Cationic mesoporous silica nanoparticles alleviate osteoarthritis by targeting multiple inflammatory mediators.

Osteoarthritis (OA) is a common and complex inflammatory disorder that is frequently compounded by cartilage degradation, synovial inflammation, and osteophyte formation. Damaged chondrocytes release multiple danger mediators that exacerbate synovial inflammation and accelerate the progression to OA. Conventional treatments targeting only a single mediator of OA have failed to achieve a strong therapeutic effect. Addressing the crucial role of multiple danger mediators in OA progression, we prepared polyethylenimine (PEI)-functionalized diselenide-bridged mesoporous silica nanoparticles (MSN-PEI) with cell-free DNA (cfDNA)-binding and anti-oxidative properties. In models of surgery-induced and collagenase-induced arthritis, we showed that these cationic nanoparticles attenuated cartilage degradation and provided strong chondroprotection against joint damage. Mechanistically, multiple target blockades alleviated oxidative stress and dampened cfDNA-induced inflammation by suppressing the M1 polarization of macrophages. This study suggests a beneficial direction for targeting multiple danger mediators in the treatment of intractable arthritis.

Polycationic PAMAM ameliorates obesity-associated chronic inflammation and focal adiposity.

As a surging public health crisis, obesity and overweight predispose individuals to various severe comorbidities contributed by the accompanying chronic inflammation. However, few options exist for tackling chronic inflammation in obesity or inhibiting depot-specific adiposity. Here, we report that polycationic polyamidoamine (PAMAM) treatment can improve both aspects of obesity. With the discovery that the plasma cell-free RNA (cfRNA) level is elevated in obese subjects, we applied the cationic PAMAM generation 3 (P-G3) scavenger to treat diet-induced obese (DIO) mice. Intraperitoneal delivery of P-G3 alleviated the chronic inflammation in DIO mice and reduced their body weight, resulting in improved metabolic functions. To further enhance the applicability of P-G3, we complexed P-G3 with human serum albumin (HSA) to attain a sustained release, which showed consistent benefits in treating DIO mice. Local injection of HSA-PG3 into subcutaneous fat completely restricted the distribution of the complex within the targeted depot and reduced focal adiposity. Our study illuminates a promising cationic strategy to ameliorate chronic inflammation in obesity and target local adiposity.

Selective targeting of visceral adiposity by polycation nanomedicine.

Obesity is a pandemic health problem with poor solutions, especially for targeted treatment. Here we develop a polycation-based nanomedicine polyamidoamine generation 3 (P-G3) that-when delivered intraperitoneally-selectively targets visceral fat due to its high charge density. Moreover, P-G3 treatment of obese mice inhibits visceral adiposity, increases energy expenditure, prevents obesity and alleviates the associated metabolic dysfunctions. In vitro adipogenesis models and single-cell RNA sequencing revealed that P-G3 uncouples adipocyte lipid synthesis and storage from adipocyte development to create adipocytes that possess normal functions but are deficient in hypertrophic growth, at least through synergistically modulating nutrient-sensing signalling pathways. The visceral fat distribution of P-G3 is enhanced by modifying P-G3 with cholesterol to form lipophilic nanoparticles, which is effective in treating obesity. Our study highlights a strategy to target visceral adiposity and suggests that cationic nanomaterials could be exploited for treating metabolic diseases.

Nanoparticulate cell-free DNA scavenger for treating inflammatory bone loss in periodontitis.

Periodontitis is a common type of inflammatory bone loss and a risk factor for systemic diseases. The pathogenesis of periodontitis involves inflammatory dysregulation, which represents a target for new therapeutic strategies to treat periodontitis. After establishing the correlation of cell-free DNA (cfDNA) level with periodontitis in patient samples, we test the hypothesis that the cfDNA-scavenging approach will benefit periodontitis treatment. We create a nanoparticulate cfDNA scavenger specific for periodontitis by coating selenium-doped hydroxyapatite nanoparticles (SeHANs) with cationic polyamidoamine dendrimers (PAMAM-G3), namely G3@SeHANs, and compare the activities of G3@SeHANs with those of soluble PAMAM-G3 polymer. Both G3@SeHANs and PAMAM-G3 inhibit periodontitis-related proinflammation in vitro by scavenging cfDNA and alleviate inflammatory bone loss in a mouse model of ligature-induced periodontitis. G3@SeHANs also regulate the mononuclear phagocyte system in a periodontitis environment, promoting the M2 over the M1 macrophage phenotype. G3@SeHANs show greater therapeutic effects than PAMAM-G3 in reducing proinflammation and alveolar bone loss in vivo. Our findings demonstrate the importance of cfDNA in periodontitis and the potential for using hydroxyapatite-based nanoparticulate cfDNA scavengers to ameliorate periodontitis.

Biomimetic matrix fabricated by LMP-1 gene-transduced MC3T3-E1 cells for bone regeneration.

Bone healing is regulated by multiple microenvironmental signals provided by the extracellular matrix (ECM). This study aimed to mimic the native osteoinductive microenvironment by developing an ECM using gene-transduced cells. The LIM mineralization protein-1 (LMP-1) gene was transferred to murine pre-osteoblast cells (MC3T3-E1) using lentiviral vectors. Western blotting assay indicated that the MC3T3-E1 cells expressed an increased level of bone morphologic protein-2, -4 and -7 (BMP-2, -4 and -7) after LMP-1 gene transduction. The transduced cells were then seeded into calcined bovine bone scaffolds and cultured for 7, 14, and 21 days to construct ECMs on the scaffolds. The ECM-scaffold composites were then decellularized using the freeze-drying method. Scaffolds without ECM deposition were used as controls. The composites and controls were implanted into critical-sized bone defects created in the distal femurs of New Zealand rabbits. Twelve weeks after the surgery, both microcomputed tomography and histologic results indicated that the 7-day-cell-modified ECM-scaffold composites induced bone regeneration with significantly larger volume, trabecular thickness and connectivity than the controls. However, the 14- and 21-day-cell-modified ECM-scaffold composites triggered sustained inflammation response even at 12 weeks after the surgery and showed less bone ingrowth and integration than their 7-day-cell-modified counterparts. In conclusion, these results highlight the viable gene transfer techniques for manipulating cells in a constructed microenvironment of ECM for bone regeneration. However, the unresolved inflammation relating to the duration of ECM modification needs to be considered.

[HYPOXIA INDUCIBLE FACTOR lα/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS].

OBJECTIVE: To observe the genes expression of hypoxia inducible factor lα (HIF-1α) and HIF- 2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. METHODS: High density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2% O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type II at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type II, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. RESULTS: Toluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group; extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type II was positive in cytoplasm; when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t = 8.345, P = 0.001; t = 7.683, P = 0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P < 0.05), but no significant difference was found at 3 weeks between the 2 groups (P > 0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P < 0.01). The mRNA expressions of collagen type II and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P < 0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P < 0.05). CONCLUSION: HIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.

Intervertebral disk degeneration: T1ρ MR imaging of human and animal models.

PURPOSE: To evaluate disk degeneration in human and animal models by using a T1ρ magnetic resonance (MR) imaging technique to help in understanding the natural history and progression of intervertebral disk degeneration. MATERIALS AND METHODS: After institutional review board approval was obtained, 80 subjects (54 men and 26 women; mean age ± standard deviation, 31.6 years ± 6.20) with 400 lumbar intervertebral disks were examined at MR imaging. With approval from the animal care committee, six rhesus monkeys received two levels of either annulus fibrosus puncture or pingyangmycin subendplate injection at L3-4 and L5-6 to mimic disk degeneration. Lumbar spines of all the animals were examined at radiography and MR imaging preoperatively and 1 day and 1, 3, 6, 9, and 13 months postoperatively. Pfirrmann grading system and T1ρ quantification were used to evaluate the degenerative degree of the disks of both humans and animals. RESULTS: The mean T1ρ values of lumbar intervertebral disks of human subjects were 136.0 msec ± 31.4 and 76.1 msec ± 14.2 at Pfirrmann grades II and III, respectively. The T1ρ values in lumbar intervertebral disks of the rhesus monkey models of disk degeneration had a rapid decrease from approximately 110 msec to 80 msec and then tended to stabilize after operation. There was a large T1ρ value decrease between Pfirrmann grades II and III in human subjects that coincided with the rapid degeneration process of lumbar intervertebral disks in the rhesus monkeys. Pfirrmann grades were significantly correlated with T1ρ values in both humans (r = -0.681, P < .001) and rhesus monkeys (r = -0.824, P < .001). CONCLUSION: The data demonstrate that rapid intervertebral disk degeneration occurs early in the degenerative cascade, between Pfirrmann grades II and III.

Short-term alendronate treatment does not maintain a residual effect on spinal fusion with interbody devices and bone graft after treatment withdrawal: an experimental study on spinal fusion in pigs.

PURPOSE: Whether alendronate treatment has a residual effect on bone ingrowth into porous biomaterial in humans or experimental animals after treatment withdrawal is still unknown. The purpose of this study was to investigate bone ingrowth into porous tantalum and carbon fiber interbody implants after discontinuing alendronate treatment in experimental spinal fusion in pigs. METHODS: Twenty-four pigs were randomly divided into two groups of each 12 pigs. The pigs underwent anterior intervertebral lumbar arthrodeses at L2-3, L4-5 and L6-7. Each level was randomly allocated to one of the three implants: a porous tantalum ring with pedicle screw fixation, a porous tantalum ring or a carbon fiber cage with anterior staple fixation. The central hole of implants was packed with an autograft. Alendronate was given orally for the first 3 months to one of the two groups. The pigs were observed for 6 months postoperatively. Histology and micro-CT scans were done at the endpoint. RESULTS: The spinal fusion rates of each implant showed no differences between two treatment groups. Furthermore, no differences were found between two groups as for bone ingrowth into the central holes of implants and bone-implant interface in each implant, or as for the pores of tantalum implants. Trabecular bone microarchitecture in the central hole of the carbon fiber cage did not differ between two treatment groups. CONCLUSION: The application of ALN, with a dose equivalent to that given to humans during the first 3 months after surgery, does not maintain a residual effect on spinal fusion with porous tantalum ring and autograft after treatment withdrawal in a porcine ALIF model.

Effects of IL-1 receptor-associated kinase-4 gene silencing on human osteoblast-like cells.

The aim of this study is to identify the effects of interleukin-1 receptor-associated kinase-4 (IRAK-4) gene silencing on human osteoblast-like cells. The siRNA sequences of the target gene, IRAK-4, were constructed and transferred into MG63 cells (control group = MG63 cells; SC group = MG63 cells transfected with scrambled IRAK-4 siRNA; KD group = MG63 cells transfected with 75 nM IRAK-4 siRNA). The morphological changes, cell growth, cell-cycle progression, apoptosis, and the expression of various cytokines and proteins were compared. Compared with the control and SC groups, IRAK-4 gene silencing in MG63 cells caused morphological changes, inhibited growth, altered the cell-cycle distribution, increased apoptosis (p < 0.05), decreased bone alkaline phosphatase and osteocalcin levels (p < 0.05), and decreased protein expression of Bcl-2/Bax and Bcl-2, p-JNK1/2, p-ERK1/2, and p-p38MAPK (p < 0.05). The results indicated that IRAK-4 gene silencing in MG63 cells inhibited cell proliferation and function and increase apoptosis, which may be related to the decreased Bcl-2/Bax ratio and inhibition of the protein expression of various components of the mitogen-activated protein kinase pathways. The results of this study may help improve the understanding of the relationship between IRAK-4 and osteoblast-like cells and the interactions between various cytokines in the periprosthetic inflammatory environment.

Ginkgolide B promotes proliferation and functional activities of bone marrow-derived endothelial progenitor cells: involvement of Akt/eNOS and MAPK/p38 signaling pathways.

Bone marrow-derived, circulating endothelial progenitor cells (EPCs) contribute to neovascularization in various diseases, and represent a very interesting alternative cell source for enhancing vasculogenesis in regenerative medicine. In this study, we investigated the effects of Ginkgolide B (GB) on proliferation and differentiation of EPCs, and the involved signaling pathway in vitro. EPC proliferation, migration, adhesion and angiogenesis activities were assessed with the WST-8 assay, Transwell chamber assay, cell counting and angiogenesis kit, respectively. Apoptosis was detected with annexin V and propidium iodide staining. The protein expression of angiogenesis-related makers was detected by Western blot, and related gene expression was determined by real-time polymerase chain reaction (RT-PCR). The results showed that GB promoted the proliferation and endothelial gene expression, and markedly enhanced vascular endothelial growth factor-induced migration response and the capability to incorporate into the vascular networks in EPCs. GB protected EPCs from H2O2-induced cell death. GB induced the phosphorylation of eNOS, Akt and p38, which in turn promoted cell proliferation and function. In conclusion, the present study demonstrates that GB, at a near medical applied dose, increases the number and functional activities of EPCs with involvement of Akt/endothelial nitric oxide synthase and mitogen-activated protein kinase (MAPK)/p38 signal pathways. These findings raise the intriguing possibility that GB may play an important role in the protection and revascularization of blood vessels.

[Construction and identification of human bone morphogenetic protein-7 recombinant adeno-associated virus type 2 vector and its expression in bone mesenchymal stem cells].

OBJECTIVES: To facilitate gene therapy research using recombinant adeno-associated virus type 2 (rAAV2) vector as gene transfer vehicle, and to construct a rAAV2 based vector carrying bone morphogenetic protein-7 (BMP7) and observe its expression in bone mesenchymal stem cells. METHODS: The coding sequence (1.3 kb) of BMP7 was amplified by polymerase chain reaction (PCR) from the pcDNA1.1(+) plasmid containing the human BMP-7 cDNA. After purified, the gene fragment was cloned into a plasmid pUC18 and termed plasmid pUC18-hBMP7. The recombinant pUC18-hBMP7 was digested by Kpn I and Sal I and further ligated to the pSNAV by T4DNA ligase. The resultant plasmid PSNAV-hBMP7 was transformed into DH5a Escherichia coli, and positive colonies were screened by PCR and digest with restriction enzyme to identify the correct recombinant clones. BHK-21 cells were transfected with the purified pSNAV-BMP7 plasmid according to a standard calcium phosphate precipitation method. The cells were then cultured in selection media containing 800 micro g/ml G418 (Gibco/BRL). G418-resistant BHK-21 cell clones were isolated and the integrity of hBMP7 gene was determined by PCR using the above PCR primers. To package the virus, stably transfected BHK-21 cells were subsequently infected with recombinant herpes simplex virus type 1 (rHSV-1). The collected cells were processed by chloroform treatment, PEG8000/NaCl precipitation and chloroform extraction for purification. The titer was determined using quantitative DNA dot blots and the purity was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Following infection with rAAV2-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, MSCs were assessed qualitatively for BMP7 production. RESULTS: Transient transfection showed an efficiency of 98.8% in MSCs. RT-PCR showed that MSCs had transcription of BMP7 that was enhanced by the gene transfer. BMP-7 expression in MSCs was identified by Western-blot. CONCLUSIONS: The hBMP7 recombinant adeno-associated virus vector is successfully constructed. The present in vitro study demonstrates that rAAV2-BMP7 could infect MSCs.