Huading Lu

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA.

Porous Chitosan Scaffolds with Embedded Hyaluronic Acid/Chitosan/Plasmid-DNA Nanoparticles Encoding TGF-β1 Induce DNA Controlled Release, Transfected Chondrocytes, and Promoted Cell Proliferation

Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-β1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-β1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering.

Oxidative stress participates in age-related changes in rat lumbar intervertebral discs

Aging is a major factor associated with lumber intervertebral disc degeneration, and oxidative stress is known to play an essential role in the pathogenesis of many age-related diseases. In this study, we investigated oxidative stress in intervertebral discs of Wistar rats in three different age groups: youth, adult, and geriatric. Age-related intervertebral disc changes were examined by histological analysis. In addition, oxidative stress was evaluated by assessing nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA), and advanced oxidation protein products (AOPPs). Intervertebral disc, but not serum, NO concentrations significantly differed between the three groups. Serum and intervertebral disc SOD activity gradually decreased with age. Furthermore, both serum and intervertebral disc MDA and AOPP levels gradually increased with age. Our studies suggest that oxidative stress is associated with age-related intervertebral disc changes.

Glatiramer acetate inhibits degradation of collagen II by suppressing the activity of interferon regulatory factor-1.

Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) is considered to be the major one contributing to the process of development of osteoarthritis (OA).Interferon regulatory factor 1 (IRF-1) is an important transcriptional factor accounting for inflammation response induced by TNF-α. The physiological function of IRF-1 in OA is still unknown. In this study, we reported that the expression levels of IRF-1 in OA chondrocytes were significantly higher compared to those in normal chondrocytes, which was reversed by treatment with Glatiramer acetate (GA), a licensed clinical drug for treating patients suffering from multiple sclerosis (MS). We also found that GA is able to attenuate the upregulation of IRF-1 induced by TNF-α. Matrix metalloproteinase13 (MMP-13) is one of the downstream target genes of IRF-1, which can induce the degradation of collagen II. Importantly, our results indicated that GA suppressed the expression of MMP-13 as well as the degradation of collagen II. In addition, GA also suppressed TNF-α-induced production of NO and expression of iNOS. Finally, we found that the inhibition of STAT1 activation played a critical role in the inhibitory effects of GA on the induction of IRF-1 and MMP-13. These data suggest that GA might have a potential effect in therapeutic OA.

c-Jun transactivates Puma gene expression to promote osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disorder in which genetic, hormonal, mechanical and ageing factors affect its progression. Current studies are focusing on chondrocytes as a key mediator of OA at a cellular level. however, the mechanism underlying chondrocyte apoptosis remains unclear. PUMA is a pro-apoptotic member of the BH3-only subgroup of the Bcl-2 family and is involved in a large number of physiological and pathological processes. In the present study, we examined whether PUMA has a role in IL-1β-induced apoptosis and whether the c-Jun N-terminal kinase (JNK)/c-Jun pathway mediates the induction of PUMA, thus contributing to chondrocyte apoptosis. The results demonstrated an increase in PUMA protein and mRNA levels in cultured mouse chondrocytes following 4 h of IL-1β treatment. Furthermore, this upregulation of PUMA was critical for chondrocyte apoptosis as knockdown of PUMA using PUMA-specific siRNA significantly reduced apoptosis in cultured cells. Upon pharmacological inhibition of the JNK/c-Jun pathway with CE11004 or SP600125, the expression of PUMA was notably suppressed with a concomitant decrease in apoptosis observed in IL-1β-treated chondrocytes. Also, immunohistochemical studies revealed that the PUMA and c-Jun proteins were upregulated in chondrocytes from the articular cartilage of OA patients. Together, these data suggest a role for PUMA and the JNK/c-Jun pathway in the regulation of chondrocyte apoptosis during OA.

Hepcidin promotes osteogenic differentiation through the bone morphogenetic protein 2/small mothers against decapentaplegic and mitogen‑activated protein kinase/P38 signaling pathways in mesenchymal stem cells

The ability of mesenchymal stem cells (MSCs) to differentiate into osteogenic lineages requires management for their future use in treating bone destruction and osteoporosis. Hepcidin is closely associated with bone metabolism, however, it remains to be elucidated whether hepcidin affects osteogenic differentiation in MSCs. The present study demonstrated that hepcidin enhanced osteoblastic differentiation and mineralization, which was manifested by an upregulation in the differentiation markers alkaline phosphatase and osteogenic genes. Furthermore, the expression levels of bone morphogenetic proteins and small mothers against decapentaplegic homologs were concomitantly increased following hepcidin treatment. In addition, the p38 mitogen-activated protein kinase may be an upstream kinase for osteoblastic differentiation. Thus, hepcidin may be important in the osteogenic differentiation of MSCs and may be considered as a target in the development of therapies for pathological bone loss.

Artificial biological ligament: Its making, testing, and experimental study on animals

By applying a novel biochemical technique using porcine tendon as the raw material with its antigen minimized, we developed an artificial biological ligament (ABL). We examined and tested its structure, mechanical properties, and biocompatibility and explored the feasibility of reconstructing the anterior cruciate ligament (ACL) with ABL. By means of treating porcine tendon with epoxy crosslinking fixation, diversified antigen minimization process, mechanic enhancement modification, and surface activating process, we fabricated the ABL samples. We then analyzed its in vivo and in vitro performances, respectively, with animal (goat) implantation, histological examination, scanning electron microscope, cells culture, and mechanical tests before and after animal implants. The appearance of ABL was similar to that of normal human ligament. Histological examination showed that the ABL was composed of collagen fibers with no cells. Electron microscope examination revealed that the ABL was composed of hair‐appearing and fiber‐like objects running uniformly in a certain direction and closely parallel‐arranged. Three weeks after xenogenic marrow matrix cells were cultured on the surface of the ABL, it was noted that cells adhered and the matrix secreted by the cells precipitated around the cells. There were no cells found inside the ABL. The average diameter of ABL was 5 mm and the mechanical test at a speed of 100 mm/min showed that its average tensile limit was 927.19 N; the tension‐resistant strength was 47.22 N/mm. Those measurements were close to the corresponding parameters of the normal goat ACL. Twelve weeks after ABL replacement of the goat ACL, synovial membrane covering with the ingrowths of small blood vessels was seen on the surface of the implant. Fifty weeks after the replacement, the ABL material was partially replaced by regenerated host ligament‐like tissue. Around the ABL material fibers in the bone tunnel close to the joint surface the direct connection mode, ligament‐fibro cartilage‐calcified cartilage‐bone, was seen. As we used the unique biochemical technique and minimized the xenogenic protein immunogenicity of the porcine tendon, ABL showed acceptable biomechanical properties and superior biocompatibility. As a substitute for ligament in the reconstruction of the ACL, ABL has a promising prospect in clinical applications.

Open Reduction Assisted With an External Fixator and Internal Fixation With Calcaneal Locking Plate for Intra-articular Calcaneal Fractures

Background: The extensile lateral approach (ELA) has been considered to be a standard approach for displaced intra-articular calcaneal fractures (DICF) because it provides excellent exposure and allows direct reduction of the depressed posterior facet fragment. But continuous retraction during surgery needs sufficient manpower and may cause ischemia. Failure of rigid fixation of DICF will not allow for early weight bearing and may lead to a loss of reduction. To avoid these disadvantages, this study presents open reduction assisted with an external fixator and internal fixation with a calcaneal locking plate. Methods: A series of 58 patients with 62 DICFs were treated over a period of 49 months. All patients were clinically and radiologically followed up with a mean follow-up of 35 (range 29-42) months. Clinical follow-up included visual analog scale (VAS), the American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score, and ability to work. Radiologic follow-up included axial and lateral radiographs and measurements of the Bohler angle and Gissane angle. Results: At the final follow-up, all fractures had healed. The mean VAS score was 2.9 (range 0 to 8, SD 1.9) and the average AOFAS score was 71 (range 55-85, SD 8.1). The mean postoperative Bohler angle immediately after the surgery was 28.3 degrees (range 13.0-44.6, SD 7.0), which decreased to 27.5 degrees (range 12.2-43.3, SD 7.0) at the final follow-up, and the mean postoperative Gissane angle after the surgery was 116.3 degrees (range 94.9-131.5, SD 9.0) which finally increased to 118.4 degrees (range 94.5-135.8, SD 9.3). No statistically significant differences regarding Bohler and Gissane angles were found between different Sanders fracture types (P>.05). Conclusion: The presented operative technique was found to provide comparable reduction of Sanders type II-IV injuries. Level of Evidence: Level III, case control study.

PGC-1α regulates the expression and activity of IRF-1.

Interferon regulatory transcription factor 1 (IRF‐1) regulates downstream signals of tumor necrosis factor α (TNF‐α). The activity of IRF‐1 is mediated by Jak/Stat signaling pathway. In this study, we found that PPAR γ coactivator‐1α (PGC‐1α) is able to suppress the induction of IRF‐1. Treatment with TNF‐α in MC3T3 cells leads to a sustainable increase in the expression of IRF‐1 and its target gene cyclooxygenase 2 (COX‐2). In contrast, TNF‐α treatment led to a sustainable reduction in expression of PGC‐1α. Interestingly, we found that overexpression of PGC‐1α attenuated the induction of IRF‐1 and COX‐2. However, silence of PGC‐1α exacerbated the induction of IRF‐1 and COX‐2. Importantly, we found that the effect of PGC‐1α on repressing IRF‐1 expression and activity is facilitated by the reduction in phosphorylation of STAT1 at position 727 (S727P), an essential transcriptional activator of IRF‐1. Finally, we found that calyculin A, a pharmacological inhibitor of protein phosphatase 2A (PP2A) and PP1 abolishes the repression of STAT1 phosphorylation mediated by PGC‐1α, suggesting a new mechanism of PGC‐1α in regulating STAT1/IRF‐1 pathway.