Xuedong Bai

Blocking the Function of Inflammatory Cytokines and Mediators by Using IL-10 and TGF-β: A Potential Biological Immunotherapy for Intervertebral Disc Degeneration in a Beagle Model

The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play key roles in the pathology of IDD. Therefore, the development of treatments that inhibit the expression and/or effects of TNF-α and IL-1β in IDD patients should be a promising therapeutic approach to consider. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD. IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by fluorescence activated cell sorting (FACS) analysis and enzyme-linked immunosorbent assay (ELISA); moreover, ELISA and real-time PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. These data suggest that IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.

BMP7-Based Functionalized Self-Assembling Peptides Protect Nucleus Pulposus-Derived Stem Cells From Apoptosis In Vitro.

Tissue engineering has shown great success in the treatment of intervertebral disk degeneration (IVDD) in the past decade. However, the adverse and harsh microenvironment associated in the intervertebral disks remains a great obstacle for the survival of transplanted cells. Although increasing numbers of new materials have been created or modified to overcome this hurdle, a new effective strategy of biological therapy is still required. In this study, bone morphogenic protein 7 (BMP7)-based functionalized self-assembling peptides were developed by conjugating a bioactive motif from BMP-7 (RKPS) onto the C-terminal of the peptide RADARADARADARADA (RADA16-I) at a ratio of 1:1 to form a new RADARKPS peptide. Human nucleus pulposus-derived stem cells (NPDCs) were cultured in the presence of RADA-RKPS or RADA16-I in an apoptosis-promoting environment that was induced by tumor necrosis factor-alpha, and cells were cultured with RADA16-I in normal medium that served as the control group. After 48 h of apoptosis induction, the viability, proliferation, apoptosis rate, and expression of apoptosis-related genes of NPDCs in the different groups were evaluated, and the differentiation of NPDCs toward nucleus pulposus-like cells was tested. The results showed that the RADA-RKPS peptide could significantly protect the survival and proliferation of NPDCs. In addition, the application of RADA-RKPS decreased the rate of cell apoptosis, as detected by TUNEL-positive staining. Furthermore, our in vitro study confirmed the apoptosis-protecting effects of RADA-RKPS peptides, which significantly reduced the BAX/BCL-2 ratio of NPDCs and upregulated the gene expression of collagen II a1, aggrecan, and Sox-9 after 48 h of apoptosis induction. Collectively, these lines of evidence suggest that RADA-RKPS peptides confer a protective effect to NPDCs in an apoptosis environment, suggesting their potential application in the development of new biological treatment strategies for IVDD.

Functional self-assembled peptide scaffold inhibits tumor necrosis factor-alpha-induced inflammation and apoptosis in nucleus pulposus cells by suppressing nuclear factor-κB signaling: RADA-KPSS INHIBITS APOPTOSIS IN TNF-α INDUCED NPCs

Although nucleus pulposus (NP) tissue engineering has achieved tremendous success, researches still face the huge obstacles in maintaining cell survival and function. A novel functional self-assembled peptide RADA-KPSS was constructed by conjugating BMP-7 short active fragment (KPSS) to the C-terminus of RADA16-I that displays anti-inflammatory and anti-apoptosis effects. However, whether this functional self-assembled RADA-KPSS peptide can alleviate inflammation and NPC apoptosis induced by tumor necrosis factor-alpha (TNF-α) has not been studied. Therefore, we cultured NPCs treated with TNF-α for 48 h with the RADA-KPSS peptide, and compared the results to those with RADA16-I peptide. The cell apoptosis rate, inflammatory mediator secretion, expression of matrix-degrading enzymes, and extracellular matrix (ECM) protein levels were evaluated. The expression of nuclear factor-κB-p65 (NF-κB-p65) protein was also tested. TNF-α-treated NPCs cultured with the RADA16-I peptide showed up-regulated gene expression for matrix-degrading enzymes, such as matrix metalloproteinases-3 (MMP-3), MMP-9, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4), and down-regulated gene expression for ECM proteins such as aggrecan, collagen II, and Sox-9. The RADA-KPSS peptide could attenuate the expression of MMP-3, MMP-9, and ADAMTS-4, promote accumulation of ECM proteins, and increase secretion of glycosaminoglycan as compared with the RADA16-I peptide. Moreover, the TNF-α-damaged NPCs was further demonstrated to inhibit NF-κB-p65, IL-1, IL-6, and prostaglandin E-2 proteins and decrease cell apoptosis in RADA-KPSS peptide. In conclusion, the functional self-assembled RADA-KPSS peptides have anti-inflammatory and anti-apoptotic effects by promoting anabolic processes and inhibiting catabolic processes in intervertebral disk degeneration. These peptides may be feasible for clinical applications in NP tissue engineering.

Biological evaluation of human degenerated nucleus pulposus cells in functionalized self-assembling peptide nanofiber hydrogel scaffold.

Nucleus pulposus (NP) tissue engineering has been proposed as a novel biological treatment for early-stage intervertebral disc degeneration. In this study, a novel functional self-assembling peptide PKP was first designed by linking the short functional motif of bone morphogenetic protein-7 (BMP7) to the C-terminal of RADA16-I, and another new functional self-assembling peptide was obtained by mixing RKP with RADA16-I. Then, the biocompatibilities and bioactivities of RKP and RAD-RKP for human degenerated nucleus pulposus cells (hNPCs) were studied in vitro. Atomic force microscopy and scanning electron microscopy (SEM) confirmed that both RKP and RAD-RKP could self-assemble into three-dimensional (3D) nanofiber hydrogel scaffolds in a culture medium at 37°C. After the hNPCs were cultured in 3D scaffolds, both RKP and RAD-RKP exhibited reliable attachment and extremely low cytotoxicities (<14%), which were verified by SEM and cytotoxity assays, respectively. Our results also showed that the functional-based scaffolds could increase the proliferation and migration of hNPCs after 7 days compared with culture plates and pure RADA16-I. Quantitative real-time polymerase chain reaction demonstrated that the expressions of collagen II α1, Sox-9, and aggrecan were upregulated, while collagen I α1 was downregulated by functional-based scaffolds after 28 days. Furthermore, we also confirmed that RAD-RKP exhibited a higher hNPC proliferation, migration, and expression of Sox-9 and aggrecan compared with pure RKP. Therefore, the results of this study indicated that the BMP7 short motif-designed functional self-assembling peptide nanofiber hydrogels could be used as excellent scaffolds in NP tissue engineering, and RAD-RKP might have further potential application in human mild degenerated NP tissue regeneration.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) could be an effective adjuvant therapy for orthopedic implant-related infections (OIRI)

Bone infections associated with foreign bodies are very difficult to treat satisfactorily. The tissue damage and circulation destruction caused by surgery make the organism susceptible to infection. Meanwhile, the presence of foreign body lowers the threshold of bacterial infection and often generates local immunosuppression. In such cases, antibiotic therapies are usually not efficacious and removing the implant is the only solution for completely eradicating the problem. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been in clinical use for about two decades, most commonly adopted for treating of various types of neutropenia. Many studies have also demonstrated that rhG-CSF play important roles in nonneutropenic infections, angiogenic response and tissue repairing. In addition to increasing the number of neutrophils, rhG-CSF helps to enhance the neutrophil microbicidal functions. More importantly, orthopedic implant-related infections (OIRI) are especially good indications for the administration of rhG-CSF in therapeutic regimens. rhG-CSF may also play important roles in the process of postsurgery revascularization and tissue repairing, which would help a lot in treating infections and preventing implant failure. So far, there has been no report of such cases in this field. Therefore, a hypothesis is put forward in the paper that application of rhG-CSF may well be an effective adjuvant therapy for OIRI.

Noninvasive cumulative axial load may induce intervertebral disc degeneration‑A potential rabbit model

Intervertebral disc degeneration (IDD) is considered to be the main cause of many spinal disorders; however, its underlying pathophysiology is not clearly understood. Recent studies indicate that excessive mechanical loading may serve a major role in the initiation of IDD. The aim of the present study was to explore the effect of noninvasive cumulative axial loading on the intervertebral discs of the lumbar spine using a novel rabbit model. Rabbits in the experimental group were placed into individual tubes specifically designed to force maintenance of an upright posture and were loaded with a heavy collar to increase the intradiscal pressure of their lumbar spine. Radiograph imaging and magnetic resonance imaging (MRI) was performed every 4 weeks to provide evidence of disc degeneration. At the end of the experiment, the animals were sacrificed and disc specimens were harvested for quantitative polymerase chain reaction and histological analysis. MRI results revealed significant and progressive reductions in the signal intensities of lumbar discs in the experimental group compared with the control group throughout the 14-week study period. The expression level of type I collagen was significantly increased and the expression levels of type II collagen and aggrecan were significantly decreased in the experimental group compared with the control group (P<0.05). Histological examination revealed marked structural changes in the experimental group, including fibrocartilage-like tissue ingrowth and accelerated fibrotic changes of the nucleus pulposus. The results of the present study indicate that noninvasive cumulative axial load is able to induce accelerated degenerative changes in rabbit lumbar discs, which may provide useful information for the establishment of a novel animal model of IDD for the research of IDD in humans.

Cell properties of nucleus pulposus progenitor cells declines with intervertebral disc degeneration

Endogenous repair of nucleus pulposus progenitor cells (NPPCs) has exhibited encouraging regenerative potential for treating intervertebral disc degeneration (IDD). However, few studies have explored the properties of NPPCs during the progression of IDD. Hence, additional studies are needed to characterize NPPCs from human degenerated intervertebral disc (IVDs) at different Pfirrmann grades. In this study, human NPPCs were isolated and identified from human IVDs with different Pfirrmann grades. Then, cell biological characteristics, including proliferation, colony formation, cell senescence, migration capacity and chondrogenic ability, were compared. NPPCs were successfully harvested from grade I to IV IVDs, but not from grade V IVD due to the marked loss of nucleus pulposus tissue at grade V, leading to poor cell cultures. All four grades of NPPCs were identified as mesenchymal stem cells (MSCs) based on the criteria of the International Society for Cellular Therapy (ISCT) and shared similar cell morphological characteristics. In addition decreasing trends in proliferation, colony-formation capacity, migration, and chondrogenic ability and increasing levels of cell senescence; were detected in cells from grade I to IV IVDs, with significant changes between NPPCs of grade II and III. In summary, compared with NPPCs from normal IVDs (grade I), cells from degenerated IVDs (grades II to IV) exhibited gradually decreased cell properties and increased cell senescence. Grade II NPPCs displayed the optimal regeneration potential, suggesting that these NPPCs are an ideal candidate for endogenous