Rat Articular Cartilage Research Articles

In Vivo Evaluation of Cartilage Regenerative Effects of CCN2 Protein.

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique growth factor that promotes the proliferation and differentiation, but not the hypertrophy of articular chondrocytes in vitro. Based on these findings, we previously evaluated the cartilage-regenerative effects of recombinant CCN2 protein (rCCN2) by using both mono-iodoacetate (MIA) injection into the rat joint cavity and formation of full-thickness defects of rat articular cartilage in vivo, and our results suggested the utility of CCN2 in the regeneration of articular cartilage. This chapter entails helpful tips to apply these two animal models for the evaluation of cartilage-regenerative effects of CCN2 or its derivatives.

Expression and Immunohistochemical Localisation of the Gβγ-activated and Calcineurin-Inhibited Adenylyl Cyclase Isoforms in Rat Articular Chondrocytes.

To determine the expression and localisation of the Gβγ-activated adenylyl cyclase (AC) isoforms 2, 4, and 7 and calcineurin-inhibited AC isoform 9 in rat articular chondrocytes. Experimental study. Jumma Research Laboratory and Histology Laboratory, The Aga Khan University, Karachi, from 2009 to 2011. Fresh slices of articular cartilage were taken from various synovial joints of rats of different age groups. The expression of AC isoforms was determined by RT-PCR and immunohistochemistry was performed to localise these isoforms in articular chondrocytes. Tissue sections were processed for immunostaining with respective antibodies. The color was developed by diaminobenzidine. All the studied AC isoforms were found to be differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms studied increased with age. The expression of the AC isoforms through PCR was almost consistent with the localisation of these isoforms by immunohistochemistry. These data add to the information about signalling cascades possibly involved in articular chondrocytes. Variable expression of AC isoforms 2, 4, 7, and 9 suggest a role for the signalling cascades regulated by the AC isoforms in articular chondrocytes.

Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage.

Hyaluronic acid (HA) is used clinically to treat osteoarthritis (OA), but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and dimethylmethylene blue (DMMB) assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α). These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses.

Effect of moderate treadmill exercise together with modified hydroxyapatite chitosan composite hydrogel implantation on repair of full-thickness defects of articular cartilage in rats

Objective To investigate the effect of moderate treadmill exercise together with modified hydroxyapatite chitosan composite hydrogel(CS/HA-g-CS)implantation on repair of full-thickness defects of articular cartilage in rats. Methods Full-thickness cartilage defects were drilled in the patellar groove of bilateral femoral condyles in a total of 24 male SD rats before they were randomly assigned into 4 groups. The control group(BC group)was subjected to no exercise or CS/HA-g-CS implantation; the chitosan group(CHI group)to CS/HA-g-CS implantation without exercise; the moderate treadmill exercise group(MIR group)to exercise 4 weeks after modeling without CS/HA-g-CS implantation; the CHI + MIR group to moderate treadmill exercise plus CS/HA-g-CS implantation 4 weeks after modeling. Half of the animals were sacrificed at week 8 and half at week 16 after operation. Femoral condyles were harvested for gross observation and his-tochemical measurement by O’ Driscoll scoring system. mRNA expressions of glycosaminoglycan, collagen typeⅡ and BMP-2 were detected by RT-PCR. Results Gross observation revealed: at 8 weeks after modeling, the CHI + MIR group was significantly better than the other 3 groups, with the BC group in the poorest(P 0. 05). O' Driscoll scoring revealed: at both 8and 16 weeks after modeling, the CHI + MIR group was significantly better than the other 2 groups and the BC group significantly poorer than the other 3 groups(P 0. 05). The expressions of collagen type Ⅱ, glycosaminoglycan and BMP-2 were significantly higher in the CHI + MIR group than in the other 3 groups(P< 0. 05)and significantly lower in the BC group than in the other 3 groups(P< 0. 05). Conclusions Moderate treadmill exercise together with CS/HA-g-CS implantation has significant positive effects on repair of full-thickness defects of articular cartilage in rats than merely moderate treadmill exercise or CS/HA-g-CS implantation alone. The defective cartilage repaired by moderate treadmill exercise together with CS/HA-g-CS implantation contains more collagen type II and glycosaminoglycan and shows morphology of nearly normal cartilage. Key words: Exercise; Cartilage, articuler; Injuries; Hydroxyapatite chitosan composite hydrogel

Chondroprotective effects of alpha-lipoic acid in a rat model of osteoarthritis

ABSTRACTObjective: The purpose of this study was to investigate whether alpha-lipoic acid (ALA) confers a chondroprotective effect on articular cartilage in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA).Methods: Fifty male SD rats were divided into five groups, including SHAM-operated, MIA-induced OA, and three experimental groups treated with 50-, 100-, or 200-mg/kg ALA. After 14 d of ALA treatment, rats were sacrificed for joint macroscopic and histology assessments. The gene and protein expressions of markers related to chondrocyte phenotype, caspase proteins, NADPH oxidase 4 (Nox4), p22phox, activation of nuclear factor-κB (NF-κB), and endoplasmic reticulum (ER) stress were measured by Western blot analyses or qRT-PCR.Results: The results showed that MIA injection successfully induced OA by causing cartilage degeneration. Morphological and histological examinations demonstrated that ALA treatment, especially 200 mg/kg of ALA, significantly ameliorated cartilage degeneration in rats with MIA-induced OA. ALA could effectively increase the levels of the collagen type II and aggrecan genes and inhibit apoptosis-related proteins expression. ALA reduced biomakers of oxidative damage and over-expression levels of Nox4 and p22phox. ALA also suppressed ER stress and inhibited the activation of NF-κB pathway. Moreover, ALA obviously inhibited TNF-α secretion and Wnt/β-catenin signaling way.Conclusion: These findings indicated that ALA might be a potential therapeutic agent for the protection of articular cartilage against progression of OA through inhibition of oxidative stress, ER stress, inflammatory cytokine secretion, and Wnt/β-catenin activation.

A novel method of RNA isolation and gene expression profiling of articular cartilage in rats by cryotome sectioning

A novel method of RNA isolation and gene expression profiling of articular cartilage in rats by cryotome sectioning

Effect of ozone on Wnt/β-catenin signaling pathway in articular cartilage of rats with osteoarthritis

Objective To investigate the effects of ozone (O3) on Wnt/β-catenin signaling pathway in the articular cartilage of rats with osteoarthritis (OA). Methods Eighteen male SPF Wistar rats, aged 3 months, weighing 200-250 g, were randomly divided into 3 groups (n=6 each) using a random number table: control group (group C), group OA, and O3 group (group O). OA was induced by injection of monosodium iodoacetate 3 mg (50 μl) into the right knee joint cavity.On 7th day after the model was established successfully, 25 μg/ml O3 1 ml were injected into the knee joint cavity, once a week for 3 consecutive weeks in group O. Behavioral changes were observed after establishment of the model.At 1 day before establishment of the model, and 1, 4, 7, 14, 21 and 28 days after establishment of the model, the mechanical paw withdrawal threshold (MWT) was measured.At 28 days after establishment of the model, the total knee joint was removed and stained with haematoxylin and eosin for examination of the pathological changes of the cartilage (under light microscope) and for determination of the expression of β-catenin and matrix metalloproteinase-13 (MMP-13) in the cartilage (by immunohistochemistry). Results The signs of OA such as hind-limb motor dysfunction, knee joint swelling, or decreased joint motion, and signs of hyperalgesia such as lickings were observed after establishment of the model in rats.Compared with group C, the MWT was significantly decreased at each time point after establishment of the model, and the expression of β-catenin and MMP-13 in the cartilage was significantly up-regulated in the other two groups (P<0.05). Compared with group OA, the MWT was significantly increased at 7-28 days after establishment of the model, and the expression of β-catenin and MMP-13 in the cartilage was significantly down-regulated in group O (P<0.05). The pathological changes of the cartilage were significantly reduced in group O as compared with group OA. Conclusion The mechanism by which O3 mitigates OA is probably related to inhibition of Wnt/β-catenin signaling pathway activation in the articular cartilage in rats. Key words: Ozone; Osteoarthritis; Wnt proteins; beta catenin

3D analysis of chondron morphology of human and rat articular cartilage from micro-computed tomography

3D analysis of chondron morphology of human and rat articular cartilage from micro-computed tomography

Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles.

Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1) are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA), whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI) system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01). By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01). Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.

Exercise-driven metabolic pathways in healthy cartilage

Exercise is vital for maintaining cartilage integrity in healthy joints. Here we examined the exercise-driven transcriptional regulation of genes in healthy rat articular cartilage to dissect the metabolic pathways responsible for the potential benefits of exercise. Transcriptome-wide gene expression in the articular cartilage of healthy Sprague-Dawley female rats exercised daily (low intensity treadmill walking) for 2, 5, or 15 days was compared to that of non-exercised rats, using Affymetrix GeneChip arrays. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO)-term enrichment and Functional Annotation analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway mapper was used to identify the metabolic pathways regulated by exercise. Microarray analysis revealed that exercise-induced 644 DEGs in healthy articular cartilage. The DAVID bioinformatics tool demonstrated high prevalence of functional annotation clusters with greater enrichment scores and GO-terms associated with extracellular matrix (ECM) biosynthesis/remodeling and inflammation/immune response. The KEGG database revealed that exercise regulates 147 metabolic pathways representing molecular interaction networks for Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Diseases. These pathways collectively supported the complex regulation of the beneficial effects of exercise on the cartilage. Overall, the findings highlight that exercise is a robust transcriptional regulator of a wide array of metabolic pathways in healthy cartilage. The major actions of exercise involve ECM biosynthesis/cartilage strengthening and attenuation of inflammatory pathways to provide prophylaxis against onset of arthritic diseases in healthy cartilage.

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11-13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro-CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro-CT following immobilization in a time-dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod-like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization-induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage.

CHI3L1 and lubricin expression in osteoarthritic cartilage

Osteoarthritis (OA) is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein 39 (GP-39) are known as a potential marker for the activation of chondrocytes and the progression of OA, whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren-Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology OARSI system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage. By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage. Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA evidenced in histomorphometric analysis of our samples, so that they could have a role in the daily clinical practice in staging the severity and progression of the disease.This work was supported by by a Grant-in-Aid provided by FIR 2014-2016, (cod. 314509), University of Catania, Italy.

참당귀 추출분말이 in vitro and in vivo model에서 MMPs 조절 기전

골관절염에 대한 참당귀 추출분말 (AGE)의 치료효과를 검토하고자 토끼연골세포와 흰쥐의 monosodium iodoacetate (MIA)로 유발된 골관절염 부위에서 시료를 채취하여 MMPs의 발현에 대한 AGE의 억제 효능을 검토하였다. 고 농도의 AGE (50 &#x3BC;g/mL) 투여에서 세포독성은 관찰되지 않았으며 면역 및 염증반응과 관련된 여러 인자의 전사인자인 NF-&#x03BA;B 활성화를 효과적으로 억제시켰다. 토끼연골 세포에서 MMP-2와 MMP-9의 활성을 확인해본 결과 AGE는 MMP-9의 활성을 효과적으로 억제시키는 것을 확인하였다. AGE를 토끼연골세포에 처리하여 분석한 결과, 주요성분인 decursin과 decursinol angelate가 3.62&#xB1;0.47 &#x3BC;g/mg protein와 2.14&#xB1;0.36 &#x3BC;g/mg protein으로 검출되었다. 동물실험을 위하여, 골관절염은 MIA를 흰쥐 무릎관절에 처리하여 동물모델을 만들었으며, 매일 25, 50와 100 mg/kg의 AGE를 3주 동안 먹인 결과 흰쥐의 연골 조직에서 MMPs가 억제되는 것을 확인하였다. 연골조직으로부터 RT-PCR을 통해 collagen Type I, collagen Type II, aggrecan 및 MMPs (MMP-3, MMP-9, MMP-13)의 mRNA를 확인해본 결과 AGE는 collagen Type I, collagen Type II, aggrecan은 증가시키며 MMPs는 감소시키는 효과를 얻었다. 결론적으로 AGE는 MMPs 억제를 통하여 골관절염의 발생을 억제한다. The precise mechanism underlying the therapeutic efficacy of an extraction powder of Angelica gigas (AGE) for the treatment of degenerative osteoarthritis was investigated in primary cultured rabbit chondrocytes and in a monosodium-iodoacetate (MIA)-induced osteoarthritis rat model. The treatment with AGE (50 &#x3BC;g/mL) effectively inhibited NF-B activation. The anti-inflammatory mechanism was clarified by gelatin zymography and western blotting measurements of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities. The AGE (50 &#x3BC;g/mL) treatment significantly reduced MMP-9 activity. The constituents of AGE&#x2014; decursinol, decursin, and decursinol angelate&#x2014;were determined by LC-MS/MS after a 24 hr treatment of rabbit chondrocytes. The contents of the major products, decursin and decursinol angelate, were 3.62&#xB1;0.47 and 2.14 &#xB1;0.36 &#x3BC;g/mg protein, respectively in AGE-treated (50 &#x3BC;g/mL) rabbit chondrocytes. An in vivo animal study on rats fed a diet containing 25, 50, and 100 mg/kg AGE for 3 weeks revealed a significant inhibition of the MMPs in the MIA-induced rat articular cartilage. The genetic expression of arthritic factors in the articular cartilage was examined by RT-PCR of collagen Type I, collagen Type II, aggrecan, and MMP (MMP3, MMP-9, MMP13). Specifically, AGE up-regulated the expression of collagen Type I, collagen Type II, and aggrecan and inhibited MMP levels at all tested concentrations. Collectively, AGE showed a strong specific site of action on MMP regulation and protected against the degeneration of articular cartilage via cellular regulation of MMP expression both in vitro and in vivo.

Culture expanded primary chondrocytes have potent immunomodulatory properties and do not induce an allogeneic immune response

Allogeneic cell therapies, such as mesenchymal stromal cells (MSC), which have potent regenerative and anti-inflammatory potential are being investigated as a therapy for osteoarthritis (OA) and cartilage injury. Here we describe another potential source of regenerative and anti-inflammatory allogeneic cells, culture expanded primary chondrocytes (CEPC). In direct comparison to allogeneic MSC, we extensively assess the immunological interactions of CEPC in an allogeneic setting. Chondrocytes were isolated from rat articular cartilage and cultured in normoxic or hypoxic conditions. In vitro co-culture assays with allogeneic lymphocytes and macrophages were used to assess the immunomodulatory capacities of the chondrocytes, followed by immune response analysis by flow cytometry, ELISA and qPCR. CEPC showed reduced induction of proliferation, activation and cytotoxic granzyme B expression in allogeneic T cells. Importantly, exposure to pro-inflammatory cytokines did not increase CEPC immunogenicity despite increases in MHC-I. Furthermore, CEPC had a potent ability to suppress allogeneic T cell proliferation, which was dependent on nitric oxide production. This suppression was contact independent in hypoxia cultured CEPC. Finally, chondrocytes were shown to have the capacity to modulate pro-inflammatory macrophage activity by reducing MHC-II expression and TNF-α secretion. These data indicate the potential use of allogeneic chondrocytes in OA and cartilage defects. The lack of evident immunogenicity, despite exposure to a pro-inflammatory environment, coupled with the immunomodulatory ability indicates that these cells have the potential to evade the host immune system and suppress inflammation, thus potentially facilitating the resolution of OA induced inflammation and cartilage regeneration.

참당귀 추출분말의 골관절염 흰쥐의 염증성 사이토카인류의 억제활성

The protective effects of extract powder of Angelica gigas on the degeneration of the articular cartilage in rats was investigated with monosodium iodoacetate (MIA)-induced osteoarthritis, The treatment of high concentration (50 µg/mL) of Angelica gigas effectively inhibited nitric oxide (NO) production induced by interleukin-1α (IL-1α) without any cytotoxicity. Specifically, mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were dose dependently reduced by extract powder of Angelica gigas. Importantly, mRNA expression in articular cartilage of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were clearly reduced. The inflammatory cytokines in blood were also reduced as well. These results suggested that the protective effects on the degeneration of the articular cartilage was derived from the inhibitory effects of mRNA and protein expression of tested inflammatory cytokines which is linked to prevent the degradation of proteoglycan (PG), the main matrix content in articular cartilage. Meanwhile, the 2 hrs incubation of decursin, a major compound of extract powder in rat whole blood rapidely converted decursin into decursinol which shows string anti-inflammatory activity. The coverted decursinol was detected after 8 hrs in whole blood by LC-MS/MS. Conclusively, the inhibitory effects of inflammatory cytokines production in osteoarthritis may be derived from the production of decursinol, which performs against inflammatroy cytokines like TNF-α, IL-1β‚ and IL-6. 요약 : 골관절염에 대한 참당귀 추출분말의 치료효과를 검토하고자 흰쥐의 monosodium iodoacetate (MIA)로 유발된 골관절염 부위에서 시료를 채취하여 염증관련 효소 및 염증성 cytokines의 발현에 대하 여 참당귀 추출분말의 억제효능을 검토하였다. 고농도의 참당귀 추출분말 (50 µg/mL) 투여에서도 독성

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.

In vivo anti-apoptosis activity of novel berberine-loaded chitosan nanoparticles effectively ameliorates osteoarthritis.

Berberine chloride (BBR) is an isoquinoline alkaloid that possesses promising protective efficacies against osteoarthritis (OA). Nevertheless, the therapeutic agent of this substance in OA is limited by its poor aqueous solubility, low bioavailability and short biological half-life. In this study, chitosan (CS)-based nanoparticles were prepared for the sustained release of BBR. Novel BBR-loaded chitosan nanoparticles (CNs) were successfully synthesized by the ionic cross-linking method. BBR-loaded CNs were spherical and homogeneous in shape. Moreover, they exhibited good stability and had ideal releasing profile in vitro. After intra-articular injection of BBR-loaded CNs, the level of BBR in rat plasma decreased and the retention time in synovial fluid increased compared with free BBR solution. In vivo evaluation of BBR-loaded CNs further showed higher anti-apoptosis activity in the treatment of OA compared with BBR solution at equivalent concentration. This result was evidenced by the changes of gross morphology and histological analyses in rat articular cartilage, TUNEL assay, quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical analyses of caspase-3, Bcl-2 and Bax expressions. Given these results, BBR-loaded CNs are potential therapeutic agents for OA.

참당귀 추출분말이 Monosodium Iodoacetate로 유발된 흰쥐의 골관절염에 대한 효과

참당귀 추출물의 골관절염에 대한 억제 또는 치료효과를 알고자 monosodium iodoacetate로 유발한 흰쥐의 골관절염 모델을 사용하여 30마리 6주령 SD흰쥐를 정상군(normal), 비처리군(untreated) 및 참당귀 처리군(treated)으로 각각 10마리씩 나누어 실험하였다. 실험기간 동안 실험 개시일부터 4일 간격으로 체중 변화를 측정하였다. 3주간 참당귀 추출물 처리 후 관절연골 내의 glycosaminoglycan (GAG)의 함량 및 proteoglycan (PG) 함량을 측정 비교하였다. 그 결과, 참당귀 추출물은 MIA로 증가한 GAG를 완화시켰으며, PG의 함량을 회복시켰다. 또한, 활액에서의 TNF-a함량은 미약하게 감소하였다. 한편, 경골 관절연골을 분리하여 Safranin-O 염색으로 관절연골 상태를 확인한 결과, MIA 처리로 유발된 관절연골 소실을 억제하였다. 이 참당귀 추출물을 HPLC로 분석한 결과, 주요 성분인 decursin 및 decursinol angelate의 총 함량은 <TEX>$10.5{\pm}0.2%$</TEX>이었다. To study the efficacy of extract powder of Angelica gigas in preventing and treating degeneration of the articular cartilage in rats with monosodium iodoacetate (MIA)-induced osteoarthritis, A total of 30 six-week-old Sprague-Dawley rats were randomly divided into normal control group, untreated group and Angelica gigas treated group, with 10 rats in each group. During the treatment period, body weight were measured in each four days interval from starting date. The rat were sacrificed at the end of 3rd week after daily administration of Angelica gigas and then rat tibia articular cartilage was removed. In articular cartilages, glycosaminoglycan (GAG) amount increased by MIA treatment were reduced while proteoglycan (PG) amount decreased by MIA treatment were fairly recovered by Angelica gigas treatment, respectively. The content of TNF-a was also slightly reduced sections of the cartilage were stained with safranin-0 were also partially recovered by Angelica gigas treatment. By HPLC analysis, the content of main compounds decursin and decursinol angelate was analyzed as <TEX>$10.5{\pm}0.2%$</TEX> of total extracts.

Expression of CHI3L1 and CHIT1 in osteoarthritic rat cartilage model. A morphological study.

Osteoarthritis is a degenerative joint disease, which affects millions of people around the world. It occurs when the protective cartilage at the end of bones wears over time, leading to loss of flexibility of the joint, pain and stiffness. The cause of osteoarthritis is unknown, but its development is associated with different factors, such as metabolic, genetic, mechanical and inflammatory ones. In recent years the biological role of chitinases has been studied in relation to different inflammatory diseases and more in particular the elevated levels of human cartilage glycoprotein 39 (CHI3L1) and chitotriosidase (CHIT1) have been reported in a variety of diseases including chronic inflammation and degenerative disorders. The aim of this study was to investigate, by immunohistochemistry, the distribution of CHI3L1 and CHIT1 in osteoarthritic and normal rat articular cartilage, to discover their potential role in the development of this disease. The hypothesis was that the expression of chitinases could increase in OA disease. Immunohistochemical analysis showed that CHI3L1 and CHIT1 staining was very strong in osteoarthritic cartilage, especially in the superficial areas of the cartilage most exposed to mechanical load, while it was weak or absent in normal cartilage. These findings suggest that these two chitinases could be functionally associated with the development of osteoarthritis and could be used as markers, so in the future they could have a role in the daily clinical practice to stage the severity of the disease. However, the longer-term in vivoand in vitro studies are needed to understand the exact mechanism of these molecules, their receptors and activities on cartilage tissue.

Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes

Ethnopharmacological relevanceYaotongning Capsule (YTNC) is a Traditional Chinese Medicinal (TCM) formula that has been demonstrated to be effective for osteoarthritis (OA) treatment in clinical use. Many compounds and 10 component medicinal materials (CMMs for short, i.e., the fundamental elements used in TCM formulas) in YTNC are challenging to study the pharmacological effects and interactions of the CMMs. Besides, it is difficult to know whether the YTNC formula is reasonable, and if YTNC formula could be improved without comparing YTNC with other TCM formulas of treating OA. Based on different combinations of the active fractions from the 10 CMMs of YTNC and eight additional herbs frequently used in the TCM formulas of treating OA, the present study evaluated systematically the in vitro effects of these active fractions and the interactions among the active fractions from YTNC on rat chondrocytes to find possible solutions of the above questions. Materials and methodsBased on the formulation of YTNC and the concept of combinatorial chemistry, the active fractions were applied to form the whole YTNC prescription (i.e., the combination of all YTNC active fractions and the extract of YTNC׳s vehicle), five disassembled formulas of YTNC (i.e., the combinations of some active fractions in YTNC) and 21 TCM samples consisted of different kinds of active fractions. The degenerated chondrocytes were induced with interleukin-1β (IL-1β), and then the half-effective concentration (EC50) value of the proliferation activity was analyzed to evaluate the 27 TCM samples. Nine samples were screened for the following evaluation on glycosaminoglycan (GAG) synthesis. Rat articular cartilage was obtained from six Sprague-Dawley rats (seven days of age), and then chondrocytes were isolated through enzymatic digestion with 0.2% Collagenase II. Proliferations of chondrocytes were examined through Cell Counting Kit-8 assay, when the intracellular levels of GAG were detected by 1,9-Dimethylmethylene blue staining. The interactions between the active fractions in YTNC were evaluated by comparing experimental EC50 values of the YTNC formulas with their additive EC50 values. The effects of every active fraction were estimated by comparing the EC50 values of the TCM sample containing the active fraction with that of the initial sample without the active fraction. ResultsThe whole formula of YTNC was very good at promoting the proliferation and GAG synthesis among all the 27 TCM samples. The vehicle of YTNC (Chinese rice wine) strengthened the two activities of YTNC. Refer to promoting the proliferation in chondrocytes, Davallia mariesii flavonoids (not belong to YTNC) were more potent than Glycyrrhiza uralensis flavonoids in YTNC, while the saponins, volatile oils and polysaccharides of YTNC were more potent than those from the eight additional herbs. Some samples including fewer active fractions were as good as YTNC. The YTNC formula and its disassembled formulas exhibited good activities both in promoting the proliferation and GAG synthesis, and the whole formula was most potent among the six YTNC formulas. ConclusionsThe YTNC formula is reasonable and has advantage in promoting the proliferation and GAG synthesis in IL-1β induced chondrocytes. YTNC׳s vehicle Chinese rice wine plays an important role in strengthening the activity of YTNC. YTNC may have the potential activity on treating chondrocytes degeneration caused by OA. However, the formula still can be simplified based on the combination of alkaloids, flavonoids and 50% of saponins from Glycyrrhiza uralensis to improve its quality controllability and safety. The present study can be a quite purposeful work for developing new YTNC-based formulas with maximal therapeutic efficacy and minimal adverse effects.