The Identification of CD163 Expressing Phagocytic Chondrocytes in Joint Cartilage and Its Novel Scavenger Role in Cartilage Degradation

Background

Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation.

Methods

Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163⁺ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated.

Results

In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163⁺ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were present in Col-II⁺ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163⁺ chondrocytes were also found in isolated Col-II⁺ chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163⁺ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II⁺ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05).

Conclusions

An increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.     

Progression of Cartilage Degradation, Bone Resorption and Pain in Rat Temporomandibular Joint Osteoarthritis Induced by Injection of Iodoacetate

Background

Osteoarthritis (OA) is an important subtype of temporomandibular disorders. A simple and reproducible animal model that mimics the histopathologic changes, both in the cartilage and subchondral bone, and clinical symptoms of temporomandibular joint osteoarthritis (TMJOA) would help in our understanding of its process and underlying mechanism.

Objective

To explore whether injection of monosodium iodoacetate (MIA) into the upper compartment of rat TMJ could induce OA-like lesions.

Methods

Female rats were injected with varied doses of MIA into the upper compartment and observed for up to 12 weeks. Histologic, radiographic, behavioral, and molecular changes in the TMJ were evaluated by light and electron microscopy, MicroCT scanning, head withdrawal threshold test, real-time PCR, immunohistochemistry, and TUNEL assay.

Results

The intermediate zone of the disc loosened by 1 day post-MIA injection and thinned thereafter. Injection of an MIA dose of 0.5 mg or higher induced typical OA-like lesions in the TMJ within 4 weeks. Condylar destruction presented in a time-dependent manner, including chondrocyte apoptosis in the early stages, subsequent cartilage matrix disorganization and subchondral bone erosion, fibrosis, subchondral bone sclerosis, and osteophyte formation in the late stages. Nociceptive responses increased in the early stages, corresponding to severe synovitis. Furthermore, chondrocyte apoptosis and an imbalance between anabolism and catabolism of cartilage and subchondral bone might account for the condylar destruction.

Conclusions

Multi-level data demonstrated a reliable and convenient rat model of TMJOA could be induced by MIA injection into the upper compartment. The model might facilitate TMJOA related researches.     

Pathology of the Calcified Zone of Articular Cartilage in Post-Traumatic Osteoarthritis in Rat Knees

Objectives

This study aimed to investigate the pathology occurring at the calcified zone of articular cartilage (CZC) in the joints afflicted with post-traumatic osteoarthritis (PTOA).

Methods

Rats underwent bilateral anterior cruciate ligament (ACL) transection and medial meniscectomy to induce PTOA. Sham surgery was performed on another five rats to serve as controls. The rats were euthanized after four weeks of surgery and tibial plateaus were dissected for histology. The pathology of PTOA, CZC area and the tidemark roughness at six pre-defined locations on the tibial plateaus were quantified by histomorphometry.

Results

PTOA developed in the knees, generally more severe at the medial plateau than the lateral plateau, of rats in the experimental group. The CZC area was unchanged in the PTOA joints, but the topographic variations of CZC areas that presented in the control knees were reduced in the PTOA joints. The tidemark roughness decreased in areas of the medial plateau of PTOA joints and that was inversely correlated with the Mankin’s score of PTOA pathology.

Conclusion

Reduced tidemark roughness and unchanged CZC area differentiate PTOA from primary osteoarthritis, which is generally believed to have the opposite pathology at CZC, and may contribute to the distinct disease progression of the two entities of arthropathy.     

Salivary Alterations in Rats with Experimental Chronic Kidney Disease

Objective

This study aimed to analyze changes in saliva composition and salivary secretion process of rats with chronic kidney disease induced by 5/6 nephrectomy to set the foundation for salivary studies related to CKD.

Methods

CKD was induced in Wistar rats via 5/6 nephrectomy. Blood and saliva samples were collected from Control, Sham and CKD groups at 8 and 12 weeks after the surgery. Salivation was stimulated via intraperitoneal injections of pilocarpine (1.0 mg/Kg body weight) or isoproterenol (5.0 mg/Kg body weight). Saliva was collected and immediately stored at -80°C until analysis. The salivary flow rate, total protein, amylase and peroxidase activities, and urea concentrations were measured. The blood urea nitrogen (BUN) and serum creatinine concentrations were also evaluated.

Results

Increases in BUN and serum creatinine concentrations were observed in the CKD groups. Amylase activity was significantly reduced in response to both stimuli in the CKD groups at 8 weeks and increased in the CKD groups at 12 weeks in response to isoproterenol stimulus. The peroxidase activities of the CKD groups were significantly reduced in response to isoproterenol stimulation and were increased at 12 weeks in response to pilocarpine stimulation. Salivary urea was significantly increased in the CKD groups at 8 weeks in response to the isoproterenol stimuli and at 12 weeks in response to both salivary agonists.

Conclusions

The pattern of alterations observed in this experimental model is similar to those observed in patients and clearly demonstrates the viability of 5/6 nephrectomy as an experimental model in future studies to understand the alterations in salivary compositions and in salivary glands that are elicited by CKD.     

Periodic knee injections of collagen tripeptide delay cartilage degeneration in rabbit experimental osteoarthritis

Introduction

Collagen peptides have been reported to possess various biological activities for various cell types. The purposes of this study were, first, to examine the therapeutic effects of collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with hyaluronan (HA).

Methods

Osteoarthritis was induced by anterior cruciate ligament transection of the right knee in 72 Japanese white rabbits and they were divided into four groups (control, Ctp, HA and Ctp/HA). Each material was injected weekly into the knee, and knee joint samples were collected 5, 10 and 15 weeks after surgery. Macroscopic and histomorphological analyses of cartilage were conducted. Expression of type II collagen and matrix metalloproteinase-13 was also analyzed immunohistochemically. A Tukey''s honestly significant difference test was used to evaluate the statistical significance of difference in the macroscopic, histological and immnohistochemical results.

Results

All treatment groups exhibited slightly higher resistance to the progression of osteoarthritis than the control group macroscopically 15 weeks after surgery. Histologically, intra-articular injection of Ctp significantly reduced cartilage degradation 10 weeks after surgery, and Ctp/HA significantly reduced it 5 weeks after surgery in comparison with the control. Immunohistochemically, both Ctp-treated and Ctp/HA-treated groups had significantly increased type II collagen-positive chondrocytes at the fifth week after the surgery, although the numbers of matrix metalloproteinase-13-positive chondrocytes were not affected.

Conclusion

Periodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be exerted by promotion of type II collagen synthesis predominantly.     

SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways

Objectives

Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored.

Methods

SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis.

Results

Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.

Conclusions

Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.     

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Introduction

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.

Methods

Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.

Results

Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.

Conclusions

The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.     

Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes

Aim

The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.

Materials and Methods

Osteoarthritic (OA) human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM). The production of aggrecan, matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-6 and nitric oxide (NO) and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC) or non-sclerotic (NSC) subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.

Results

In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008). MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01). TIMP-1 production was slightly increased at 3 μM (p = 0.02) and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05). IL-6 and PGE₂ production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.

Conclusions

Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in osteoblasts as similar results were obtained with anti-IL-6 antibody.     

Sequential Change in T2* Values of Cartilage,Meniscus, and Subchondral Bone Marrow in a Rat Model of Knee Osteoarthritis

Background

There is an emerging interest in using magnetic resonance imaging (MRI) T2* measurement for the evaluation of degenerative cartilage in osteoarthritis (OA). However, relatively few studies have addressed OA-related changes in adjacent knee structures. This study used MRI T2* measurement to investigate sequential changes in knee cartilage, meniscus, and subchondral bone marrow in a rat OA model induced by anterior cruciate ligament transection (ACLX).

Materials and Methods

Eighteen male Sprague Dawley rats were randomly separated into three groups (n = 6 each group). Group 1 was the normal control group. Groups 2 and 3 received ACLX and sham-ACLX, respectively, of the right knee. T2* values were measured in the knee cartilage, the meniscus, and femoral subchondral bone marrow of all rats at 0, 4, 13, and 18 weeks after surgery.

Results

Cartilage T2* values were significantly higher at 4, 13, and 18 weeks postoperatively in rats of the ACLX group than in rats of the control and sham groups (p<0.001). In the ACLX group (compared to the sham and control groups), T2* values increased significantly first in the posterior horn of the medial meniscus at 4 weeks (p = 0.001), then in the anterior horn of the medial meniscus at 13 weeks (p<0.001), and began to increase significantly in the femoral subchondral bone marrow at 13 weeks (p = 0.043).

Conclusion

Quantitative MR T2* measurements of OA-related tissues are feasible. Sequential change in T2* over time in cartilage, meniscus, and subchondral bone marrow were documented. This information could be potentially useful for in vivo monitoring of disease progression.     

Administration of a CXCL12 Analog in Endotoxemia Is Associated with Anti-Inflammatory,Anti-Oxidative and Cytoprotective Effects In Vivo

Background

The chemokine receptor CXCR4 is a multifunctional receptor which is activated by its natural ligand C-X-C motif chemokine 12 (CXCL12). As CXCR4 is part of the lipopolysaccharide sensing complex and CXCL12 analogs are not well characterized in inflammation, we aimed to uncover the systemic effects of a CXCL12 analog in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by using a sublethal LPS dose.

Methods

The plasma stable CXCL12 analog CTCE-0214D was synthesized and administered subcutaneously shortly before LPS treatment. After 24 hours, mice were sacrificed and blood was obtained for TNF alpha, IFN gamma and blood glucose evaluation. Oxidative stress in the liver and spleen was assessed and liver biotransformation capacity was determined. Finally, CXCR4, CXCL12 and TLR4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for apoptosis and oxidative stress were determined by means of immunohistochemistry.

Results

CTCE-0214D distinctly reduced the LPS mediated effects on TNF alpha, IFN gamma, ALAT and blood glucose levels. It attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. Furthermore, in all three organs investigated, CTCE-0214D diminished the LPS induced expression of CXCR4, CXCL12, TLR4, NF-κB, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity was induced above average. Additionally, TUNEL staining revealed anti-apoptotic effects of CTCE-0214D.

Conclusions

In summary, CTCE-0214D displayed anti-inflammatory, anti-oxidative and cytoprotective features. It attenuated reactive oxygen species, induced heme oxygenase 1 activity and mitigated apoptosis. Thus, the CXCR4/CXCL12 axis seems to be a promising target in the treatment of acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals.     

Amelioration of Liver Injury by Continuously Targeted Intervention against TNFRp55 in Rats with Acute-on-Chronic Liver Failure

Background

Acute-on-chronic liver failure (ACLF) is an acute deterioration of established liver disease. Blocking the TNF (tumor necrosis factor)/TNFR (tumor necrosis factor receptor) 1 pathway may reduce hepatocyte apoptosis/necrosis, and subsequently decrease mortality during development of ACLF. We demonstrated that a long-acting TNF antagonist (soluble TNF receptor: IgG Fc [sTNFR:IgG-Fc]) prevented/reduced development of acute liver failure by blocking the TNF/TNFR1 (TNFRp55) pathway. However, it is still unclear if sTNFR:IgG-Fc can inhibit hepatocyte damage during development of ACLF.

Methodology

Chronic liver disease (liver fibrosis/cirrhosis) was induced in Wistar rats by repeatedly challenging with human serum albumin (HSA), and confirmed by histopathology. ACLF was induced with D-galactosamine (D-GalN)/lipopolysaccharide (LPS) i.p. in the rats with chronic liver disease. Serum and liver were collected for biochemical, pathological and molecular biological examinations.

Principal Findings

Reduced mortality was observed in sTNFR:IgG-Fc treated ACLF rats, consistent with reduced interleukin (IL)-6 levels in serum and liver, as well as reduced hepatic caspase-3 activity, compared to that of mock treated group. Reduced hepatic damage was confirmed with histopathology in the sTNFR:IgG-Fc treated group, which is consistent with reduced Bcl-2 and Bax, at mRNA and protein levels, but increased hepatocyte proliferation (PCNA). This is also supported by the findings that caspase-3 production was up-regulated significantly in ACLF group compared to the mock treated group. Moreover, up-regulated caspase-3 was inhibited following sTNFR:IgG-Fc treatment. Finally, there was up-regulation of hepatic IL-22R in sTNFR:IgG-Fc treated ACLF rats.

Conclusions

sTNFR:IgG-Fc improved survival rate during development of ACLF via ameliorating liver injury with a potential therapeutic value.     

miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4

Introduction

miR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis.

Methods

Primary chondrocytes from Sprague-Dawley rats were treated with IL-1β before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-β1 was quantified by a luciferase reporter harboring TGF-β1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry.

Results

IL-1β treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-β pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3''-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-β and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group.

Conclusion

IL-1β responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage.     

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells

Purpose

To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.

Methods

Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.

Results

Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.

Conclusions

Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.     

Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain

Background

Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant.

Methodology

The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints.

Principal Findings

Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair.

Conclusion and Significance

The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.     

Multiparametric MRI of Epiphyseal Cartilage Necrosis (Osteochondrosis) with Histological Validation in a Goat Model

Purpose

To evaluate multiple MRI parameters in a surgical model of osteochondrosis (OC) in goats.

Methods

Focal ischemic lesions of two different sizes were induced in the epiphyseal cartilage of the medial femoral condyles of goats at 4 days of age by surgical transection of cartilage canal blood vessels. Goats were euthanized and specimens harvested 3, 4, 5, 6, 9 and 10 weeks post-op. Ex vivo MRI scans were conducted at 9.4 Tesla for mapping the T₁, T₂, T_1ρ, adiabatic T_1ρ and T_RAFF relaxation times of articular cartilage, unaffected epiphyseal cartilage, and epiphyseal cartilage within the area of the induced lesion. After MRI scans, safranin O staining was conducted to validate areas of ischemic necrosis induced in the medial femoral condyles of six goats, and to allow comparison of MRI findings with the semi-quantitative proteoglycan assessment in corresponding safranin O-stained histological sections.

Results

All relaxation time constants differentiated normal epiphyseal cartilage from lesions of ischemic cartilage necrosis, and the histological staining results confirmed the proteoglycan (PG) loss in the areas of ischemia. In the scanned specimens, all of the measured relaxation time constants were higher in the articular than in the normal epiphyseal cartilage, consistently allowing differentiation between these two tissues.

Conclusions

Multiparametric MRI provided a sensitive approach to discriminate between necrotic and viable epiphyseal cartilage and between articular and epiphyseal cartilage, which may be useful for diagnosing and monitoring OC lesions and, potentially, for assessing effectiveness of treatment interventions.     

Chronic Testosterone Replacement Exerts Cardioprotection against Cardiac Ischemia-Reperfusion Injury by Attenuating Mitochondrial Dysfunction in Testosterone-Deprived Rats

Background

Although testosterone deficiency is associated with increased risks of heart disease, the benefits of testosterone therapy are controversial. Moreover, current understanding on the cardiac effect of testosterone during cardiac ischemia-reperfusion (I/R) periods is unclear. We tested the hypothesis that testosterone replacement attenuates the impairment of left ventricular (LV) function and heart rate variability (HRV), and reduces the infarct size and arrhythmias caused by I/R injury in orchiectomized (ORX) rats.

Methodology

ORX or sham-operated male Wistar rats (n = 24) were randomly divided and received either testosterone (2 mg/kg, subcutaneously administered) or the vehicle for 8 weeks. The ejection fraction (EF) and HRV were determined at baseline and the 4^th and 8^th week. I/R was performed by left anterior descending coronary artery ligation for 30 minutes, followed by a 120-minute reperfusion. LV pressure, arrhythmia scores, infarct size and cardiac mitochondrial function were determined.

Results

Prior to I/R, EF and HRV were impaired in the ORX group, but were restored in the testosterone-treated group. During I/R, arrhythmia scores and the infarct size were greater, and cardiac mitochondrial function was impaired, whereas the time to 1^st VT/VF onset and the LV end-systolic pressure were decreased in the ORX group when compared to the sham group. Testosterone replacement attenuated the impairment of these parameters in ORX rats during I/R injury, but did not show any benefit or adverse effect in non-ORX rats.

Conclusions

Testosterone replacement restores cardiac function and autonomic regulation, and exerts cardioprotective effects during the I/R period via mitochondrial protection in ORX rats.     

Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells

Background

Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.

Methods

OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H₂O₂) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.

Results

The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H₂O₂ or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H₂O₂ or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).

Conclusion

Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H₂O₂ or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.