Platelet-rich plasma (PRP) is a promising strategy for intervertebral disc degeneration (IDD). However, the short half-life of growth factors released from PRP cannot continuously stimulate the degenerated discs. Thus, the present study hypothesized that the combined use of PRP and bone marrow-derived mesenchymal stem cells (BMSCs) may repair the early degenerated discs in the long term for their synergistic reparative effect. In the present study, following the induction of early IDD by annular puncture in rabbits, PRP was prepared and mixed with BMSCs (PRP-BMSC group) for injection into the early degenerated discs. As controls, phosphate-buffered saline (PBS; PBS group) and PRP (PRP group) were similarly injected. Rabbits without any intervention served as a control group. At 8 weeks following treatment, histological changes of the injected discs were assessed. Magnetic resonance imaging (MRI) was used to detect the T2-weighted signal intensity of the targeted discs at weeks 1, 2 and 8 following treatment. Annular puncture resulted in disc narrowing and decreased T2-weighted signal intensity. At weeks 1 and 3, MRI examinations showed regenerative changes in the PRP-BMSC group and PRP group, whereas the PBS group exhibited a continuous degenerative process of the discs. At 8 weeks post-injection, the PRP-BMSCs induced a statistically significant restoration of discs, as shown by MRI (PRP-BMSCs, vs.PRP and PBS; P<0.05), which was also confirmed by histological evaluations. Thus, compared with PRP, the administration of PRP-containing BMSCs resulted in a superior regenerative effect on the early degenerated discs, which may be a promising therapeutic strategy for the restoration of early degenerated discs.

Abstract. This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro.

Treatment for multiple myeloma (MM) with a combined strategy of bone and tumor targeting remains a crucial technical challenge due to the incorporation of various functional components into one single system. Here, we developed dioleoyl phosphatidic acid (DOPA)-based paclitaxel (PTX)-loaded liposomes with modifications of alendronate and transferrin (Ald-/Tf-modified PTX-L), which were capable of bone affinity mediated by phosphate groups in DOPA and alendronate, and tumor targeting offered by transferrin. Ald-/Tf-modified PTX-L had clear and well-defined spherical shape with an intermediated size of 118.8 ± 4.8 nm, a highly negative surface charge of À46.9 ± 6.8 mV and a drug entrapment efficiency (DEE) of approximately 80%. When the pH was changed from pH 7.4 to pH 6.5, the accumulative release of PTX from Ald-/Tf-modified PTX-L significantly increased from 26.7 ± 3.7% to 41.7 ± 4.9%. Importantly, liposomes based on DOPA displayed an obviously stronger affinity with hydroxyapatite (HAp) than 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-based liposomes. Compared to PTX-L, Ald-/Tf-modified PTX-L exhibited obvious improvement of cytotoxicity (IC 50 ¼ 1.25 ± 0.09 mg/mL), significant enhancement on PTX intracellular accumulation (16.58 ± 0.62 mg/mg) and notable promotion to apoptosis induction (45.21 ± 3.10%) toward myeloma (MM1s) cells. In this study of antitumor efficacy, Ald-/Tf-modified PTX-L with bone-specific targeting showed a significant effect on extending the median survival time (48 days) and terminal survival time (458 days) against the MM1S-injected nude mice among all formulations. The results suggested that Ald-/Tf-modified PTX-L had potential as an efficient anticancer drug delivery system for MM therapy.

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.

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 miR-654-5p 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 ECM-related proteins in NP cells. Luciferase reporter and RNA immunoprecipitation assays were conducted to identify the binding between RNAs. The results demonstrated that miR-654-5p was significantly upregulated in degenerated NP tissues from patients with IDD and high miR-654-5p expression was positively associated with disc degeneration grade. Functional assays suggested that miR-654-5p facilitated ECM degradation by increasing the expression levels of MMP-3, MMP-9 and MMP-13, as well as decreasing collagen I, collagen II, SOX9 and aggrecan expression by inhibiting autophagy. Furthermore, autophagy-related gene 7 (ATG7) was verified as a direct downstream target gene of miR-654-5p. miR-654-5p 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 miR-654-5p inhibitor on ECM degradation and autophagy regulation. Furthermore, miR-654-5p inhibited autophagy in NP cells by increasing the protein expression levels of phosphorylated (p)-PI3K, p-AKT and p-mTOR in an ATG7-dependent manner. In conclusion, the results of the present study revealed that miR-654-5p 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.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) 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-β1 on the differentiation of LDSC. We developed a BMP-2/TGF-β1 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-β1, 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-β1-, and BMP-2/TGF-β1-transduced. At 4 and 8 weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-β1-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-β1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-β1 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. K E Y W O R D Santerior cruciate ligament (ACL)-derived stem cells, anterior cruciate ligament reconstruction, bone morphogenetic protein-2, differentiation, tendon-bone healing, transforming growth factor-β1Abbreviations: ACL, anterior cruciate ligament; ACLR, ACL reconstruction; Ad, adenoviral vectors; BMP-2, bone morphogenetic protein-2; BMSC, bone marrow mesenchymal stem cell; BV/TV, bone volume/total volume fraction; ECM, extracellular matrix; FTGT, femur-tendon graft-tibia; GFP, green fluorescent protein; H&E, hematoxylin and eosin; IHC, immunohistochemistry; LDSC, ACL-derived stem cell; TGF-β1, transforming growth factor-β1.

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 -1β) 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.

<abstract> <p>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.</p> </abstract>