Xiaolei Zhang

Apoptosis, senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration†‡

This research was aimed to study the mechanisms by which diabetes aggravates intervertebral disc degeneration (IDD) and to discuss the relationship between autophagy and IDD in nucleus pulposus (NP) cells. Sixteen weeks after injecting streptozotocin (STZ), the intervertebral discs (IVDs) were studied by histology, Alcian blue, 1,9‐dimethylmethylene blue (DMMB), immunohistochemistry, and RT‐PCR to explore the IDD. The apoptosis and senescence of NP cells was investigated by terminal deoxyribonucleotidyl transferase (TDT)‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) assay, immunohistochemistry, and Western blot for caspase3, caspase8, caspase9, and p16lnk4A (increased in cellular senescence). The level of autophagy in NP cells was detected by Western blot, immunohistochemistry, and transmission electron microscopy (TEM). The proteoglycan and collagen II in the extracellular matrix and the aggrecan and collagen II mRNA expression in NP cells of diabetic rats were decreased compared with the control group. Diabetes increased apoptosis of NP cells and led to activations of initiators of intrinsic (caspases‐9) and extrinsic (caspase‐8) pathways as well as their common executioner (caspase‐3). Cellular senescence was increased about twofold in NP of diabetic rats. In addition, the Western blot, immunohistochemistry, and TEM demonstrated higher level of autophagy in NP cells of diabetic rats than control rats to a statistically significant extent. These findings support that diabetes induced by STZ can cause IDD by accelerating the apoptosis and senescence of NP cells excluding the overweight influence. And the results suggest that the autophagy may be a response mechanism to the change of NP cells in diabetic rats.

Inhibition of Rac1 activity by controlled release of NSC23766 from chitosan microspheres effectively ameliorates osteoarthritis development in vivo

Background Osteoarthritis (OA) is a degenerative joint disease characterised by cartilage degradation and chondrocyte hypertrophy. A recent study showed that Rac1 promoted expression of MMP13 and chondrocyte hypertrophy within the growth plate. These findings warrant further investigations on the roles of Rac1 in OA development and therapy in animal models. Objective To investigate the role and mechanistic pathway of Rac1 involvement in pathological changes of OA chondrocytes in vitro and OA development in vivo, as well as to develop a strategy of modulating Rac1 activity for OA treatment. Material and methods OA and normal cartilage from human or mice were used for immunohistochemical study and Rac1 activity assay. Chondrocytes treated with IL1β and the untreated control were subjected to the Rac1 activity assay. Chondrocytes transfected with CA-Rac1, DN-Rac1 or GFP were cultured under conditions for inducing calcification. To evaluate the effect of Rac1 in OA development, an OA model was created by anterior cruciate ligament transection in mice. CA-Rac1, DN-Rac1 and GFP lentivirus, or NSC23766, were injected intra-articularly. Joints were subjected to histological analysis. Results It was found that there is aberrant Rac1 activation in human OA cartilage. Rac1 activity could also be elevated by IL1β. Additionally, activated Rac1 promoted expression of MMP13, ADAMTS-5 and COLX by chondrocytes, partially through the β-catenin pathway. Moreover, activation of Rac1 in knee joints by CA-Rac1 lentivirus accelerated OA progression, while inhibition of Rac1 activity by DN-Rac1 lentivirus or Rac1 inhibitor NSC23766 delayed OA development. Therefore, we developed a strategy of controlled release of NSC23766 from chitosan microspheres to OA joints, which effectively protected cartilage from destruction. Conclusions These findings demonstrated that Rac1 activity is implicated in OA development. Also, controlled release of Rac1 inhibitor is a promising strategy for OA treatment.

Fusion versus Nonfusion for Surgically Treated Thoracolumbar Burst Fractures: A Meta-Analysis

Background Posterior pedicle screw fixation has become a popular method for treating thoracolumbar burst fractures. However, it remains unclear whether additional fusion could improve clinical and radiological outcomes. This meta-analysis was performed to evaluate the effectiveness of fusion as a supplement to pedicle screw fixation for thoracolumbar burst fractures. Methodology/Principal Findings MEDLINE, OVID, Springer, and Google Scholar were searched for relevant randomized and quasi-randomized controlled trials that compared the clinical and radiological efficacy of fusion versus nonfusion for thoracolumbar burst fractures managed by posterior pedicle screw fixation. Risk of bias in included studies was assessed using the Cochrane Risk of Bias tool. We generated pooled risk ratios or weighted mean differences across studies. Based on predefined inclusion criteria, 4 eligible trials with a total of 220 patients were included in this meta-analysis. The mean age of the patients was 35.1 years. 96.8% of the fractures were located at T12 to L1 level. Baseline characteristics were similar between the fusion and nonfusion groups. No significant difference was identified between the two groups regarding radiological outcome, functional outcome, neurologic improvement, and implant failure rate. The pooled data showed that the nonfusion group was associated with significantly reduced operative time (p<0.0001) and blood loss (p  = 0.0003). Conclusions/Significances The results of this meta-analysis suggested that fusion was not necessary when thoracolumbar burst fracture was treated by posterior pedicle screw fixation. More randomized controlled trials with high quality are still needed in the future.

3D‐Printed Atsttrin‐Incorporated Alginate/Hydroxyapatite Scaffold Promotes Bone Defect Regeneration with TNF/TNFR Signaling Involvement

High expression levels of pro-inflammatory tumor necrosis factor (TNF)-α within bone defects can decelerate and impair bone regeneration. However, there are few available bone scaffolds with anti-inflammatory function. The progranulin (PGRN)-derived engineered protein, Atsttrin, is known to exert antagonistic effects on the TNF-α function. Hence, this study investigates whether 3D-printed Atsttrin-incorporated alginate(Alg)/hydroxyapatite(nHAp) scaffolds can facilitate bone healing through affecting the TNF/TNFR signaling. A 3D bioprinting system is used to fabricate Atsttrin-Alg/nHAp composite scaffolds, and the Atsttrin release from this scaffold is characterized, followed by evaluation of its efficacy on bone regeneration both in vitro and in vivo. The 3D-printed Atsttrin-Alg/nHAp scaffold exhibits a precisely defined structure, can sustain Atsttrin release for at least 5 days, has negligible cytotoxicity, and supports cell adhesion. Atsttrin can also attenuate the suppressive effects of TNF-α on BMP-2-induced osteoblastic differentiation in vitro. The 3D-printed Atsttrin-Alg/nHAp scaffold significantly reduces the number of TNF-α positive cells within wound sites, 7 days after post-calvarial defect surgery. Additionally, histological staining and X-ray scanning results also show that the 3D-printed Atsttrin-Alg/nHAp scaffold enhances the regeneration of mice calvarial bone defects. These findings thus demonstrate that the precise structure and anti-inflammatory properties of 3D-printed Atsttrin-Alg/nHAp scaffolds may promote bone defect repair.

Pooled Analysis of Non-Union, Re-Operation, Infection, and Approach Related Complications after Anterior Odontoid Screw Fixation

Background Anterior odontoid screw fixation (AOSF) has been one of the most popular treatments for odontoid fractures. However, the true efficacy of AOSF remains unclear. In this study, we aimed to provide the pooled rates of non-union, reoperation, infection, and approach related complications after AOSF for odontoid fractures. Methods We searched studies that discussed complications after AOSF for type II or type III odontoid fractures. A proportion meta-analysis was done and potential sources of heterogeneity were explored by meta-regression analysis. Results Of 972 references initially identified, 63 were eligible for inclusion. 54 studies provided data regarding non-union. The pooled non-union rate was 10% (95% CI: 7%–3%). 48 citations provided re-operation information with a pooled proportion of 5% (95% CI: 3%–7%). Infection was described in 20 studies with an overall rate of 0.2% (95% CI: 0%–1.2%). The main approach related complication is postoperative dysphagia with a pooled rate of 10% (95% CI: 4%–17%). Proportions for the other approach related complications such as postoperative hoarseness (1.2%, 95% CI: 0%–3.7%), esophageal/retropharyngeal injury (0%, 95% CI: 0%–1.1%), wound hematomas (0.2%, 95% CI: 0%–1.8%), and spinal cord injury (0%, 95% CI: 0%–0.2%) were very low. Significant heterogeneities were detected when we combined the rates of non-union, re-operation, and dysphagia. Multivariate meta-regression analysis showed that old age was significantly predictive of non-union. Subgroup comparisons showed significant higher non-union rates in age ≥70 than that in age ≤40 and in age 40 to <50. Meta-regression analysis did not reveal any examined variables influencing the re-operation rate. Meta-regression analysis showed age had a significant effect on the dysphagia rate. Conclusions/Significances This study summarized the rates of non-union, reoperation, infection, and approach related complications after AOSF for odontoid factures. Elderly patients were more likely to experience non-union and dysphagia.

Lactate down‐regulates matrix systhesis and promotes apoptosis and autophagy in rat nucleus pulposus cells

The intervertebral disk (IVD) is avascular and anaerobic glycolysis has been recognized as the main source of energy. Due to anaerobic glycolysis, there are high levels of lactate production in disk. Previous study shows lactate concentration is elevated in the degenerated IVD. However, it is not clear how lactate causes degeneration of disks. In this study, we found that 2 mM lactate promote proliferation of NP cells, while 6 mM lactate slightly inhibit their proliferation. By detection under transmission electron microscopy, and western bolt for autophagy related protein beclin‐1, LC3 and p62, we demonstrated that 6 mM lactate leads to autophagy induction of NP cells. TUNEL results showed that the apoptosis incidence was increased. High lactate concentration induced the degradation in protein expression and mRNA level of GAG content, type II collagen and slight increase of type I collagen. Based on these observations, we conclude that high lactate concentration is a pathogenic factor for IVD degeneration, and lactate metabolism may be a new therapeutic target for IVD degeneration.

The effects of lactate and acid on articular chondrocytes function: Implications for polymeric cartilage scaffold design.

UNLABELLED Poly (lactic-co-glycolic acid) (PLGA) and poly-l-lactate acid (PLLA) are biodegradable polymers widely utilized as scaffold materials for cartilage tissue engineering. Their acid degradation products have been widely recognized as being detrimental to cell function. However, the biological effects of lactate, rather than lactic acid, on chondrocytes have never been investigated. This is the major focus of this study. The amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100mM and exposure duration of 8h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds. STATEMENT OF SIGNIFICANCE Lactic acid is a widely used substrate for polymers synthesis, PLGA and PLLA in particular. Although physical and biological modifications have been made on these polymers to make them be better cartilage scaffolds, little concern has been given on the biological effect of lactic acid, the main degradation product of these polymers, on chondrocytes. Our finding illustrates the differential biological function of lactate and acid on chondrocytes matrix synthesis. These results can facilitate future design of lactate polymers-based cartilage scaffolds.

Kdm6b regulates cartilage development and homeostasis through anabolic metabolism

Objectives Epigenetic mechanisms have been reported to play key roles in chondrogenesis and osteoarthritis (OA) development. Here, we sought to identify specific histone demethylases that are involved and delineate the underlying mechanisms. Methods We screened the expression of 17 distinct histone demethylases by quantitative real time PCR (qRT-PCR) during chondrogenic differentiation of C3H10T1/2 cells. The role of Kdm6b in cartilage development was then analysed with transgenic Col2a1-CreERT2;Kdm6bf/f . RNA-Seq was applied to explore the underlying changes in chondrocytes upon knockdown of Kdm6b. Experimental OA in mice was induced by destabilisation of the medial meniscus in C57BL/6J (wild type, Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f ) mice, either with intra-articular injection of shKdm6b lentivirus or after tamoxifen treatment. Mouse joints and human cartilage samples were used for histological analysis. Results Kdm6b expression was significantly increased during cartilage development. Col2a1-CreERT2;Kdm6bf/f mice displayed obvious skeletal abnormalities at E16.5 and E18.5 with intraperitoneal injection of tamoxifen at E12.5. RNA-Seq and qRT-PCR analyses revealed decreased expression of chondrocyte anabolic genes in Col2a1-CreERT2;Kdm6bf/f chondrocytes. The histological OA score was significantly higher in mice injected with Kdm6b short hairpin RNA lentivirus. Col2a1-CreERT2;Kdm6bf/f mice exhibited accelerated OA development at 8 and 12 weeks following surgical induction. The number of Kdm6b-positive chondrocytes was lower in both mice and human OA cartilage samples. Conclusions These findings indicate that knockdown of Kdm6b in chondrocytes leads to abnormal cartilage development and accelerated OA progression via inhibition of the anabolic metabolism of chondrocytes. Understanding the epigenetic mechanism of joint cartilage development and homeostasis would be useful for development of new therapeutic modalities for OA.

Incidence of heterotopic ossification after implantation of interspinous process devices

OBJECT This study aimed to investigate the incidence rate of heterotopic ossification (HO) after implantation of Coflex interspinous devices. Possible risk factors associated with HO were evaluated. METHODS The authors retrospectively analyzed patients who had undergone single-level (L4-5) implantation of a Coflex device for the treatment of lumbar spinal stenosis. Patient data recorded were age, sex, height, weight, body mass index, smoking habits, and surgical time. Heterotopic ossification was identified through lumbar anteroposterior and lateral view radiographs. The authors developed a simple classification for defining HO and compared HO-positive and HO-negative cases to identify possible risk factors. RESULTS Among 32 patients with follow-up times of 24-57 months, HO was detectable in 26 (81.2%). Among these 26 patients, HO was in the lateral space of the spinous process but not in the interspinous space in 8, HO was in the interspinous space but did not bridge the adjacent spinous process in 16, and interspinous fusion occurred at the level of the device in 2. Occurrence of HO was not associated with patient age, sex, height, weight, body mass index, smoking habits, or surgical time. CONCLUSIONS A high incidence of HO has been detected after implantation of Coflex devices. Clinicians should be aware of this possible outcome, and more studies should be conducted to clarify the clinical effects of HO.

Therapeutic effects of gefitinib-encapsulated thermosensitive injectable hydrogel in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is one of the most widespread musculoskeletal diseases worldwide, which remains an intractable clinical challenge. The aim of this study is to investigate the therapeutic potential of the small molecule gefitinib (an epidermal growth factor receptor (EGFR) inhibitor) in ameliorating IVD degeneration. Aberrant EGFR activation levels were detected in both human and rat degenerative IVDs, which prompted us to investigate the functional roles of EGFR by utilizing inducible cartilage-specific EGFR-deficient mice. We demonstrated that conditional EGFR deletion in mice increased nucleus pulposus (NP) extracellular matrix (ECM) production and autophagy marker activation while MMP13 expression decreased. These outcomes are comparable to the use of a controlled-release injectable thermosensitive hydrogel of gefitinib to block EGFR activity in a puncture-induced rat model. We also conducted a case series study involving patients with non-small cell lung cancer and IVD degeneration who received gefitinib treatment from 2010 to 2015. Gefitinib-treated patients displayed a relative slower disc degenerating progression, in contrast to control subjects. These findings thus provide evidence that suppression of EGFR by the FDA-approved drug gefitinib can protect IVD degeneration in rats, implying the potential application of gefitinib as a small molecule drug for treating IVD degeneration.