Kaige Ma

Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells

Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.

CsA attenuates compression-induced nucleus pulposus mesenchymal stem cells apoptosis via alleviating mitochondrial dysfunction and oxidative stress

Aims: This study aims to investigate the protective effects and potential mechanisms of cyclosporine A (CsA), which efficiently inhibits mitochondrial permeability transition pore (MPTP) opening, on compression‐induced apoptosis of human nucleus pulposus mesenchymal stem cells (NP‐MSCs). Materials and methods: Human NP‐MSCs were subjected to various periods of 1.0 MPa compression. Cell viability was evaluated using cell counting kit‐8 (CCK‐8) assay. The cellular ultrastructure and ATP level were analyzed via transmission electron microscopy (TEM) and ATP detection kit respectively. The apoptosis ratio was determined using Annexin V/PI dual staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays. The levels of apoptosis‐associated molecules (cleaved caspase‐3, Bax and Bcl‐2) were analyzed by western blot and qRT‐PCR. Additionally, MPTP opening, mitochondrial membrane potential (MMP) and the levels of oxidative stress‐related indicators (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) were monitored. Key findings: Annexin V/PI dual staining and detection of apoptosis‐associated molecules demonstrated that compression significantly up‐regulated apoptosis level of NP‐MSCs in a time‐dependent manner. CsA greatly down‐regulated compression‐mediated NP‐MSC apoptosis and the cell death ratio. Compression also notably exacerbated mitochondrial dysfunction, ATP depletion and oxidative stress in NP‐MSCs, all of which were rescued by CsA. Significance: Our results demonstrated that CsA efficiently inhibited compression‐induced NP‐MSCs apoptosis by alleviating mitochondrial dysfunction and oxidative stress. These findings provide new insights into intervertebral disc (IVD) degeneration (IVDD), and suggest CsA treatment as a potential strategy for delaying or even preventing IVDD.

Encapsulation of mesenchymal stem cells in chitosan/β-glycerophosphate hydrogel for seeding on a novel calcium phosphate cement scaffold

Due to its moldability, biocompatibility, osteoconductivity and resorbability, calcium phosphate cement (CPC) is a highly promising scaffold material for orthopedic applications. However, pH changes and ionic activity during the CPC setting reaction may adversely affect cells seeded directly on CPC. Moreover, a lack of macropores in CPC limits ingrowth of new bone. The objectives of this study were to prepare macroporous CPC scaffolds via porogen leaching, using mannitol crystals as the porogen and to evaluate the in vitro proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) encapsulated in chitosan/β-glycerophosphate (C/GP) hydrogel prior to exposure to the novel CPC scaffold. MSCs were found to be adhered to the surfaces of CPC macropores via scanning electron microscopy. The viability and osteogenic differentiation of MSCs in C/GP hydrogel with or without exposure to CPC constructs containing mannitol crystals indicated that coating with C/GP hydrogel protected the cells during cement mixing and setting. In conclusion, novel, macroporous CPC scaffolds were prepared, and our data indicate that a hydrogel encapsulation-based strategy can be used to protect cells during scaffold formation. Thus, the MSC-laden CPC scaffolds show promise for the delivery of stem cells to promote bone regeneration.

Hydrogen Peroxide Induces Programmed Necrosis in Rat Nucleus Pulposus Cells through the RIP1/ RIP3-PARP-AIF Pathway†

This study aimed to systematically investigate whether programmed necrosis contributes to H2O2‐induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H2O2 for various time periods. The cell viability was measured using a cell counting kit‐8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis‐related molecules receptor‐interacting protein 1 (RIP1), receptor‐interacting protein 3 (RIP3), poly (ADP‐ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real‐time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin‐1 (Nec‐1), the PARP inhibitor diphenyl‐benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H2O2 induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec‐1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3‐mediated programmed necrosis, executed through the PARP‐AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration.

Effect of Compression Loading on Human Nucleus Pulposus-Derived Mesenchymal Stem Cells

Purpose Mechanical loading plays a vital role in the progression of intervertebral disc (IVD) degeneration, but little is known about the effect of compression loading on human nucleus pulposus-derived mesenchymal stem cells (NP-MSCs). Thus, this study is aimed at investigating the effect of compression on the biological behavior of NP-MSCs in vitro. Methods Human NP-MSCs were isolated from patients undergoing lumbar discectomy for IVD degeneration and were identified by immunophenotypes and multilineage differentiation. Then, cells were cultured in the compression apparatus at 1.0 MPa for different times (0 h, 24 h, 36 h, and 48 h). The viability-, differentiation-, and differentiation-related genes (Runx2, APP, and Col2) and colony formation-, migration-, and stem cell-related proteins (Sox2 and Oct4) were evaluated. Results The results showed that the isolated cells fulfilled the criteria of MSC stated by the International Society for Cellular Therapy (ISCT). And our results also indicated that compression loading significantly inhibited cell viability, differentiation, colony formation, and migration. Furthermore, gene expression suggested that compression loading could downregulate the expression of stem cell-related proteins and lead to NP-MSC stemness losses. Conclusions Our results suggested that the biological behavior of NP-MSCs could be inhibited by compression loading and therefore enhanced our understanding on the compression-induced endogenous repair failure of NP-MSCs during IVDD.

Icariin Improves the Viability and Function of Cryopreserved Human Nucleus Pulposus-Derived Mesenchymal Stem Cells

Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.

Mechanisms of endogenous repair failure during intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is frequently associated with Low back pain (LBP), which can severely reduce the quality of human life and cause enormous economic loss. However, there is a lack of long-lasting and effective therapies for IVD degeneration at present. Recently, stem cell based tissue engineering techniques have provided novel and promising treatment for the repair of degenerative IVDs. Numerous studies showed that stem/progenitor cells exist naturally in IVDs and could migrate from their niche to the IVD to maintain the quantity of nucleus pulposus (NP) cells. Unfortunately, these endogenous repair processes cannot prevent IVD degeneration as effectively as expected. Therefore, theoretical basis for regeneration of the NP in situ can be obtained from studying the mechanisms of endogenous repair failure during IVD degeneration. Although there have been few researches to study the mechanism of cell death and migration of stem/progenitor cells in IVD so far, studies demonstrated that the major inducing factors (compression and hypoxia) of IVD degeneration could decrease the number of NP cells by regulating apoptosis, autophagy, and necroptosis, and the particular chemokines and their receptors played a vital role in the migration of mesenchymal stem cells (MSCs). These studies provide a clue for revealing the mechanisms of endogenous repair failure during IVD degeneration. This article reviewed the current research situation and progress of the mechanisms through which IVD stem/progenitor cells failed to repair IVD tissues during IVD degeneration. Such studies provide an innovative research direction for endogenous repair and a new potential treatment strategy for IVD degeneration.

Tauroursodeoxycholic Acid Protects Nucleus Pulposus Cells from Compression-Induced Apoptosis and Necroptosis via Inhibiting Endoplasmic Reticulum Stress

Tauroursodeoxycholic acid (TUDCA) is a kind of hydrophilic bile acid, which could protect cells from death via inhibiting endoplasmic reticulum (ER) stress. However, the role of TUDCA in compression-induced intervertebral disc degeneration (IVDD) has not been elucidated. Here, we used a previously described device to mimic in vivo compression conditions. NP cells treated with DMSO or TUDCA were exposed to compression. Then, cell viability, morphology, and apoptosis were detected. Furthermore, apoptosis-related proteins and necroptosis markers were detected too. To investigate the specific cytoprotective mechanisms of TUDCA in IVDD, we detected the ER morphology by electron microscopy. In addition, the ER stress of nucleus pulposus (NP) cells was quantitatively evaluated by analyzing the level of ER-stress-associated proteins. Our results revealed that TUDCA could protect NP cells from excessive compression-induced death by reducing the apoptosis and necroptosis. In addition, ER stress is involved in pathogenesis of IVDD induced by excessive compression and plays a detrimental role. TUDCA exerts its protective functions by inhibiting ER stress. In conclusion, TUDCA could protect NP cells from compression-induced death, which suggested that treatment by TUDCA may be a potential method to retard IVDD.

Prognostic value of Kindlin-2 expression in patients with solid tumors: a meta-analysis

BackgroundKindlin-2 is one of the Kindlin family members which are evolutionarily conserved focal adhesion proteins with integrin β-binding affinity. Recently, accumulative studies have suggested that Kindlin-2 plays important roles in tumor biology. However, the prognostic significance of Kindlin-2 in patients with solid tumors remains controversial. Therefore, this study aimed to clarify the prognostic value of Kindlin-2 in solid tumors via meta-analysis.MethodsA comprehensive search was performed in PubMed, Embase, Web of Science and EBSCO for all relevant studies reporting the prognostic significance of Kindlin-2 expression in solid cancer patients. The summary hazard ratio (HR) and corresponding 95% confidence interval (CI) were calculated to estimate the association between Kindlin-2 expression with survival of solid cancer patients.ResultsWe included 14 eligible studies containing 1869 patients in our meta-analysis. The pooled results indicated that high Kindlin-2 expression was significantly associated with poor overall survival (OS) (pooled HR 1.66, 95% CI 1.44–1.92, P < 0.0001), disease-free survival (DFS)/recurrence-free survival (RFS)/progression-free survival (PFS) (pooled HR 1.73, 95% CI 1.16–2.57, P = 0.0067). For certain tumor types, high Kindlin-2 expression was significantly correlated with a poor outcome in patients with solid tumors, including pancreatic ductal adenocarcinoma (DFS/RFS/PFS), esophageal squamous cell carcinoma (OS, DFS/RFS/PFS), hepatocellular carcinoma (OS), clear cell renal cell carcinoma (OS), bladder cancer (OS, DFS/RFS/PFS), chondrosarcoma (OS), osteosarcoma (OS), gastric cancer (DFS/RFS/PFS), and glioma (OS).ConclusionsOur meta-analysis demonstrated that high Kindlin-2 expression might indicate poor outcome in patients with solid tumors and could serve as a prognostic biomarker for solid cancer patients.

Association between Fas/FasL gene polymorphism and musculoskeletal degenerative diseases: a meta-analysis

BackgroundIt was reported that Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) gene polymorphism might be related to the risk of musculoskeletal degenerative diseases (MSDD), such as osteoarthritis (OA), intervertebral disc degeneration (IVDD) and rheumatoid arthritis (RA). However, data from different studies was inconsistent. Here we aim to elaborately summarize and explore the association between the Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) and MSDD.MethodsLiteratures were selected from PubMed, Web of Science, Embase, Scopus and Medline in English and VIP, SinoMed, Wanfang and the China National Knowledge Infrastructure (CNKI) in Chinese up to August 21, 2017. All the researches included are case-control studies about human. We calculated the pooled odds ratios (ORs) with 95% confidence intervals (95% CI) to evaluate the strengths of the associations of Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) polymorphisms with MSDD risk.ResultsEleven eligible studies for rs1800682 with 1930 cases and 1720 controls, 6 eligible studies for rs2234767 with 1794 cases and 1909 controls, 3 eligible studies for rs5030772 with 367 cases and 313 controls and 8 eligible studies for rs763110 with 2010 cases and 2105 controls were included in this analysis. The results showed that the G allele of Fas (rs1800682) is associated with an increased risk of IVDD in homozygote and recessive models. The G allele of Fas (rs2234767) is linked to a decreased risk of RA but an enhanced risk of OA in allele and recessive models. In addition, the T allele of FasL (rs763110) is correlated with a reduced risk of IVDD in all of models. However, no relationship was found between FasL (rs5030772) and these three types of MSDD in any models.ConclusionsFas (rs1800682) and FasL (rs763110) polymorphism were associated with the risk of IVDD and Fas (rs2234767) was correlated to the susceptibility of OA and RA. Fas (rs1800682) and Fas (rs2234767) are more likely to be associated with MSDD for Chinese people. FasL (rs763110) is related to the progression of MSDD for both Caucasoid and Chinese race groups. But FasL (rs5030772) might not be associated with any types of MSDD or any race groups statistically.