Gene Expression Of Collagen Type Research Articles

Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

Introduction Notochordal cell-conditioned medium (NCCM) has shown to stimulate matrix production by nucleus pulposus cells (NPCs) in alginate bead cultures.1,2 This cell culture method, however, provides a markedly different environment than the nucleus pulposus (NP) tissue, in which the bioactive factors should exert their effect. Therefore, the main objective of this study was to test the stimulatory effect of NCCM in an NP explant culture system. Materials and Methods NCCM was generated by incubating porcine NC-rich NP tissue in plain high glucose DMEM (hgDMEM) at 37°C, 5% CO2 and 5% O2 for 4 days. The medium was filtered and the filtrate was resuspended in fresh hgDMEM. NP tissue samples were isolated from the caudal IVDs of 2-year old cows ( n = 5), excluding the annulus fibrosus (AF) tissue. The NP tissues were cultured, according to a previously established method,3 in a fiber jacket, constraining the tissue and thereby acting as an AF. Samples were cultured in base medium (BM: hgDMEM supplemented with pen/strep, ITS, ascorbic acid, proline, bovine serum albumin, and sodium pyruvate), NCCM, supplemented with the same factors as BM, or positive control medium (Pos, BM supplemented with 1 µg/mL Link-N). After 4 weeks, NP tissue explants were assayed for water, GAG, DNA, and hydroxyproline content, histology with safranin O/fast green staining and gene expression of collagen type 1 and 2, aggrecan, and SOX9. Results The GAG content of tissue explants treated with NCCM significantly increased ( p < 0.05) compared with day 0 and BM ( Fig. 1a ). GAG content in the positive control group tended to increase compared with BM ( p = 0.086). This was confirmed by safranin O/fast green staining, showing a more intense red staining for the NCCM culture group ( Fig. 1b ). No significant differences were observed between groups regarding water, DNA, and hydroxyproline content. Gene expression results did not confirm the finding of the biochemical content assays. The relative expression of collagen type 1 and 2, aggrecan and SOX9 did not differ between groups, and only minor changes were observed compared with day 0. NCCM may have an initial and a transient stimulatory effect on transcriptional level, which could explain why the stimulatory effect of NCCM after 4 weeks of culture was not observed at the gene level. Nevertheless, we cannot exclude that GAGs originating from the NCCM may have been incorporated into the NP tissue explant during culture. Conclusion These results confirm the stimulatory effect of NCCM in an NP tissue explant culture. Moreover, NCCM appears to be at least as potent as Link-N, of which the stimulatory effect on NP tissue has been previously established in vivo.4 This study, therefore provides further evidence for the promising role of NCCM in the treatment of IVD regeneration. Identification of the bioactive factors in NCCM will be helpful to further develop a treatment method and achieve maximal anabolic effects. Acknowledgment This work was supported by AOSpine International through an AOSpine Research Network grant (SRN2011_11). [Figure: see text] References Potier E, de Vries S, van Doeselaar M, Ito K. Potential application of notochordal cells for intervertebral disc regeneration: an in vitro assessment. Eur Cell Mater 2014;28:68–80, discussion 80–81 Abbott RD, Purmessur D, Monsey RD, Iatridis JC. Regenerative potential of TGFb3 + Dex and notochordal cell conditioned media on degenerated human intervertebral disc cells. J Orthop Res 2012;30(3):482–488 van Dijk BG, Potier E, Ito K. Long-term culture of bovine nucleus pulposus explants in a native environment. Spine J 2013;13(4):454–463 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120

618. bFGF or VEGF Gene Transfer Via AAV2 Vectors To the Tendon Activates Multiple Genes Critical To Tendon Growth and Regeneration: An In Vivo Study

Purpose: Tendon healing is innately weak and transfer of growth factor gene therapy may activate multiple genes necessary or specific for tendon growth and regeneration. In this study we transferred bFGF or VEGF gene by AAV2 vectors to the healing tendon in a chicken flexor tendon model. We investigated how endogenous tendon healing related genes were activated after the delivery of AAV2-bFGF or AAV2-VEGF and the differences between epitenon and endotenon using a laser capture microdissection ((LCM).Methods: Eighteen digital flexor tendons from the big toes of 9 chickens were used. The tendons were completely transected and divided into three groups: AAV-bFGF injection, AAV-VEGF injection and no-injection control. After AAV injection, the tendons were surgically repaired. At 4 weeks after surgery, the tendons were harvested. The formalin-fixed, paraffin-embedded tendons were sectioned. LCM was used to dissect epitenon and endotenon separately. The tissue was subsequently analyzed for gene expression of collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), proliferating cell nuclear antigen (PCNA), fibronectin 1 (FN1), scleraxis (SCX) and tenascin C (TNC) using RT-qPCR analysis.Results: In AAV-VEGF and AAV-bFGF injected tendons, we found significant increases in expression of FN1, PCNA, SCX, COL1A1 and COL3A1 genes (p < 0.05 or p<0.01) compared with no-injection control. In the AAV-bFGF injected tendon, SCX was expressed 5-fold higher in the endotenon than in the epitenon. The increases in TNC gene expression did not reach statistical significance, but interestingly, TNC gene was expressed highly in the endotenon (10-fold greater than in the epitenon) in AAV-VEGF injected tendon. The TNC in the endotenon of AAV-VEGF-injected tendon was raised by 55-fold compared with that of the non-injected tendon.Discussion: Our results demonstrated a drastic increase in expression of studied genes specific to tendon growth and regeneration after AAV2-bFGF or AAV2-VEGF therapy. We also found precisely the epitenon and endotenon are equally responsive to the gene therapy. This study provides evidence at genetic levels to support the effective changes of the gene expression by the gene therapy, which may be key mechanistic findings to support the use of these gene therapies.

Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner.

Icariin, a flavonoid isolated from Epimedium pubescens, has previously been identified to exert beneficial effects on preventing bone loss and promoting bone regeneration. However, molecular mechanisms for its anabolic action have, up to now, remained largely unknown. Effects of icariin on cell proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) were systematically evaluated. To characterize underlying mechanisms, its effects on mitogen-activated protein kinase (MAPK) signalling pathways were determined. Results showed that icariin might not have enhanced effects on cell proliferation. However, it seemed to significantly enhance osteogenic differentiation of BMSCs, demonstrated by increasing alkaline phosphatase (ALP) activity and gene expression of collagen type I (Col I), osteocalcin (OCN) and osteopotin (OPN). It was demonstrated that icariin rapidly phosphorylated extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N terminal kinase (JNK). Furthermore, icariin-stimulated osteogenic effects on BMSCs were dramatically attenuated by treatment with either specific ERK inhibitor of PD98059, p38 inhibitor of SB202190 or JNK inhibitor SP600125. These results provide a potential mechanism of anabolic activity of icariin on BMSCs involving ERK, p38 and JNK MAPK pathways.

Carbon nanotube array inducing osteogenic differentiation of human mesenchymal stem cells.

Carbon nanotubes (CNTs) are a kind of nanomaterials which have been shown a promising application for biomedicine. There are a lot of studies to use CNTs to induce the differentiation of mesenchymal stem cells (MSCs). However, the cellular behavior of MSCs on the top layer of CNT array was still not well understood. In this study, we evaluated the morphology, the gene expressions of the osteogenic differentiation related markers, and the gene expressions of collagen type II (Col II, a marker of chondrogenesis), PPARγ (a marker of adipogenesis) and scleraxis (SCX, a marker of tenogenesis) in human mesenchymal stem cells (hMSCs) cultured on multi-walled carbon nanotube (MWCNT) array. The effect of MWCNT array on the mineralization of hMSCs which were cultured in osteogenic differentiation medium (ODM) was further assayed. Our results showed that the hMSCs cultured on MWCNT array spread well, formed numerous spiral shaped cell colons and showed perinuclear morphology. Compared to hMSCs cultured on dish, the gene expression of osteocalcin (OCN) was increased while the gene expressions of collagen type II (Col II), PPARγ and scleraxis (SCX) were decreased in hMSCs which were cultured on MWCNT array without any differentiation factors. Furthermore, compared with hMSCs on dish, the gene expressions of collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and RUNX2, and the mineralization of hMSCs on MWCNT array were enhanced when they were cultured in osteogenic differentiation medium (ODM). Our results indicated that MWCNT array was able to promote the osteogenesis of hMSCs.

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease.

Iron accumulation is associated with the pathophysiology of chronic kidney disease (CKD). Renal fibrosis is a final common feature that contributes to the progression of CKD; however, little is known about the association between renal iron accumulation and renal interstitial fibrosis in CKD. Here we investigate the effects of iron chelation on renal interstitial fibrosis in a rat model of CKD. CKD was induced by 5/6 nephrectomy in Sprague-Dawley rats. At 8 weeks after operation, 5/6 nephrectomized rats were administered an oral iron chelator, deferasirox (DFX), in chow for 8 weeks. Other CKD rats were given a normal diet. Sham-operative rats given a normal diet served as a control. CKD rats exhibited hypertension, glomerulosclerosis and renal interstitial fibrosis. Iron chelation with DFX did not change hypertension and glomerulosclerosis; however, renal interstitial fibrosis was attenuated in CKD rats. Consistent with these findings, renal gene expression of collagen type III and transforming growth factor-β was increased in CKD rats compared with the controls, while iron chelation suppressed these increments. In addition, a decrease in vimentin along an increase in E-cadherin in renal gene expression was observed in CKD rats with iron chelation. CKD rats also showed increased CD68-positive cells in the kidney, whereas its increase was attenuated by iron deprivation. Similarly, increased renal gene expression of CD68, tumor necrosis factor-α and monocyte chemoattractant protein-1 was suppressed in CKD rats with iron chelation. Renal iron accumulation seems to be associated with renal interstitial fibrosis in a rat model of CKD.

Biomechanics and gene expression in abdominal aortic aneurysm

The aim of the study was to detect inter-relations between the mechanical conditions and material properties of abdominal aortic aneurysm (AAA) wall and the underlying local gene expression of destabilizing inflammatory, proteolytic, and structural factors. During open surgery, 51 tissue samples from 31 AAA patients were harvested. Gene expression of collagen types I and III, inflammatory factors CD45 and MSR1, proteolytic enzymes matrix metalloproteinases 2 and 9, and tissue inhibitor of matrix metalloproteinase 1 was analyzed by reverse transcription-polymerase chain reaction. Material properties of corresponding AAA tissue samples were assessed by cyclic sinusoidal and destructive testing. Local mechanical conditions of stress and strain were determined by advanced nonlinear finite element analysis based on patient-specific three-dimensional AAA models derived from preoperative computed tomography data. In the AAA wall, all parameters analyzed were significantly expressed at the messenger RNA level. With respect to mechanical properties of the aneurysmatic wall, expression of collagen III correlated with the stiffness parameter α (r = -0.348; P = .017), and matrix metalloprotease 2 correlated with the stiffness parameter β and wall strength (r = -0.438 and -0.593; P = .005 and P < .001). Furthermore, significant relationships were observed between local AAA diameter and the expression of CD45, MSR1, and tissue inhibitor of matrix metalloproteinase 1 (r = 0.285, 0.551, 0.328; P < .05). However, we found no inter-relation of local calculated wall stresses and strains with gene expression. Our results show for the first time that gene expressions of destabilizing factors within AAA tissue might be correlated to geometric and mechanical properties of the AAA wall. However, we found no influence of local mechanical conditions on gene expression of these factors. Therefore, these preliminary results are still ambiguous.

PLGA/gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration.

The novel strategies of skin regenerative treatment are aimed at the development of biologically responsive scaffolds capable of delivering multiple bioactive agents and cells to the target tissues. In this study, nanofibers of poly(lactic-co-glycolic acid) (PLGA) and gelatin were electrospun and the effect of parameters viz polymer concentration, acid concentration, flow rate and voltage on the morphology of the fibers were investigated. PLGA nanofibers encapsulating epidermal growth factor were also prepared through emulsion electrospinning. The core-sheath structure of the nanofibers was verified by transmission electron microscopy. The hemostatic attributes and the biocompatibility of the scaffolds for human fibroblast cell were scrutinized. Furthermore, gene expression of collagen type I and type III by the cells on the scaffolds was quantified using real-time reverse transcriptase polymerase chain reaction. The results indicated desirable bioactivity and hemostasis of the scaffolds with the capability of encapsulation and controlled release of the protein which can be served as skin tissue engineering scaffolds and wound dressings.

Transdentinal cell photobiomodulation using different wavelengths.

The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23). Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level. Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2). The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Effect of parathyroid hormone on early chondrogenic differentiation from mesenchymal stem cells.

BackgroundTreatment of articular cartilage injuries remains a difficult challenge due to the limited capacity for intrinsic repair. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes under certain culture conditions. This study focused on the modulatory effects of parathyroid hormone (PTH) on chondrogenic differentiation from MSCs.MethodsMSCs were treated with various concentrations of PTH under chondrogenic pellet culture condition. RNA was isolated for real-time polymerase chain reaction (PCR) and gene expressions of collagen type II α1 chain (Col2a1), collagen type X α1 chain, collagen type I α1 chain, SRY-box9 (Sox9), and type 1 PTH/PTHrP receptor (PTH1R) were examined. Chondrogenic differentiation was also evaluated by histological findings.ResultsPTH had opposite effects on chondrogenesis, depending on the concentration. A low to moderate concentration of PTH promoted chondrogenic differentiation of MSCs with increased expression of Sox9, Col2a1, and PTH1R, whereas chondrogenesis of MSCs was inhibited rather than stimulated with a higher concentration of PTH.ConclusionThis study provides insights into the modulatory effect of PTH on chondrogenic differentiation from MSCs and the therapeutic potential for cartilage regeneration. Based on clinical experience regarding the efficacy and safety of PTH for bone metabolism, PTH may also be useful clinically for cartilage repair.

Nanosecond pulsed electric fields (nsPEFs) regulate phenotypes of chondrocytes through Wnt/β-catenin signaling pathway.

Nanosecond pulsed electric fields (nsPEFs) characterized by high voltage, low energy and non-thermal effects, have been broadly investigated as a potential tumor therapy; however, little is known about their effects on somatic cells. In this current study, we evaluated effects of nsPEFs on the phenotype of chondrocytes (morphology, glycosaminoglycan (GAG) content, proliferation and gene expression) and explored the mechanisms involved. Our results demonstrated that exposing chondrocytes to nsPEFs led to enhanced proliferation and dedifferentiation, evidenced by the upregulated gene expression of collagen type I (COL I) and downregulated gene expression of Sox9, collagen type II (COL II) and aggrecan (AGG) with activation of the wnt/β-catenin signaling pathway. Inhibition of the wnt/β-catenin pathway partially blocked these effects. Thus we concluded that nsPEFs induce dedifferentiation of chondrocytes partially through transient activation of the wnt/β-catenin signaling pathway.

Chondrogenic potential of rat mesenchymal stem cells in vitro under the influence of electromagnetic fields

Objective To determine the chondrogenic potential of mesenchymal stem cells (BMSCs) under exposure of extremely low frequency electromagnetic fields (ELF-EMF) and discuss the application prospect of EMF in cartilage tissue engineering.Methods Cell pellets of rat bone marrow-derived BMSCs were cultured in vitro under the addition of fibroblast growth factor 2 (FGF-2) and transforming growth factor-β3 (TGF-β3).Pellet cultures were exposed to sinusoidal ELF-EMF (1.0 mT,50 Hz).After 3 weeks of inductive differentiation culture,chondrogenesis was detected by alcian blue staining,and quantitative real-time polymerase chain reaction (real-time PCR) was used for detection of chondrogenesis related proteins.Glycosaminoglycan (GAG) contents of pellet cultures were determined using the dimethylmethylene blue (DMMB) dye-binding assay.Results With the effects of growth factors,EMF could significantly promote chondrogenic differentiation of BMSCs pellet cultures,and the gene expression of collagen type Ⅱ,X and aggrecan in rat BMSCs was also significantly up-regulated.The gene expression of sex determining region Y box 2 (SOX9) did not show significant difference between EMF exposed groups and nonexposed groups.The glycosaminoglycan (GAG)/DNA ratio of EMF exposed pellet cultures reached 3.108 ±0.341,which was significantly higer than those unexposed pellet cultures.Conclusion Increased expression of collagen type Ⅱ,X and GAG content in pellet cultures under EMF exposure may contribute to the chonderogenic differentiation of rat BMSCs.EMF was able to stimulate and maintain chondrogenic differentiation of rat BMSCs under influence of growth factors.EMF alone,however,could not induce chondrogenic differentiation. Key words: Electromagnetic fields; Mesenchymal stem cells; Pellet culture; Chondrogenic differentiation

Silk fibroin‐chondroitin sulfate‐alginate porous scaffolds: Structural properties and in vitro studies

ABSTRACTThe development of porous biodegradable scaffolds is of great interest in tissue engineering. In this regard, exploration of novel biocompatible materials is needed. Silk fibroin‐chondroitin sulfate‐sodium alginate (SF‐CHS‐SA) porous hybrid scaffolds were successfully prepared via lyophilization method and crosslinked by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide‐ethanol treatment. According to the scanning electron microscopy studies, mean pore diameters of the scaffolds were in the range of 60–187 μm. The porosity percentage of the scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (w/w/w %) was 92.4 ± 3%. Attenuated total reflectance Fourier transform infrared spectroscopy, X‐ray diffraction, and differential scanning calorimetry results confirmed the transition from amorphous random coil to crystalline β‐sheet in treated SF‐CHS‐SA scaffold. Compressive modulus was significantly improved in hybrid scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (3.35 ± 0.15 MPa). Cytotoxicity assay showed that the scaffolds have no toxic effects on chondrocytes. Attachment of chondrocytes was much more improved within the SF‐CHS‐SA hybrid scaffold. Real‐time polymerase chain reaction analyses showed a significant increase in gene expression of collagen type II, aggrecan, and SOX9 and decrease in gene expression of collagen type I for SF‐CHS‐SA compared with SF scaffold. This novel hybrid scaffold can be a good candidate to be utilized as an efficient scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41048.

AB0770 Differential Extracellular Protein Profiles of Osteoarthritic and Normal Mesenchymal Stem Cells Undergoing Chondrogenesis by the Double-Silac Technique

BackgroundChondrogenesis occurs as a result of mesenchymal stem cells (MSCs) condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage. A major...

Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo: laboratory investigation.

The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo. Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses. Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II. Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

Correlation between 3D microstructural and 2D histomorphometric properties of subchondral bone with healthy and degenerative cartilage of the knee joint.

Cartilage degeneration of the knee joint is considered to be a largely mechanically driven process. We conducted a microstructural and histomorphometric analysis of subchondral bone samples of intact cartilage and in samples with early and higher- grade arthritic degeneration to compare the different states and correlate the findings with the condition of hyaline cartilage. These findings will enable us to evaluate changes in biomechanical properties of subchondral bone during the evolution of arthritic degeneration, for which bone density alone is an insufficient parameter. From a continuous series of 80 patients undergoing implantation of total knee endoprosthesis 30 osteochondral samples with lesions macroscopically classified as ICRS grade 1b (group A) and 30 samples with ICRS grade 3a or 3b lesions (group B) were taken. The bone samples were assessed by 2D histomorphometry (semiautomatic image analysis system) and 3D microstructural analysis (high-resolution micro-CT system). The cartilage was examined using the semiquantitative real-time PCR gene expression of collagen type I and II and aggrecan. Both histomorphometry and microstructural and biomechanical analysis of subchondral bone in groups A and B consistently revealed progressive changes of both bone and cartilage compared with healthy controls. The severity of cartilage degeneration as assessed by RT PCR was significantly correlated with BV/TV (Bone Volume Fraction), Tb.Th (Trabecular Thickness) showed a slight increase. Tb.N (Trabecular Number), Tb.Sp (Trabecular separation) SMI (Structure Model Index), Conn.D (Connectivity Density) and DA (Degree of Anisotropy) were inversely correlated. We saw sclerotic transformation and phagocytic reticulum cells. Bone volume fraction decreased with an increasing distance from the cartilage with the differences compared with healthy controls becoming greater in more advanced cartilage damage. The density of subchondral bone alone is considered an unreliable parameter for classifying changes evolving over time. The progressive damage of subchondral bone seen in the present study correlates well with cartilage changes. Trabecular orientation is also impaired, which explains the changes in biomechanical parameters and the inadequate load transfer and excessive loading of cartilage. Besides subchondral bone density, which in turn correlates with cartilage thickness, other parameters such as structure model index and grade of anisotropy best reflect mechanical properties such as Young modulus, compressive strength, tensile stress, and failure energy. However, it remains unclear whether the mechanical interaction of the mineralized subchondral tissues with articular cartilage works vice versa. The possibility of a biochemical signalling from the degenerating cartilage via the synovial fluid and bone- cartilage crosstalks via subchondral pores may indeed explain a certain depth-dependency of subchondral bone changes.

Low-level laser therapy for osteonecrotic lesions: effects on osteoblasts treated with zoledronic acid.

Clinical studies have shown that low-level laser therapy (LLLT) can improve local tissue healing of bisphosphonate-induced osteonecrosis of the jaw. However, the effects of laser irradiation on bisphosphonate-treated osteoblasts have not been completely elucidated. Human osteoblasts were cultured in plain culture medium (DMEM). After 48 h, plain DMEM was replaced by DMEM with no fetal bovine serum, for a 24-h incubation followed by addition of zoledronic acid (5 μM) for additional 48 h. Cells were subjected to LLLT (InGaAsP; 780 ± 3 nm; 0.025 W) at 0.5, 1.5, 3, 5, and 7 J/cm(2), three times every 24 h. Cell viability, total protein production, alkaline phosphatase activity (ALP), mineral nodule formation, gene expression of collagen type I and ALP, and cell morphology were evaluated. LLLT at 0.5 J/cm(2) increased cell viability of cultured osteoblasts. ALP activity and gene expression, in addition to mineral nodule formation and Col-I gene expression, were not increased by LLLT. LLLT applied to ZA-treated cells increased Col-I expression at 0.5, 1.5, and 3 J/cm(2) but did not improve any other cell activity assessed. LLLT showed limited effects on bisphosphonate-treated osteoblasts.

An evaluation of the delayed effect of intra-articular injections of lidocaine (2 %) on articular cartilage: an experimental study in rabbits

Lidocaine is commonly injected into the joints as an analgesic. The aim of the present study was to evaluate the delayed effect of intra-articular injections of lidocaine (2%) on articular cartilage in rabbit knees. Ten rabbits were divided into two groups, each group containing five animals. Two milliliters of normal saline solution was injected into both knee joints of animals in group one (control group), and 2 ml of lidocaine was injected into both knee joints of animals in group two (case group). After 8 weeks, the articular cartilage of the distal femur was harvested and analyzed through confocal microscopy and real-time polymerase chain reaction to evaluate the viability and function of chondrocytes, respectively. Confocal microscopy showed a significant decrease in the number of live cells caused by lidocaine (P ≤ 0.001). The changes in gene expression of collagen types II (COL II) and aggrecan were significant in group two (P = 0.008 and P = 0.002, respectively). According to the results, the delayed in vivo effect of lidocaine on chondrocyte is to reduce live chondrocytes and change in the gene expression of COL II and aggrecan.

Quantitative proteomic analysis revealed differentially extracellular protein profiles of osteoarthritic and normal mesenchymal stem cells undergoing chondrogenesis

Purpose: Chondrogenesis occurs as a result of mesenchymal stem cells (MSCs) condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage. A major challenge for the osteoarthitic (OA) cartilage reparation by stem cell-based approaches is the understanding of this multi-step process. In this work, we have analyzed the extracellular protein expression profile of human bone marrow MSCs (hBMSCs) of osteoarthritic patients and control donors undergoing chondrogenesis, in order to compare the mechanisms involved in the cartilage extracellular matrix (ECM) remodeling that occurs during the chondrogenic differentiation process. Methods: hBMSCs isolated from 3 OA patients and 3 healthy controls were grown with different isotope variants of lysine and arginine (Arg6, Lys4 for the control population and Arg10, Lys8 for the OA population) during 4-6 weeks, until achievement of full protein labeling. The labeled populations were then subjected to differentiation in 3D cultures (micromasses) supplemented with chondrogenic inducers for 14 days. Proteins in the conditioned media from the two cell populations were combined, separated by 1D-SDS-PAGE and subjected to in-gel trypsin digestion using an automatic digestor. The resulting peptide mixtures were analyzed by nanoLC coupled on-line to an LTQ-Orbitrap XL mass spectrometer and quantified using the MaxQuant software and the Perseus tool. Results: Real-Time PCR assays showed a relevant difference in the gene expression of collagen type II in the normal donors when compared to the OA patients. Moreover, the chondrocyte phenotype was confirmed in both cases by the proteglycan immunostainings such as aggrecan and chondroitin-6-sulfate after 14 days in chondrogenesis. Using the proteomic approach, we compared the extracellular protein profiles of OA and normal hBMSC at the same time of differentiation. Among the 531 proteins, 56 had significantly altered levels. 35 proteins displayed consistently higher levels in the OA samples compared to normal donors. Many of these proteins are cartilage specific proteoglycans such as hyaluronan and proteoglycan link protein 1, aggrecan core protein or lumican as well as some proteins with a well-known role in the pathogenesis of OA like COMP or MMP3. On the other hand, 21 proteins exhibited a significantly reduced abundance in OA patients when compared to controls. Interestingly, we detected several proteins which belong to the tenascin protein family, like tenascin-X, which accelerates collagen fibril formation. We also found WISP2 decreased at day 14, suggesting a lower activity of the Wnt signaling pathway in OA cells. Conclusions: The identification and quantification of these secreted proteins enhance our knowledge on the extracellular regulation of the chondrogenesis and allow the identification of extracellular markers of this process. Moreover, the lower expression of some of them in OA patients (like tenascin-X or WISP2), suggest their putative usefulness for the molecular monitorization of the chondrogenesis in cell therapy-based approaches for cartilage repair.

Effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct

The aim of this study was to determine the mechanism by which the avian infectious bronchitis virus (IBV) affects eggshell formation. Attenuated IBV (aIBV group) or vehicle (control group) was injected into the oviductal magnum lumen of White Leghorn laying hens. The changes in the expression of genes related to eggshell formation (collagen types I and V, and CaBP-D28K), densities of cytotoxic cells (CD8+ and TCR-γδ+ T cells), and gene expression of molecules related to cytotoxic immunoreaction (B-NK, perforin, granzyme, and IL-2) and proinflammatory cytokines (IL-1β, IL-6 and IFN-γ) were examined by quantitative reverse transcriptase polymerase chain reaction or immunohistochemistry in the isthmus and uterus. Gene expression of IL-1β and IL-6receptors in the tubular gland cells of the isthmus and uterus was analyzed by reverse transcriptase polymerase chain reaction. Gene expression of collagen type I, but not collagen type V, in the isthmus and CaBP-D28K in the uterus was decreased in the aIBV group compared with that in the control. The frequencies of CD8+ cells and TCR-γδ+ T cells in the isthmus and uterus were significantly higher in the aIBV group than in the control group. The expression of cytotoxic molecular and proinflammatory cytokines was also higher in the aIBV group than in the control. The expression of IL-6 receptor, but not IL-1β receptor, was identified in the tubular gland cells in the isthmus and uterus. These results suggest that IBV infection causes disorder of eggshell formation by disturbing gene expression of collagen type I in the isthmus and CaBP-D28K in the uterus, probably via the effects of substances from cytotoxic cells and proinflammatory cytokines.