Nucleus pulposus cell-derived efficient microcarrier for intervertebral disc tissue engineering.

Adipose-derived stem cells (ADSCs) show great potential for the treatment of intervertebral disc (IVD) degeneration. An ideal carrier is necessary to transplant ADSCs into degenerated IVDs without influencing cell function. Nucleus pulposus cells (NPCs) can synthesize and deposit chondroitin sulfate and type II collagen which are NP-specific extracellular matrix (ECM) and can also regulate the NP-specific differentiation of stem cells. Bioscaffolds fabricated based on the ECM synthesis functions of NPCs have possible roles in cell transplantation and differentiation induction, but it has not been studied. In this study, we first aggregated NPCs into pellets, and then, NPC-derived efficient microcarriers (NPCMs) were fabricated by pellet cultivation under specific conditions and optimized decellularization. Thirdly, we evaluated the microstructure, biochemical composition, biostability and cytotoxicity of the NPCMs. Finally, we investigated the NP-specific differentiation of ADSCs induced by the NPCMsin vitroand NP regeneration induced by the ADSC-loaded NPCMs in a rabbit model. The results indicated that the injectable NPCMs retained maximal ECM and minimal cell nucleic acid after optimized decellularization and had good biostability and no cytotoxicity. The NPCMs also promoted the NP-specific differentiation of ADSCsin vitro. In addition, the results of MRI, x-ray, and the structure and ECM content of NP showed that the ADSCs-loaded NPCMs can partly restored the degenerated NPin vivo. Our injectable NPCMs regenerated the degenerated NP and provide a simplified and efficient strategy for treating IVD degeneration.

Micro Fragmented Adipose Tissue Promotes the Matrix Synthesis Function of Nucleus Pulposus Cells and Regenerates Degenerated Intervertebral Disc in a Pig Model.

Intervertebral disc (IVD) degeneration and consequent lower back pain is a common disease. Micro fragmented adipose tissue (MFAT) is promising for a wide range of applications in regenerative medicine. In this study, MFAT was isolated by a nonenzymatic method and co-cultured with nucleus pulposus cells (NPCs) using an indirect co-culture system in vitro. A pig disc degeneration model was used to investigate the regenerative effect of MFAT on degenerated IVDs in vivo. The mRNA expression of Sox9, Acan, and Col2 in NPCs was significantly increased, while no significant increase was observed in the mRNA expression of proinflammatory cytokine genes after the NPCs were co-cultured with MFAT. Nucleus pulposus (NP)-specific markers were increased in MFAT cells after co-culture with NPCs. After injection of MFAT, the disc height, water content, extracellular matrix, and structure of the degenerated NP were significantly improved. MFAT promoted the matrix synthesis function of NPCs, and NPCs stimulated the NP-like differentiation of MFAT cells. In addition, MFAT also partly regenerated degenerated IVDs in the pig model.

Small Molecule-Based Strategy Promotes Nucleus Pulposus Specific Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising for regenerating degenerated intervertebral discs (IVDs), but the low efficiency of nucleus pulposus (NP)-specific differentiation limits their clinical applications. The Sonic hedgehog (Shh) signaling pathway is important in NP-specific differentiation of ADSCs, and Smoothened Agonist (SAG) is a highly specific and effective agonist of Shh signaling. In this study, we proposed a new differentiation strategy with the use of the small molecule SAG. The NP-specific differentiation and extracellular matrix (ECM) synthesis of ADSCs were measured in vitro , and the regenerative effects of SAG pretreated ADSCs in degenerated IVDs were verified in vivo . The results showed that the combination of SAG and transforming growth factor-ß3 (TGF-ß3) is able to increase the ECM synthesis of ADSCs. In addition, the gene and protein expression levels of NP-specific markers were increased by treatment with SAG and TGF-ß3. Furthermore, SAG pretreated ADSCs can also improve the disc height, water content, ECM content, and structure of degenerated IVDs in vivo . Our new differentiation scheme has high efficiency in inducing NP-specific differentiation of ADSCs and is promising for stem cell-based treatment of degenerated IVDs.

Intradiscal Injection of Induced Pluripotent Stem Cell-Derived Nucleus Pulposus-Like Cell-Seeded Polymeric Microspheres Promotes Rat Disc Regeneration.

BACKGROUND: Cell replacement therapy is an attractive alternative for treating degenerated intervertebral discs (IVDs), which are related to the reduction of nucleus pulposus-like cells (NP-lCs) and the loss of the extracellular matrix. Induced pluripotent stem cells (iPSCs) which resemble embryonic stem cells are considered to be a potential resource for restoring NP-lCs and disc homeostasis. Here, we proposed an efficient two-step differentiation protocol of human iPSCs into NP-lCs and continuously tested their in vivo ability to regenerate IVDs. METHODS: A polymeric gelatin microsphere (GM) was generated for sustained release of growth and differentiation factor-5 (GDF-5) and as a cell delivery vehicle of NP-lCs. By injecting NP-lC-seeded GDF-5-loaded GMs into the rat coccygeal intervertebral discs, the disc height and water content were examined with the molybdenum target radiographic imaging test and magnetic resonance imaging examination. Histology and immunohistochemistry results were shown with H&E, S-O-Fast Green, and immunohistochemistry staining. RESULTS: We demonstrated that the injection of NP-lC-seeded GDF-5-loaded GMs could reverse IDD in a rat model. The imaging examination indicated that disc height recovered and water content increased. Histology and immunohistochemistry results indicated that the NP cells as well as their extracellular matrix were partially restored. CONCLUSIONS: The results suggest that NP-lC-seeded GDF-5-loaded GMs could partially regenerate degenerated intervertebral discs after transplantation into rat coccygeal intervertebral discs. Our study will help develop a promising method of stem cell-based therapy for IDD.

Sustained release of GDF5 from a designed coacervate attenuates disc degeneration in a rat model.

Low back pain is often caused by intervertebral disc degeneration, which is characterized by nucleus pulposus (NP) and extracellular matrix (ECM) degeneration. Human adipose-derived stem cells (hADSCs) induced by growth and differentiation factor-5 (GDF5) can differentiate into an NP-like phenotype. Although stem cell-based therapy with prolonged exposure to growth factors is regarded as a promising treatment, the efficacy of this approach in attenuating the disc degeneration process is limited by the short lifespan of growth factors. In our study, a unique growth factor delivery vehicle composed of heparin and the synthetic polycation poly(ethylene argininylaspartate diglyceride) (PEAD) was used to sustain GDF5 release. The results showed that sustained release of GDF5 by the PEAD:heparin delivery system promoted hADSC differentiation to an NP-like phenotype in vitro. After injection of the PEAD:heparin:GDF5 delivery platform and hADSCs into intervertebral spaces of coccygeal (Co) vertebrae Co7/Co8 and Co8/Co9 of the rat, the disc height, water content, and structure of the NPs decreased more slowly than other treatment groups. This new strategy may be used as an alternative treatment for attenuating intervertebral disc degeneration with hADSCs without the need for gene therapy. STATEMENT OF SIGNIFICANCE: Low back pain is often caused by intervertebral disc degeneration, which is characterized by nucleus pulposus (NP) and extracellular matrix (ECM) degeneration. Human adipose-derived stem cells (hADSCs) induced by growth and differentiation factor-5 (GDF-5) can differentiate into an NP-like phenotype. Although stem cell-based therapy with prolonged exposure to growth factor is regarded as a promising treatment, the efficacy of this approach in the disc regeneration process is limited by the short life of growth factors. In our study, a unique growth factor delivery vehicle comprised of heparin and the synthetic polycation poly(ethylene argininylaspartate diglyceride) (PEAD) was used to sustain the release of GDF-5. Numerous groups have explored IDD regeneration methods in vitro and in vivo. Our study differs in that GDF5 was incorporated into a vehicle through charge attraction and exhibited a sustained release profile. Moreover, GDF-5 seeded coacervate combined with hADSC injection could be a minimally invasive approach for tissue engineering that is suitable for clinical application. We investigated the stimulatory effects of our GDF-5 seeded coacervate on the differentiation of ADSCs in vitro and the reparative effect of the delivery system on degenerated NP in vivo.

Wogonin mitigates intervertebral disc degeneration through the Nrf2/ARE and MAPK signaling pathways.

Intervertebral disc degeneration (IVDD) is a prevalent disease characterized by the progressive loss of the extracellular matrix in the local nucleus pulposus region. Metalloproteinases and pro-inflammatory cytokines play an important role in this process. Thus, anti-inflammatory strategies are an important component of IVDD treatment. Wogonin, a naturally existing monoflavonoid, has been reported to have potential anti-inflammatory effects in some inflammatory diseases. Hence, in our present study we investigated the protective effects and potential mechanisms of wogonin in rat nucleus pulposus cells that had been treated with interleukin-1beta (IL-1ß) to induce severe IVDD. An in vivo rat caudal vertebrae needle-stab model was also designed and its validity was evaluated as an IVDD model. The results demonstrated that wogonin suppressed IL-1ß-induced inflammatory mediators (iNOS, IL-6 and COX2) and matrix-degrading proteinases (MMP1, MMP3, MMP13 and ADAMTS4). Wogonin also upregulated some of the key components of the extracellular matrix, such as collagen II. Furthermore, we discovered that wogonin exerted anti-inflammatory effects by activating the Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis. Moreover, the IL-1ß-induced stimulation of the MAPK signaling pathway was reversed by wogonin treatment. The in vivo MRI and histological results also revealed that wogonin protected the nucleus pulposus from the progression of IVDD. Therefore, wogonin may be a potential agent for the treatment of IVDD.

Injectable decellularized nucleus pulposus-based cell delivery system for differentiation of adipose-derived stem cells and nucleus pulposus regeneration.

Stem cell-based tissue engineering is a promising treatment for intervertebral disc (IVD) degeneration. A bio-scaffold that can maintain the function of transplanted cells and possesses favorable mechanical properties is needed in tissue engineering. Decellularized nucleus pulposus (dNP) has the potential to be a suitable bio-scaffold because it mimics the native nucleus pulposus (NP) composition. However, matrix loss during decellularization and difficulty in transplantation limit the clinical application of dNP scaffolds. In this study, we fabricated an injectable dNP-based cell delivery system (NPCS) and evaluated its properties by assessing the microstructure, biochemical composition, water content, biosafety, biostability, and mechanical properties. We also investigated the stimulatory effects of the bio-scaffold on the NP-like differentiation of adipose-derived stem cells (ADSCs) in vitro and the regenerative effects of the NPCS on degenerated NP in an in vivo animal model. The results showed that approximately 68% and 43% of the collagen and sGAG, respectively, remained in the NPCS after 30 days. The NPCS also showed mechanical properties similar to those of fresh NP. In addition, the NPCS was biocompatible and able to induce NP-like differentiation and extracellular matrix (ECM) synthesis in ADSCs. The disc height index (almost 81%) and the MRI index (349.05 ±â€¯38.48) of the NPCS-treated NP were significantly higher than those of the degenerated NP after 16 weeks. The NPCS also partly restored the ECM content and the structure of degenerated NP in vivo. Our NPCS has good biological and mechanical properties and has the ability to promote the regeneration of degenerated NP. STATEMENT OF SIGNIFICANCE: Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration. Stem cell-based tissue engineering is a promising treatment for NP regeneration. Bio-scaffolds which have favorable biological and mechanical properties are needed in tissue engineering. Decellularized NP (dNP) scaffold is a potential choice for tissue engineering, but the difficulty in balancing complete decellularization and retaining ECM limits its usage. Instead of choosing different decellularization protocols, we complementing the sGAG lost during decellularization by cross-linking via genipin and fabricating an injectable dNP-based cell delivery system (NPCS) which has similar components as the native NP. We also investigated the biological and mechanical properties of the NPCS in vitro and verified its regenerative effects on degenerated IVDs in an animal model.

Complications in transorbital penetrating injury by bamboo branch: A case report.

RATIONALE: Wooden transorbital penetrating injury is an uncommon and serious trauma that may cause multiply complications. PATIENT CONCERNS: Here we describe a 62-year-old Chinese woman with a transorbital penetrating injury caused by a long bamboo branch. DIAGNOSIS: Computed tomography scan and magnetic resonance imaging showed the presence of a wooden foreign body. INTERVENTIONS: Cerebrovascular digital subtraction angiography and temporary balloon occlusion were performed with general anesthesia. Anti-inflammatory therapy was subsequently administered. OUTCOMES: Retention of wooden foreign body, orbital cellulitis, and traumatic aneurysm at the right internal carotid artery were diagnosed 1 month later. Coil embolization of the right internal carotid artery aneurysm and endoscopic sinus surgery were then performed, and postoperative condition was monitored and recorded. LESSONS: Penetrating transorbital injury complications may occur because of retained wooden foreign bodies near the intracranial arteries. Reasonable surgical intervention and special attention should be performed in this kind of trauma.

Genipin cross-linked type II collagen/chondroitin sulfate composite hydrogel-like cell delivery system induces differentiation of adipose-derived stem cells and regenerates degenerated nucleus pulposus.

Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Although adipose-derived stem cell (ADSC)-based therapy is regarded to be promising for the treatment of degenerated NP, there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. The induction effect of the scaffold on ADSC differentiation was studied in vitro, and a rat coccygeal vertebrae degeneration model was used to investigate the regenerative effect of the CCSA system on the degenerated NP in vivo. The results showed that the CCSA delivery system cross-linked with 0.02% genipin was biocompatible and promoted the expressions of NP-specific genes. After the injection of the CCSA system, the disc height, water content, extracellular matrix synthesis, and structure of the degenerated NP were partly restored. Our CCSA delivery system uses minimally invasive approaches to promote the regeneration of degenerated NP and provides an exciting new avenue for the treatment of degenerative disc disease. STATEMENT OF SIGNIFICANCE: Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Stem cell-based tissue engineering is a promising method in NP regeneration, but there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. Although several research groups have studied the fabrication of injectable hydrogel with biological matrix, our study differs from other works. We chose type II collagen and CS, the two primary native components in the NP, as the main materials and combined them according to the natural ratio of collagen and sGAG in the NP. The delivery system is preloaded with ADSCs and can be injected into the NP with a needle, followed by in situ gelation. Genipin is used as a cross-linker to improve the bio-stability of the scaffold, with low cytotoxicity. We investigated the stimulatory effects of our scaffold on the differentiation of ADSCs in vitro and the regenerative effect of the CCSA delivery system on degenerated NP in vivo.

[Quantitative analysis of enhanced MRI features for predicting epidermal growth factor receptor gene amplification in glioblastoma multiforme with radiomic method].

OBJECTIVE: To assess the value of contrast enhanced MRI features for predicting epidermal growth factor receptor (EGFR) gene amplification in glioblastoma multiforme (GBM) with radiomic method. METHODS: Eighty patients with EGFR status examined GBM were retrospectively reviewed. The data were randomly divided into a training dataset (60%) and test dataset (40%). Texture features of each case were extracted from the enhanced region and the edema region in contrast enhanced MR images. Principal component analysis was used for dimension reduction. Random forest model, support vector machine model and neural network model were built. Area under the curve (AUC) of the receiver operating characteristics curve was used to assess the performance of models with test dataset. RESULTS: A total of 542 features were extracted from the enhanced region and the edema region. Forty-eight principal components were obtained, which accounted for 100% accumulation contribution rate, and the first 31 principal components were selected for models building, which accounted for 98.5% accumulation contribution rate. The values of AUCs were 0.74, 0.69 and 0.63 for random forest model, support vector machine model and neural network model in the test dataset, respectively. CONCLUSIONS: Radiomic method with proper model may have a potential role in predicting the EGFR gene status with enhanced MRI features derived from the enhanced region and the edema region in patients with glioblastoma multiforme.

Application of MRI in the diagnosis of glomus tumor / 中华整形外科杂志

<p><b>OBJECTIVE</b>To discuss the necessity of MRI as a routine examination for diagnosis of glomus tumor.</p><p><b>METHODS</b>From Nov. 2013 to July 2014, 7 cases of glomus tumor were treated in our department. All patients had typical clinical symptoms of glomus tumor and received preoperative X-ray and/or MRI examination. The diagnosis was confirmed by postoperative histopathologic examination. All the patients were retrospectively analyzed by reviewing the preoperative examination results and PubMed search results.</p><p><b>RESULTS</b>2 cases underwent only X-ray examination before operation with no positive results. 3 cases underwent both X-ray and MRI. No positive finding happened in X-ray, while MRI showed glomus tumor characteristic on T1- and T2-weighted images which demonstreated a more intense signal after injection of gadolinium. The last 2 cases underwent only MRI examination, which revealed positive images of glomus tumor.</p><p><b>CONCLUSIONS</b>MRI plays an important role in diagnosis of glomus tumor and should be adopted as a routine examination.</p>

Dual release of dexamethasone and TGF-ß3 from polymeric microspheres for stem cell matrix accumulation in a rat disc degeneration model.

Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported that a new nanostructured three-dimensional poly(lactide-co-glycolide) (PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(l-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSC)-seeded PLGA microspheres into the rat intervertebral disc could regenerate the degenerated disc. Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix-associated gene expression were evaluated in normal controls (NCs) (without operations), a degeneration control (DC) group (with needle puncture, injected only with Dulbecco's modified Eagle's medium), a PLGA microspheres (PMs) treatment group (with needle puncture, PLGA microspheres only injection), and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSC injection) for a 24-week period. The results showed that at 24 weeks post-transplantation, the PM and PMA groups regained disc height values of ∼63% and 76% and MRI signal intensities of ∼47% and 76%, respectively, compared to the NC group. Biochemistry, immunohistochemistry and gene expression analysis also indicated the restoration of proteoglycan accumulation in the discs of the PM and PMA groups. However, there was almost no restoration of proteoglycan accumulation in the discs of the DC group compared with the PM and PMA groups. Taken together, these data suggest that ADSC-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat degenerative intervertebral disc.

Effects of methylphenidate on resting-state brain activity in normal adults: an fMRI study.

Methylphenidate (MPH) is one of the most commonly used stimulants for the treatment of attention deficit hyperactivity disorder (ADHD). Although several studies have evaluated the effects of MPH on human brain activation during specific cognitive tasks using functional magnetic resonance imaging (fMRI), few studies have focused on spontaneous brain activity. In the current study, we investigated the effect of MPH on the intra-regional synchronization of spontaneous brain activity during the resting state in 18 normal adult males. A handedness questionnaire and the Wechsler Adult Intelligence Scale were applied before medication, and a resting-state fMRI scan was obtained 1 h after medication (20 mg MPH or placebo, order counterbalanced between participants). We demonstrated that: (1) there were no significant differences in the performance of behavioral tasks between the MPH and placebo groups; (2) the left middle and superior temporal gyri had stronger MPHrelated regional homogeneity (ReHo); and (3) the left lingual gyrus had weaker MPH-related ReHo. Our findings showed that the ReHo in some brain areas changes with MPH compared to placebo in normal adults, even though there are no behavioral differences. This method can be applied to patients with mental illness who may be treated with MPH, and be used to compare the difference between patients taking MPH and normal participants, to help reveal the mechanism of how MPH works.

Estimation of total body skeletal muscle mass in Chinese adults: prediction model by dual-energy X-ray absorptiometry.

BACKGROUND: There are few reports on total body skeletal muscle mass (SM) in Chinese. The objective of this study is to establish a prediction model of SM for Chinese adults. METHODOLOGY: Appendicular lean soft tissue (ALST) was measured by dual energy X-ray absorptiometry (DXA) and SM by magnetic resonance image (MRI) in 66 Chinese adults (52 men and 14 women). Images of MRI were segmented into compartments including intermuscular adipose tissue (IMAT) and IMAT-free SM. Regression was used to fit the prediction model SM = c + k × ALST. Age and gender were adjusted in the fitted model. The piece-wise linear function was performed to further explore the effect of age on SM. 'Leave-One-Out Cross Validation' was utilized to evaluate the prediction performance. The significance of observed differences between predicted and actual SM was tested by t test and the level of agreement was assessed by the method of Bland and Altman. RESULTS: Men had greater ALST and IMAT-free SM than women. ALST was the primary predictor and highly correlated with IMAT-free SM (R(2) = 0.94, SEE = 1.11 kg, P<0.001). Age was an additional predictor (SM prediction model with age adjusted R(2) = 0.95, SEE = 1.05 kg, P<0.001). There was a piece-wise linear relationship between age and IMAT-free SM: IMAT-free SM = 1.21×ALST-0.98, (Age <45 years) and IMAT-free SM = 1.21×ALST-0.98-0.04× (Age-45), (Age ≥45 years). The prediction performance of this age-adjusted model was good due to 'Leave-One-Out Cross Validation'. No significant difference between measured and predicted IMAT-free SM was detected. CONCLUSION: Previous SM prediction model developed in multi-ethnic groups underestimated SM by 2.3% and 3.4% for Chinese men and women. A new prediction model by DXA has been established to predict SM in Chinese adults.

Imaging findings of splenic hamartoma.

AIM: To assess CT and MR manifestations and their diagnostic value in splenic hamartoma with review of literatures. METHODS: We described a woman who was accidentally found to have a splenic tumor by ultrasound of the abdomen. CT and MR findings of this splenic hamartoma were proved by pathology retrospectively. RESULTS: The CT and MR findings in this case included a ball-like mass with homogeneous mild-hypodensity lesions on non-enhanced CT scans or isointensity on T1-weighted images and mild hypointensity on T2-weighted images, progressive homogeneous enhancement on multiple-phase spiral CT and MR enhanced scans, and isodense enhancement on delayed post-contrast CT scans and obvious hyperintensity relative to the spleen on delayed MR images. CONCLUSION: Splenic hamartoma has some specific radiological features. However, the diagnosis of this disease must be based on clinical features and confirmed by pathology.