m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation.

BACKGROUND: Dynamic N6-methyladenosine (m6A) RNA modification generated and erased by N6-methyltransferases and demethylases regulates gene expression, alternative splicing and cell fate. Ocular melanoma, comprising uveal melanoma (UM) and conjunctival melanoma (CM), is the most common primary eye tumor in adults and the 2nd most common melanoma. However, the functional role of m6A modification in ocular melanoma remains unclear. METHODS: m6A assays and survival analysis were used to explore decreased global m6A levels, indicating a late stage of ocular melanoma and a poor prognosis. Multiomic analysis of miCLIP-seq, RNA-seq and Label-free MS data revealed that m6A RNA modification posttranscriptionally promoted HINT2 expression. RNA immunoprecipitation (RIP)-qPCR and dual luciferase assays revealed that HINT2 mRNA specifically interacted with YTHDF1. Furthermore, polysome profiling analysis indicated a greater amount of HINT2 mRNA in the translation pool in ocular melanoma cells with higher m6A methylation. RESULTS: Here, we show that RNA methylation significantly inhibits the progression of UM and CM. Ocular melanoma samples showed decreased m6A levels, indicating a poor prognosis. Changes in global m6A modification were highly associated with tumor progression in vitro and in vivo. Mechanistically, YTHDF1 promoted the translation of methylated HINT2 mRNA, a tumor suppressor in ocular melanoma. CONCLUSIONS: Our work uncovers a critical function for m6A methylation in ocular melanoma and provides additional insight into the understanding of m6A modification.

Growth factors and platelet-rich plasma: promising biological strategies for early intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a complex process with the mechanism not fully elucidated. The current clinical treatments for IDD are mainly focused on providing symptomatic relief without addressing the underlying cause of the IDD. Biological therapeutic strategies to repair and regenerate the degenerated discs are drawing more attention. Growth factor therapy is one of the biological strategies and holds promising prospects. As a promising bioactive substance, platelet-rich plasma (PRP) is considered to be an ideal growth factor "cocktail" for intervertebral disc (IVD) restoration. Results from many in vitro and in vivo studies have confirmed the efficacy of growth factors and PRP in IVD repair and regeneration. It is essential to advance the research on growth factor therapy and associated mechanism for IDD. This article reviews the background of IDD, current concepts in growth factor and PRP-related therapy for IDD. Future research perspectives and clinical directions are also discussed.

A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.

MALAT1 participates in the role of platelet-rich plasma exosomes in promoting wound healing of diabetic foot ulcer.

Exosomes isolated from platelet-rich plasma (PRP-exos) have been recently deemed as an optimized therapeutic strategy in diabetic foot ulcer (DFU) treatment. Herein, we aimed to explore whether MALAT1 participates in DFU wound healing by PRP-exos treatment and the related preliminary mechanism. Fibroblasts were isolated from healthy donors and DFU patients, and the expression of MALAT1, miR-374a-3p and DNMT3A were detected by RT-PCR. The effect of MALAT1 and miR-374a-3p on DFU fibroblast function was verified by gain/loss of function experiment. The targeted binding of MALAT and miRNA was verified by double luciferase reporter gene assay. PRP-exos were isolated from normal human blood and characterized, and then co-cultured with DFU fibroblasts. The MALAT1 expression was donwregulated while the miR-374a-5p expression was upregulated in DFU fibroblasts. Double luciferase reporter gene assay demonstrated the targeted binding of MALAT and miR-374a-5p. Overexpression of MALAT1 or knockdown of miR-374a-5p could increase viability and inhibit apoptosis and pyroptosis of DFU fibroblast. And overexpression of miR-374a-5p reversed the effect of PRR-exos or MALAT1 overexpression on cell viability, apoptosis and pyroptosis. Collectively, MALAT1 mediated signal axis participates in the role of PRP-exos in promoting DFU wound healing, which may help identify optimal targets and effective therapies for DFU treatment.

BMP-2/TGF-ß1 gene insertion into ligament-derived stem cells sheet promotes tendon-bone healing in a mouse.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-ß1 (TGF-ß1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)-derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP-2/TGF-ß1 on the differentiation of LDSC. We developed a BMP-2/TGF-ß1 gene insertion into an LDSC cell sheet that promotes tendon-bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP-2 or TGF-ß1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP-2-, TGF-ß1-, and BMP-2/TGF-ß1-transduced. At 4 and 8 weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-ß1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-ß1-transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon-bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP-2/TGF-ß1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-ß1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon-bone healing after ACLR using autografts wrapped with cell sheets in a mouse model.

Tailored biomedical materials for wound healing.

Wound healing is a long-term, multi-stage biological process that mainly includes haemostatic, inflammatory, proliferative and tissue remodelling phases. Controlling infection and inflammation and promoting tissue regeneration can contribute well to wound healing. Smart biomaterials offer significant advantages in wound healing because of their ability to control wound healing in time and space. Understanding how biomaterials are designed for different stages of wound healing will facilitate future personalized material tailoring for different wounds, making them beneficial for wound therapy. This review summarizes the design approaches of biomaterials in the field of anti-inflammatory, antimicrobial and tissue regeneration, highlights the advanced precise control achieved by biomaterials in different stages of wound healing and outlines the clinical and practical applications of biomaterials in wound healing.

Mitochondrial transfer from bone mesenchymal stem cells protects against tendinopathy both in vitro and in vivo.

BACKGROUND: Although mesenchymal stem cells (MSCs) have been effective in tendinopathy, the mechanisms by which MSCs promote tendon healing have not been fully elucidated. In this study, we tested the hypothesis that MSCs transfer mitochondria to injured tenocytes in vitro and in vivo to protect against Achilles tendinopathy (AT). METHODS: Bone marrow MSCs and H2O2-injured tenocytes were co-cultured, and mitochondrial transfer was visualized by MitoTracker dye staining. Mitochondrial function, including mitochondrial membrane potential, oxygen consumption rate, and adenosine triphosphate content, was quantified in sorted tenocytes. Tenocyte proliferation, apoptosis, oxidative stress, and inflammation were analyzed. Furthermore, a collagenase type I-induced rat AT model was used to detect mitochondrial transfer in tissues and evaluate Achilles tendon healing. RESULTS: MSCs successfully donated healthy mitochondria to in vitro and in vivo damaged tenocytes. Interestingly, mitochondrial transfer was almost completely blocked by co-treatment with cytochalasin B. Transfer of MSC-derived mitochondria decreased apoptosis, promoted proliferation, and restored mitochondrial function in H2O2-induced tenocytes. A decrease in reactive oxygen species and pro-inflammatory cytokine levels (interleukin-6 and -1ß) was observed. In vivo, mitochondrial transfer from MSCs improved the expression of tendon-specific markers (scleraxis, tenascin C, and tenomodulin) and decreased the infiltration of inflammatory cells into the tendon. In addition, the fibers of the tendon tissue were neatly arranged and the structure of the tendon was remodeled. Inhibition of mitochondrial transfer by cytochalasin B abrogated the therapeutic efficacy of MSCs in tenocytes and tendon tissues. CONCLUSIONS: MSCs rescued distressed tenocytes from apoptosis by transferring mitochondria. This provides evidence that mitochondrial transfer is one mechanism by which MSCs exert their therapeutic effects on damaged tenocytes.

Microneedle Patches with O2 Propellant for Deeply and Fast Delivering Photosensitizers: Towards Improved Photodynamic Therapy.

Photodynamic therapy (PDT) is an emerging technique for treating tumors. Especially, topical administration of photosensitizers (PSs) is more favorable for superficial tumor treatments with low systematic phototoxicity. Yet, ineffective migration of PSs to targeted tumor tissues and rapid consumption of O2 during PDT greatly limit their effects. Herein, PS-loaded microneedle (MN) patches with O2 propellant for a deeper and faster transdermal delivery of PS and improved PDT by embedding sodium percarbonate (SPC) into dissolving poly(vinyl pyrrolidone) MNs are presented. It is shown that SPC in the MNs can react with surrounding fluid to generate gaseous oxygen bubbles, forming vigorous fluid flows and thus greatly enhancing PS of chlorin e6 (Ce6) penetration in both hydrogel models and skin tissues. Reactive oxygen species (ROS) in hypoxic breast cancer cells (4T1 cells) are greatly increased by rapid penetration of PS and relief of hypoxia in vitro, and Ce6-loaded SPC MNs show an excellent cell-killing effect. Moreover, lower tumor growth rate and tumor mass after a 20-d treatment in tumor-bearing mice model verify the improved PDT in gaseous oxygen-droved delivery of PS. This study demonstrates a facile yet effective route of MN delivery of PSs for improved PDT in hypoxic tumor treatment.

The m6A reading protein YTHDF3 potentiates tumorigenicity of cancer stem-like cells in ocular melanoma through facilitating CTNNB1 translation.

N6-methyladenosine (m6A) is the most universal internal RNA modification on messenger RNAs and regulates the fate and functions of m6A-modified transcripts through m6A-specific binding proteins. Nevertheless, the functional role and potential mechanism of the m6A reading proteins in ocular melanoma tumorigenicity, especially cancer stem-like cell (CSC) properties, remain to be elucidated. Herein, we demonstrated that the m6A reading protein YTHDF3 promotes the translation of the target transcript CTNNB1, contributing to ocular melanoma propagation and migration through m6A methylation. YTHDF3 is highly expressed in ocular melanoma stem-like cells and abundantly enriched in ocular melanoma tissues, which is related to poor clinical prognosis. Moreover, YTHDF3 is required for the maintenance of CSC properties and tumor initiation capacity in ocular melanoma both in vitro and in vivo. Ocular melanoma cells with targeted YTHDF3 knockdown exhibited inhibitory tumor proliferation and migration abilities. Transcriptome-wide mapping of m6A peaks and YTHDF3 binding peaks on mRNAs revealed a key target gene candidate, CTNNB1. Mechanistically, YTHDF3 enhances CTNNB1 translation through recognizing and binding the m6A peaks on CTNNB1 mRNA.

lncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived from Adipose-Derived Stem Cells for the Treatment of Osteoporosis.

BACKGROUND: Osteoporosis is a worldwide medical and socioeconomic burden characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study was aimed at investigating the role of ADSCs-Exos and a novel long noncoding RNA KCNQ1OT1 played in osteoporosis as well as the underlying mechanism. METHODS: Primary osteoblasts were treated with different doses of tumor necrosis factor-α (TNF-α) (0, 1, 2.5, 5, and 10 ng/ml) and then cocultured with ADSCs-Exos or exosome-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3, and Bax was determined by Western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. RESULTS: TNF-α dose-dependently increased miR-141-5p expression, inhibited viability, and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or cocultured with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of KCNQ1OT1 by luciferase reporter assay. CONCLUSIONS: ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment.

miR­654­5p inhibits autophagy by targeting ATG7 via mTOR signaling in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a common chronic disease characterized by the loss of extracellular matrix (ECM) in the nucleus pulposus (NP). Accumulating evidence has revealed that abnormal expression of microRNAs (miRs) is closely associated with IDD development. The present study aimed to investigate the precise role and possible mechanism underlying the effects of miR­654­5p in the pathogenesis of IDD. NP cells were isolated from patients with IDD. Monodansylcadaverine staining was conducted to reveal cell autophagy, while western blotting was performed to detect the expression of ECM­related proteins in NP cells. Luciferase reporter and RNA immunoprecipitation assays were conducted to identify the binding between RNAs. The results demonstrated that miR­654­5p was significantly upregulated in degenerated NP tissues from patients with IDD and high miR­654­5p expression was positively associated with disc degeneration grade. Functional assays suggested that miR­654­5p facilitated ECM degradation by increasing the expression levels of MMP­3, MMP­9 and MMP­13, as well as decreasing collagen I, collagen II, SOX9 and aggrecan expression by inhibiting autophagy. Furthermore, autophagy­related gene 7 (ATG7) was verified as a direct downstream target gene of miR­654­5p. miR­654­5p could bind to the 3' untranslated region of ATG7 to inhibit its mRNA expression and further reduce its translation. Notably, ATG7 knockdown abrogated the effects of the miR­654­5p inhibitor on ECM degradation and autophagy regulation. Furthermore, miR­654­5p inhibited autophagy in NP cells by increasing the protein expression levels of phosphorylated (p)­PI3K, p­AKT and p­mTOR in an ATG7­dependent manner. In conclusion, the results of the present study revealed that miR­654­5p may enhance ECM degradation via inhibition of autophagy by targeting ATG7 to activate the PI3K/AKT/mTOR signaling pathway. These findings may provide novel insights into the treatment of IDD.

Integrative network analysis of N6 methylation-related genes reveal potential therapeutic targets for spinal cord injury.

The diagnosis of the severity of spinal cord injury (SCI) and the revelation of potential therapeutic targets are crucial for urgent clinical care and improved patient outcomes. Here, we analyzed the overall gene expression data in peripheral blood leukocytes during the acute injury phase collected from Gene Expression Omnibus (GEO) and identified six m6A regulators specifically expressed in SCI compared to normal samples. LncRNA-mRNA network analysis identified AKT2/3 and PIK3R1 related to m6A methylation as potential therapeutic targets for SCI and constructed a classifier to identify patients of SCI to assist clinical diagnosis. Moreover, FTO (eraser) and RBMX (reader) were found to be significantly down-regulated in SCI and the functional gene co-expressed with them was found to be involved in the signal transduction of multiple pathways related to nerve injury. Through the construction of the drug-target gene network, eight key genes were identified as drug targets and it was emphasized that fostamatinib can be used as a potential drug for the treatment of SCI. Taken together, our study characterized the pathogenesis and identified a potential therapeutic target of SCI providing theoretical support for the development of precision medicine.

[Intra-articular injection of compound betamethasone and hyaluronic acid for the treatment of moderate-severe knee osteoarthritis:a randomized controlled trial].

OBJECTIVE: To compare clinical effects of compound betamethasone and compound betamethasone with hyaluronic acid in treating moderate-severe knee osteoarthritis (KOA). METHODS: A prospective randomized controlled study was conducted in 116 patients with unilateral moderate-severe KOA patients from February 2017 to November 2017 and divided into observation group and control group, 58 patients in each group. In observation group, there were 15 males and 43 females aged from 45 to 80 years old with an average of (66.45±6.31) years old;according to Kellgren-Lawrence(K-L) classification, 42 patients were type Ⅲ and 16 patients were type Ⅳ;the courses of disease ranged from 4 to 8 years with an average of (5.25±2.21) years;the patients were treated by injecting 1 ml compound betamethasone into knee joint. In control group, there were 13 males and 45 females aged from 45 to 80 years old with an average of (64.89±6.41) years old;according to K-L classification, 43 patients were type Ⅲ and 15 patients were type Ⅳ;the courses of disease ranged from 4 to 10 years with an average of (5.41±2.35) years;the patients were treated by knee joint injection of 4 ml hyaluronic acid and 1 ml compound betamethasone. Visual analog scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC) were used to evaluate clinical effects before treatment and 1 week, 1, month, 3 and 6 months after treatment. RESULTS: Totally 55 patients in observation group were followed up for 6 months, and 3 patients were quit at 3 months after treatment for poor efficacy. Totally 56 patients in control group were followed up for 6 months, and 2 patients were withdrew from the follow-up on the first and third month respectively for poor efficacy. There were no statistical difference in VAS and WOMAC between two groups before treatment and different time points after treatment (P>0.05), and no significant difference in VAS and WOMAC before treatment and 6 months after treatment between two groups(P>0.05). CONCLUSION: For patients with moderate-severe KOA, there is no significant difference in therapeutic effect between compound betamethasone injection and compound betamethasone combined with hyaluronic acid injection, and long-term effect of two methods is not good.

Distinctive roles of tumor necrosis factor receptor type 1 and type 2 in a mouse disc degeneration model.

BACKGROUND: Elevated tumor necrosis factor alpha (TNF-α) expression is correlated with the progression of intervertebral disc degeneration (IVDD). Progranulin binding to tumor necrosis factor receptor (TNFR) and its derivative Atsttrin are effective for treating inflammatory arthritis. We hypothesize that Atsttrin has a protective effect in IVDD through different roles of TNFR receptor type 1 (TNFR1) and TNFR receptor type 2 (TNFR2) in degenerated discs. METHODS: IVDD models were established in TNFR1-/-, TNFR2-/- mice and their control littermates. Nucleus Pulpous (NP) samples from human patients and IVDD murine models were evaluated by X-ray, micro-MRI, µCT, histological staining and immunofluorescence staining. NP cells isolated from wild-type (WT), TNFR1-/- and TNFR2-/- mice were treated with TNF-α or Atsttrin and then assayed by Western blotting, qRT-PCR, and ELISA. RESULTS: TNFR1 and TNFR2 expression was significantly elevated in the disc tissues of both human patients and IVDD murine models. TNFR1 knockout contributed to reduced disc degeneration. In contrast, TNFR2 knockout was associated with enhanced IVDD severity, including degraded cellular composition, increased cell apoptosis and elevated vertebral destruction. Atsttrin protected against IVDD in WT and TNFR1-/- mouse models but had no effect in TNFR2-/- IVDD models. Additionally, in vitro NP cell-based assays demonstrated that TNF-α-stimulated catabolism and Atsttrin-activated anabolism depended on TNFR1 and TNFR2, respectively. CONCLUSION: TNFR1 is associated with the degenerative progression of IVDD, while TNFR2 contributes to the protective effect on the discs. Atsttrin protects against IVDD at least partially by inhibiting the TNFα/TNFR1 inflammatory/catabolic pathway and activating the TNFR2 protective/anabolic pathway. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study demonstrates that TNFR1 and TNFR2 have disparate roles in disc degeneration and hlights the potential use of Atsttrin as a therapeutic agent against IVDD in mice.

[Study on pain control of continuous adductor block analgesia after primary knee replacement].

OBJECTIVE: To investigate the effect of continuous adductor block on pain control after bilateral knee joint Ⅰ stage replacement. METHODS: A retrospective analysis was made of the data of 24 patients with bilateral knee joint I stage replacement who were treated in our hospital from January 2018 to January 2019, and who underwent continuous adductor block analgesia. There were 6 males and 18 females, aged 60 to 72 (65.05±5.82) years old. The patients underwent continuous block of adductor canal with patient-controlled analgesia system. At 4, 6, 12, 24, 36 and 48 hours after operation, visual analogue score(VAS) of resting state and passive motion state was performed;the knee joint activity was followed up for 1 week, 1, 3 and 6 months after operation;the knee joint function was scored at 6 months after operation, using the knee joint scoring standard of American Special Surgery Hospital(HSS);adverse reactions and complications were recorded. RESULTS: The VAS scores under resting state and passive motion state at each time point were less than 3 points in patients with continuous adductor block. The patients had better postoperative exercise of knee joint activity. The score of HSS was excellent in 20 cases, good in 2 cases, fair in 1 case and poor in 1 case. There were only 4 cases of nausea and vomiting, none of them had serious adverse reactions and complications such as bradycardia and deep vein thrombosis. CONCLUSION: Continuous adductor block has a significant effect on pain control and less adverse reactions after bilateral knee jointⅠ -stage replacement.

The Differential Effects of Leukocyte-Containing and Pure Platelet-Rich Plasma on Nucleus Pulposus-Derived Mesenchymal Stem Cells: Implications for the Clinical Treatment of Intervertebral Disc Degeneration.

BACKGROUND: Platelet-rich plasma (PRP) is a promising strategy for intervertebral disc degeneration. However, the potential harmful effects of leukocytes in PRP on nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have seldom been studied. This study aimed at comparatively evaluating effects of pure platelet-rich plasma (P-PRP) and leukocyte-containing platelet-rich plasma (L-PRP) on rabbit NPMSCs in vitro. METHODS: NPMSCs isolated from rabbit NP tissues were treated with L-PRP or P-PRP in vitro, and then cell proliferation and expression of stem cell markers, proinflammatory cytokines (TNF-α, IL-1ß), production of ECM (extracellular matrix-related protein), and NF-κB p65 protein were validated by CCK-8 assay, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and western blot respectively. RESULTS: NPMSCs differentiate into nucleus pulposus-like cells after treatment of PRPs (P-PRP and L-PRP), and NPMSCs exhibited maximum proliferation at a 10% PRP dose. L-PRP had observably higher concentration of leukocytes, TNF-α, and IL-1ß than P-PRP. Furthermore, compared to P-PRP, L-PRP induced the differentiated NPMSCs to upregulate the expression of TNF-α and IL-1ß, enhanced activation of the NF-κB pathway, increased the expression of MMP-1 and MMP-13, and produced less ECM in differentiated NPMSCs. CONCLUSIONS: Both P-PRP and L-PRP can induce the proliferation and NP-differentiation of NPMSCs. Compared to L-PRP, P-PRP can avoid the activation of the NF-κB pathway, thus reducing the inflammatory and catabolic responses.

Is exclusion of leukocytes from platelet-rich plasma (PRP) a better choice for early intervertebral disc regeneration?

BACKGROUND: Platelet-rich plasma (PRP) is becoming a promising strategy to treat early intervertebral disc degeneration (IDD) in clinics. Pure PRP without leukocytes (P-PRP) may decrease the catabolic and inflammatory changes in the early degenerated intervertebral discs. The aim of this study was to investigate the effects of P-PRP on nucleus pulposus-derived stem cells (NPSCs) isolated from early degenerated intervertebral discs in vitro. METHODS: NPSCs isolated from early degenerated discs of rabbits were treated with P-PRP or leukocyte-platelet-rich PRP (L-PRP) in vitro, followed by measuring cell proliferation, stem cell marker expression, inflammatory gene expression, and anabolic and catabolic protein expression by immunostaining, quantitative real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. RESULTS: Cell proliferation was induced by P-PRP in a dose-dependent manner with maximum proliferation at 10% P-PRP dose. P-PRP induced differentiation of NPSCs into active nucleus pulposus cells. P-PRP mainly increased the expression of anabolic genes and relative proteins, aggrecan (AGC), collagen types II (Col II), while L-PRP predominantly increased the expression of catabolic and inflammatory genes, matrix metalloproteinase-1 (MMP-1), MMP-13, interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor alpha (TNF-α), and protein production of IL-1ß and TNF-α. CONCLUSIONS: Leukocytes in PRP activate inflammatory and catabolic effects on NPSCs from early degenerated intervertebral discs. Hence, P-PRP may be a more suitable therapeutic strategy for early IDD.

Intra-articular, single-shot co-injection of hyaluronic acid and corticosteroids in knee osteoarthritis: A randomized controlled trial.

The aim of the present study was to investigate whether the co-injection of hyaluronic acid (HA) and corticosteroids (CS) was superior to HA alone in the treatment of knee OA. A total of 120 participants with symptomatic knee OA were recruited and formed the intention-to-treat population for a 6-month follow-up. In the HA group, patients received a single-shot injection of 4 ml HA. In the HA&CS group, patients received a co-injection of 3 ml compound betamethasone solution and 4 ml HA. Visual analog scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC) and knee flexion motion were assessed as primary outcomes. Patients in the HA&CS group exhibited better pain relief and knee function at the time points of week 1, month 1 and month 3 (P<0.05). For the last follow-up at month 6, the values did not differ significantly between these two groups. Patients in both groups exhibited improvement in pain, knee function, and range of motion following injection. For the final follow-up at month 6, the mean VAS score, WOMAC score and knee flexion motion were still superior to that prior to treatment, but the values did not differ significantly. The co-injection of HA and CS provided a rapid improvement in pain relief, knee function, and range of motion, but did not differ significantly from that of HA alone in the long term effect.

[Establishment of obliterative bronchiolitis in allo-trachea transplant model of rat and detection of its pathogenesis preliminarily].

OBJECTIVE: To establish an animal model of obliterative bronchiolitis (OB) after lung transplantation and investigate the pathogenesis preliminarily. METHODS: Tracheal segments (5 cartilaginous rings each) were transplanted from SD rats to SD rats (Group I) or to Wistar rats (Group II and III). Grafts were implanted into an abdominal cavity and wrapped in the omentum. Animals in Group I and II did not receive CsA, animals in Group III received CsA daily by gastro-tube at 10 mg.kg(-1).d(-1) from beginning to end. Grafts were harvested on day 3, 14, 28 after transplantation as representative time points for 3 phases of injury in the evolution of allograft airway obliteration, then examined histological changes and gene expression of T-helper 1-and T-helper 2-type cytokines [Th1: interleukin-2 (IL-2), interferon-gamma (IFN-gamma); Th2: interleukin-4 (IL-4), interleukin-10 (IL-10)] in grafts. At the same time, effects of CsA were observed on the above-mentioned indices. RESULTS: There was no significant difference in histological changes on day 3 after transplantation among 3 groups (P > 0.05). Tracheas in Group I approached to normal morphology on day 14 after transplantation. Airway epithelium of Group II and III almost lost completely on day 14 after transplantation. There was no significant difference between Group II and Group III (P > 0.05), but there were significant differences between Group I and Group II or Group III. The cross-sectional area of the tracheal lumen was narrowed by approximately (5.0 +/- 1.2)%, (28.5 +/- 5.0)% and (19.4 +/- 2.9)% respectively on day 14 after transplantation in Group I, II and III, there were significant differences among 3 groups. On day 14 after transplantation, tracheas in Group I revealed few lymphocytic infiltration, but it showed dense lymphocytic infiltration in Group II. Tracheas in Group III have much more lymphocyte infiltration than that in Group I, but much less than that in Group II. There were significant differences among 3 groups, too (P < 0.01). The tracheal lumen revealed almost total luminal obstruction (94.8 +/- 3.6)% on day 28 after transplantation in Group II. The cross-sectional area of the tracheal lumen was narrowed by approximately (3.7 +/- 0.8)% and (36.6 +/- 7.6)% respectively in Group I and III on day 28. There were significant differences among 3 groups (P < 0.01). Compared with that on day 14, lymphocytic infiltration had decreased gradually on day 28 in Group II and III. There were significant differences among 3 groups all the same (P < 0.01). In Group II, expression of IL-2, IFN-gamma, IL-4, and IL-10 were much higher than that in Group I. Expression of Th1 cytokines was increased to a greater extent than that of Th2 cytokines in Group II compared with Group I. Allografts in Group III expressed significantly less IL-2 gene transcripts than that in Group II over all the points. There was no significant difference between Group II and III in IFN-gamma, IL-4, and IL-10 gene expression. CONCLUSIONS: Compared with isografts, allografts have more obvious changes, such as epithelial damage, fibroproliferation and lymphocytic infiltration. Th1 and Th2 lymphocyte subtypes contribute to the development of obliterative bronchiolitis in heterotopic trachea transplant model of rat, and changes of their cytokines gene expression may be involved in the pathogenesis. CsA could reduce the development of fibroproliferation and lymphocyte infiltration markedly, but it could not protect airway epithelium. CsA inhibits IL-2 gene transcripts, so it can reduce development of the pathologic lesion of obliterative bronchiolitis to a certain degree.