Cobalt-doped bioceramic scaffolds fabricated by 3D printing show enhanced osteogenic and angiogenic properties for bone repair.

BACKGROUND: The bone regeneration of artificial bone grafts is still in need of a breakthrough to improve the processes of bone defect repair. Artificial bone grafts should be modified to enable angiogenesis and thus improve osteogenesis. We have previously revealed that crystalline Ca10Li(PO4)7 (CLP) possesses higher compressive strength and better biocompatibility than that of pure beta-tricalcium phosphate (ß-TCP). In this work, we explored the possibility of cobalt (Co), known for mimicking hypoxia, doped into CLP to promote osteogenesis and angiogenesis. METHODS: We designed and manufactured porous scaffolds by doping CLP with various concentrations of Co (0, 0.1, 0.25, 0.5, and 1 mol%) and using 3D printing techniques. The crystal phase, surface morphology, compressive strength, in vitro degradation, and mineralization properties of Co-doped and -undoped CLP scaffolds were investigated. Next, we investigated the biocompatibility and effects of Co-doped and -undoped samples on osteogenic and angiogenic properties in vitro and on bone regeneration in rat cranium defects. RESULTS: With increasing Co-doping level, the compressive strength of Co-doped CLP scaffolds decreased in comparison with that of undoped CLP scaffolds, especially when the Co-doping concentration increased to 1 mol%. Co-doped CLP scaffolds possessed excellent degradation properties compared with those of undoped CLP scaffolds. The (0.1, 0.25, 0.5 mol%) Co-doped CLP scaffolds had mineralization properties similar to those of undoped CLP scaffolds, whereas the 1 mol% Co-doped CLP scaffolds shown no mineralization changes. Furthermore, compared with undoped scaffolds, Co-doped CLP scaffolds possessed excellent biocompatibility and prominent osteogenic and angiogenic properties in vitro, notably when the doping concentration was 0.25 mol%. After 8 weeks of implantation, 0.25 mol% Co-doped scaffolds had markedly enhanced bone regeneration at the defect site compared with that of the undoped scaffold. CONCLUSION: In summary, CLP doped with 0.25 mol% Co2+ ions is a prospective method to enhance osteogenic and angiogenic properties, thus promoting bone regeneration in bone defect repair.

Visualize and quantify the structural alteration of the rat spinal cord injury based on multiphoton microscopy.

The development of imaging technique to visualize and quantify the structural alteration of the spinal cord injury (SCI) may lead to better understanding and treatments of the injuries. In this work, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was tentatively applied to quantitatively visualize the cellular microstructures of SCI to demonstrate the feasibility and superiority of MPM in SCI imaging. High-contrast MPM images of normal and injured spinal cord tissue were obtained for comparison. Moreover, the changes of injured spinal cord were characterized by the quantitative analysis of the MPM images. These results showed that MPM combined with quantitative method has the ability to identify the characteristics of spinal cord injury including the changes in the contents of nerve fibers and extracellular matrix. With the advancement of MPM, we believe that this technique has great potential to provide the histological diagnosis for the monitoring and evaluation of SCI.

FUNDC1-induced mitophagy protects spinal cord neurons against ischemic injury.

Local ischemia and hypoxia are the most important pathological processes in the early phase of secondary spinal cord injury (SCI), in which mitochondria are the main target of ischemic injury. Mitochondrial autophagy, also known as mitophagy, acts as a selective autophagy that specifically identifies and degrades damaged mitochondria, thereby reducing mitochondria-dependent apoptosis. Accumulating evidence shows that the mitophagy receptor, FUN14 domain-containing 1 (FUNDC1), plays an important role in ischemic injury, but the role of FUNDC1 in SCI has not been reported. In this study, we aimed to investigate whether FUNDC1 can enhance mitophagy and inhibit neuronal apoptosis in the early stage of SCI. In a rat SCI model, we found that FUNDC1 overexpression enhanced neuronal autophagy and decreased neuronal apoptosis in the early stage of injury, thereby reducing spinal cord damage. In vitro studies showed that the neuroprotective effects of FUNDC1 were achieved by inhibiting mitochondria-dependent apoptosis and improving mitochondrial function. In addition, FUNDC1 enhanced mitophagy. The protective effects of FUNDC1 against apoptosis and mitochondrial dysfunction were reversed by 3-methyladenine (3-MA), an autophagy inhibitor. Taken together, our results confirm that FUNDC1 can protect against neuronal loss after SCI by inducing mitophagy, inhibiting mitochondria-dependent apoptosis, and improving mitochondrial function.

Carboxymethyl cellulose/quaternized chitosan hydrogel loaded with polydopamine nanoparticles promotes spinal cord injury recovery by anti-ferroptosis and M1/M2 polarization modulation.

Spinal cord injury (SCI) represents a catastrophic neurological condition resulting in long-term loss of motor, autonomic, and sensory functions. Recently, ferroptosis, an iron-regulated form of cell death distinct from apoptosis, has emerged as a potential therapeutic target for SCI. In this study, we developed an injectable hydrogel composed of carboxymethyl cellulose (CMC), and quaternized chitosan (QCS), loaded with modified polydopamine nanoparticles (PDA NPs), referred to as CQP hydrogel. This hydrogel effectively scavenged reactive oxygen species (ROS), prevented the accumulation of Fe2+ and lipid peroxidation associated with ferroptosis, and restored mitochondrial functions in primary neuronal cells. When administered to animal models (rats) with SCI, the CQP hydrogels improved motor function by regulating iron homeostasis, inhibiting ferroptosis, and mitigating oxidative stress injury. Both in vitro and in vivo studies corroborated the capacity of CQP hydrogels to promote the shift from M1 to M2 polarization of microglia/macrophages. These findings suggest that CQP hydrogels, functioning as a localized iron-chelating system, have potential as biomaterials to enhance recovery from SCI by targeting ferroptosis and modulating anti-inflammatory macrophages activity.

An injectable and photocurable methacrylate-silk fibroin/nano-hydroxyapatite hydrogel for bone regeneration through osteoimmunomodulation.

Tissue engineering has emerged as a promising approach for addressing bone defects. Most of the traditional 3D printing materials predominantly relying on polymers and ceramics. Although these materials exhibit superior osteogenic effects, their gradual degradation poses a limitation. Digital light processing (DLP) 3D bioprinting that uses natural biomaterials as bioinks has become one of the promising strategies for bone regeneration. In this study, we introduce a hydrogel biomaterial derived from silk fibroin (SF). Notably, we present the novel integration of nano-hydroxyapatite (nHA) into the hydrogel, forming a composite hydrogel that rapidly cross-links upon initiation. Moreover, we demonstrate the loading of nHA through non-covalent bonds in SilMA. In vitro experiments reveal that composite hydrogel scaffolds with 10 % nHA exhibit enhanced osteogenic effects. Transcriptomic analysis indicates that the composite hydrogel promotes bone regeneration by inducing M2 macrophage polarization. Furthermore, rat femoral defect experiments validate the efficacy of SilMA/nHA10 in bone regeneration. This study synthesis of a simple and effective composite hydrogel bioink for bone regeneration, presenting a novel strategy for the future implementation of digital 3D printing technology in bone tissue engineering.

METTL3 Affects Spinal Cord Neuronal Apoptosis by Regulating Bcl-2 m6A Modifications After Spinal Cord Injury.

OBJECTIVE: Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. METHODS: After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. RESULTS: In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. CONCLUSION: Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

3D printing monetite-coated Ti-6Al-4V surface with osteoimmunomodulatory function to enhance osteogenesis.

Titanium and its alloys are widely used in orthopedic implant surgery due to their good mechanical properties and biocompatibility. Recent studies have shown that the healing process of fractures involve not only the calcification of osteoblasts but also the regulation of the immune system. The functionalization of titanium surface coatings is one of the most important methods for solving implant failures. In this study, monetite (CaHPO4) was coated on the Ti-6Al-4V porous scaffold by hydrothermal method. SEM, XRD and EDS were used to characterize the morphology, phase constitutes, elemental content of the coating, respectively. The results indicated that a well bonded and uniformly distributed monetite coating obtained, and the degradation performance and Ca2+ release of the surface coating were also studied. In terms of biology, live/dead staining and CCK8 methods showed the coating had good biocompatibility and BMSCs can adhere and proliferate on the surface. Flow cytometry and ELISA indicated that the surface monetite-coating had good anti-inflammatory properties. Through RNA-seq analysis, it was shown in KEGG that the osteoclast-related pathway was inhibited. In vitro, monetite induced osteogenic gene expression in BMSCs and inhibited the activity of osteoclasts. In vivo experiments showed that the monetite-coating increased bone formation. In summary, monetite-coating can effectively promote the osteogenesis in BMSCs, which may be achieved through bone immune regulation.

Does spinal sagittal imbalance lead to future vertebral compression fractures in osteoporosis patients?

BACKGROUND CONTEXT: Many risk factors for osteoporotic vertebral compression fractures (OVCFs) have been reported. However, there are few reports on the relationship between spine sagittal parameters in patients with osteoporosis. PURPOSE: To explore whether: spinal sagittal imbalance is associated with future vertebral compression fractures in osteoporosis patients; spinal sagittal parameters in patients with osteoporosis can predict the occurrence of vertebral compression fractures. STUDY DESIGN: A retrospective cohort study. PATIENT SAMPLE: Patients with osteoporosis. OUTCOME MEASURES: Occurrence of OVCFs during the follow-up period. METHODS: From January 2017 to October 2019, eligible patients with osteoporosis at the initial visit were enrolled. They were followed up to November 1, 2020. Based on whether OVCFs occurred during the follow-up, the patients were divided into two groups: the experimental group (vertebral compression fracture group) and the control group (no vertebral compression fracture group). Intragroup analysis was performed as follows: Pearson and Spearman correlation coefficients were used to calculate the correlation between each parameter. Intergroup analysis was performed as follows. For categorical variables, the chi-square test was used; for normally distributed continuous variables, an independent sample t-test was used; and for non-normally distributed variables, a two-sample nonparametric test was used. Binary logistic regression analysis and receiver operating characteristic (ROC) curves were used to determine independent risk factors and critical values, respectively. RESULTS: A total of 340 patients with osteoporosis were enrolled. The longest and shortest follow-up periods were 44 months and 12 months, respectively, with an average of 25.2±10.2 months. There were significant differences in age, bone mineral density (femur and lumbar), smoking history, medication treatment regularity, Thoracolumbar Kyphosis (TLK), Pelvic Tilt (PT), C7-S1 Sagittal Vertical Axis (C7-S1 SVA), and C2-7 Sagittal Vertical Axis (C2-7 SVA) between the experimental and control groups. There were no significant differences in sex, body mass index (BMI), alcohol consumption history, hypertension, diabetes, coronary heart disease, family history of osteoporosis, physical activity, Thoracic Kyphosis (TK), Lumbar Lordosis (LL), Pelvic Incidence (PI), Sacral Slope (SS), C2-C7 Cobb Angle (CL), T1 slope (T1S) or blood parameters. Through binary logistic regression analysis, we found that BMD, medication treatment regularity and C7-S1 SVA were independent risk factors for future vertebral compression fractures. According to the ROC curve, the prediction accuracy of C7-S1 SVA was the highest. Through the calculation of critical values, we found that when C7-S1 SVA was more than 3.81 cm, future OVCFs were more likely to occur, and for every 1cm increase in C7-S1 SVA, the incidence of future OVCFs would increase by 0.324 times (p<.001, OR=1.324). Through intragroup analysis, we further found that C7-S1 SVA was positively correlated with the percentage of vertebral body wedging. CONCLUSIONS: For patients with osteoporosis, a C7-S1 SVA more than 3.81cm is significantly associated with a greater risk for vertebral compression fractures in the future.

Lysine-specific demethylase 1 inhibition enhances autophagy and attenuates early-stage post-spinal cord injury apoptosis.

Neuron death in spinal cords is caused primarily by apoptosis after spinal cord injury (SCI). Autophagy can act as a cellular response to maintain neuron homeostasis that can reduce apoptosis. Although more studies have shown that an epigenetic enzyme called Lysine-specific demethylase 1 (LSD1) can negatively regulate autophagy during cancer research, existing research does not focus on impacts related to LSD1 in nerve injury diseases. This study was designed to determine whether inhibiting LSD1 could enhance autophagy against apoptosis and provide effective neuroprotection in vitro and vivo after SCI. The results showed that LSD1 inhibition treatment significantly reduced spinal cord damage in SCI rat models and was characterized by upregulated autophagy and downregulated apoptosis. Further research demonstrated that using both pharmacological inhibition and gene knockdown could enhance autophagy and reduce apoptosis for in vitro simulation of SCI-caused damage models. Additionally, 3-methyladenine (3-MA) could partially eliminate the effect of autophagy enhancement and apoptosis suppression. These findings demonstrated that LSD1 inhibition could protect against SCI by activating autophagy and hindering apoptosis, suggesting a potential candidate for SCI therapy.

An aneurysmal bone cyst ruptured and compressed the spinal cord: a case report.

Spinal aneurysmal bone cyst (ABC) is a rare benign bone lesion with various prognosis. Common clinical symptoms of spinal ABCs include local pain, swelling. But we presented a case of a teenager girl who exhibited symptoms of acute thoracic cord compression after a slight trauma and was then diagnosed with ABC in her thoracic spine. A unique aspect is that this patient did not have symptoms before she fell down on her hip, and had an acute worsening of her neurological deficits. In the vast majority of cases, for a teenager, the trauma on the spine is tiny after falling down on the hips. That is the reason we initially felt confused before she had an urgent CT scan. In order to achieve early decompression of the thoracic cord and stabilization of the local spine around T4, we proceeded with urgent lesion resection and pedicle screws fixation from T2 to T5 to remove the liquid containing cyst and achieve spinal stability. Postoperative pathology indicated the lesion was an ABC. The patient gained good neurological recovery without any adverse effect in the final follow-up. We believe spinal ABC of teenagers can have no symptoms until a slight trauma leading to acute neurological deficits. Careful preparing for emergency surgery, prompt resection of the lesion as well as spinal stability reconstruction can promote good recovery and minimal adverse effect.

High neural precursor cell-expressed developmentally down-regulated 9 expression is a poor prognostic indicator for osteosarcoma.

BACKGROUND: This study detected neural precursor cell-expressed developmentally down-regulated 9 (NEDD9) expression in osteosarcoma tissue samples. It also examined the association between NEDD9 expression and clinicopathological features of osteosarcoma. METHODS: The prognostic value of NEDD9 has been verified in multiple solid cancers, but not osteosarcoma. In this study, 45 osteosarcoma paraffin-embedded specimens were accumulated and applied based on immunohistochemistry to identify NEDD9 expression. A correlation linking NEDD9 expression, clinicopathological qualities, and its prognostic significance was assessed to verify whether NEDD9 could be a poor prognosis biomarker. RESULTS: High NEDD9 expression compared to its low expression in osteosarcoma tissue was shown in the immunohistochemical assay. The percentage of high and low NEDD9 expression was 62.2% and 37.8% respectively. NEDD9 expression level was significantly associated with metastasis and Enneking stage (P<0.05), while gender, age, tumor size, sites, and histopathological types were not. Univariate analysis showed that metastasis with a hazard ratio (HR) of 2.63 (P=0.003), Enneking stage III (HR =5.44, P=0.009), chondroblastic osteosarcoma (HR =2.59, P=0.014), and high NEDD9 expression (HR =2.79, P=0.002) were prognostic factors contributing to poor overall survival (OS). Moreover, multivariate analysis revealed that nonconventional pathological osteosarcoma (HR =0.17, P=0.010) and NEDD9 expression (HR =6.03, P<0.001) were independent prognostic factors. CONCLUSIONS: High NEDD9 expression in osteosarcoma independently indicated poor clinical prognosis, indicating the possibility that NEDD9 could be a therapeutic target for osteosarcoma.

4-PBA Enhances Autophagy by Inhibiting Endoplasmic Reticulum Stress in Recombinant Human Beta Nerve Growth Factor-Induced PC12 cells After Mechanical Injury via PI3K/AKT/mTOR Signaling Pathway.

OBJECTIVE: To investigate mechanism of endoplasmic reticulum (ER) stress-mediated autophagy in spinal cord injury (SCI). METHODS: An in vitro model of spinal cord injury (SCI) was established by recombinant human beta nerve growth factor (NGF)-induced PC12 cells. Immunofluorescence was used to detect properties of PC12 cells induced by NGF. Western blot assay was used to detect expressions of the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)I/II, the ER stress-related protein (HSPA5/GRP78), as well as the PI3K/AKT/mTOR signaling pathway-related proteins after mechanical injury at different time points. Then the sample assigned into sham, SCI, LY294002, SCI+LY294002, 4-PBA (4-phenylbutyric acid), and SCI+4-PBA groups. The expressions of the LC3I/II and PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blot assay. RESULTS: NGF-induced PC12 cells have neurophysiological characteristics. After administration of the PI3K-specific inhibitor LY294002, phosphorylation levels of AKT and mTOR decreased, and the ratio of LC3II/I was higher in the inhibitor-treated injury group than the simple-injury group. After administration of the ER stress inhibitor 4-PBA, the results were similar to LY294002 group's results compared with SCI group. CONCLUSIONS: Our study showed that NGF-induced PC12 cells can induce autophagy and ER stress after mechanical injury. ER stress inhibitor 4-PBA obtained similar effects to PI3K inhibitor LY294002, enhanced autophagy via PI3K/AKT/mTOR signaling pathway.

Is Cervical Traction Effective in Chronic Nonspecific Neck Pain Patients With Unsatisfactory NSAID Control? A Nomogram to Predict Effectiveness.

OBJECTIVE: In this retrospective study, our objective was to establish a nomogram to predict the effectiveness of cervical traction in young and middle-aged chronic nonspecific neck pain (CNNP) patients with unsatisfactory nonsteroidal anti-inflammatory drug (NSAID) control. For CNNP patients with unsatisfactory NSAID control, the effectiveness of cervical traction varies. Neck muscle fat infiltration and clinical features may associate with the effectiveness. METHODS: A total of 186 suitable patients were classified into a training data set (from August 2015 to July 2018, n = 118) and a validation data set (from August 2018 to June 2019, n = 68) with time sequence. All patients were included to receive magnetic resonance imaging scan to calculate posterior cervical fat and muscle features, then undergoing unified cervical traction in an outpatient clinic. The least absolute shrinkage and selection operator (LASSO) regression model was used to select potentially relevant features to predict effectiveness possibility of cervical traction. Multivariable logistic regression analysis was used to develop the predicting model, presenting with a nomogram. The performance of the nomogram was assessed based on its calibration, discrimination, and clinical utility. RESULTS: Through the LASSO regression model, we identified 4 predictors including sex, good exercise compliance, the ratio of the cross-sectional area (CSA) between fat and muscle on C5 level (C5 fat CSA/muscle CSA), the ratio of CSA between fat and centrum on C5 level (C5 fat CSA/centrum muscle CSA). The nomogram provided good calibration and discrimination in the training cohort, showing an area under the curve (AUC) of 0.704 (95% CI, 0.608-0.799) and good concordance between the predicted and actual probabilities with Spiegelhalter's Z-test (P = 0.835). Discrimination of the model in the validation data set was acceptable, with AUC of 0.691 (95% CI, 0.564-0.817). Decision curve analysis revealed the nomogram to be clinically useful. CONCLUSIONS: Male sex, good exercise compliance, lower C5 fat CSA/centrum CSA, and and lower C5 fat CSA/muscle CSA could be favorable features to predict the effectiveness of cervical traction in CNNP patients with unsatisfactory NSAID control.

Effects of dexamethasone on autophagy and apoptosis in acute spinal cord injury.

Previous studies have indicated that spinal cord injury can induce autophagy. To a certain extent, increased autophagy has a protective effect on neurons. Early hormone therapy is well recognized as a treatment for spinal cord injury. However, whether the protective effect of autophagy is important in recovery from spinal cord injury remains unclear. In this study, we established an in-vitro model of spinal cord injury to study the effects of dexamethasone on mechanical injury, autophagy, and apoptosis in spinal cord neurons. The results showed that dexamethasone inhibited the level of autophagy in the injured nerve cells in a dose-dependent manner. High doses of dexamethasone protected the damaged spinal cord neurons by inhibiting apoptosis, but a protective effect from low hormone concentrations was not obvious. When autophagy was inhibited in damaged spinal cord neurons, apoptosis decreased significantly; in contrast, impairment of autophagy-induced activation of spinal cord neurons and apoptosis levels were significantly increased.

Autophagy protects against PI3K/Akt/mTOR-mediated apoptosis of spinal cord neurons after mechanical injury.

Many studies have indicated that autophagy and apoptosis play an important role in the pathogenesis of spinal cord injury. In recent years, research on autophagy-related signal transduction pathways has demonstrated that the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is closely associated with the initiation of autophagy. However, the mechanism of the pathological relationship between this signaling pathway and apoptosis in spinal cord injury is unclear. In this study, we used an in vitro model of spinal cord injury to observe the mechanism of the PI3K/Akt/mTOR signaling pathway and the apoptosis of neurons via the mitochondrial pathway. Mitochondrial pathway apoptosis-related proteins were detected by western blot. Akt and mTOR phosphorylation levels peaked 4h after mechanical damage and then decreased. Following administration of the PI3K-specific inhibitor LY294002, the phosphorylation levels of Akt and mTOR decreased, and the ratio of autophagy-specific protein microtubule-associated protein 1 light chain (LC3)II/I was higher in the inhibitor-treated injury group than in the simple-injury group. TUNEL staining was used to detect apoptosis, and apoptosis was significantly reduced after the inhibition of the PI3K/Akt/mTOR signaling pathway. In summary, the PI3K/Akt/mTOR signaling pathway is involved in the apoptosis of neurons after mechanical injury and can induce apoptosis through the mitochondrial pathway.