) and temperature-induced cloud point extraction (TICPE) using PEG-base aqueous solution as the extractant. Based on screening different molecular weights of PEGs, PEG-400/water was used as the extractant, and the effects of key factors on extraction yields of flavonoids were investigated by single-factor experiments and response surface methodology (RSM). The optimum conditions of UAE were as follows: PEG-400 concentration of 16% (w/w), particle size of 80 mesh, solvent-to-material ratio of 60:1, extraction temperature of 90 °C and extraction time of 15 min. The results obtained by validation experiments were consistent with the values predicted by RSM. Temperature-induced formation of the aqueous two-phase system (ATPS) and TICPE process were further investigated by controlling temperature and adding (NH4)2SO4. In the presence of (NH4)2SO4, the ATPS formed at 75 â„ƒ and pH 3.5 could effectively improve separation and recovery of flavonoids with enrichment factor of above five times. Gallic acid, catechin, dihydromyricetin and ellagic acid in the extract were identified and confirmed by UPLC-Q-TOF-MS and the corresponding standards. The UAE-TICPE coupled to HPLC was successfully applied for extraction and determination of flavonoids in two batches of Euonymus alatus. The extraction yields of catechin, dihydromyricetin and total flavonoids were 0.377-0.684 mg/g, 1.091-1.353 mg/g and 2.612-3.146 mg/g, respectively. Compared to conventional extraction methods, PEG-based UAE integrated with TICPE in one-step procedure exhibited higher extraction efficiency and better extraction selectivity.