Dimethylmethylene Blue Dye-binding Assay Research Articles

Comparison of urine glycosaminoglycan excretion between children with autism spectrum disorder and typically developed children

BackgroundAbnormalities pertaining to glycosaminoglycan metabolism have been demonstrated in children with autism spectrum disorder. The aim of the present study was to compare urine excretion of sulfated glycosaminoglycans in children with autism spectrum disorder, with neurotypical controls and explore its association with co-occurring symptoms. MethodRandom urine samples were collected from children with autism spectrum disorder (n = 61) between the ages of 2 and 6 years, and age- and sex-matched neurotypical controls. Urine glycosaminoglycan levels were quantified by the dimethylmethylene blue (DMMB) dye-binding assay. ResultsMean glycosaminoglycan/creatinine ratio of children with autism spectrum disorder was 22.279 ± 13.044 mg/mmol while that of neurotypical controls was 19.121 ± 7.319 mg/mmol. Eight patients with autism spectrum disorder (13.11%) exhibited abnormally high glycosaminoglycan excretion. Unstandardized urine glycosaminoglycan levels are significantly higher (p = 0.019) in the autism spectrum disorder group when covariates such as age, urinary creatinine, and height are taken into consideration by ANCOVA. However, the outcome showed a trend towards significance when glycosaminoglycan/creatinine ratio was used in ANCOVA (p = 0.058). In neurotypical subjects, the urine glycosaminoglycan levels appear to decline with age, height, and weight while this trend was not apparent in subjects with autism spectrum disorder. Glycosaminoglycan excretion did not correlate with the presence of co-occurring symptoms of autism spectrum disorder; frequent gastrointestinal symptoms, self-injurious behaviors, food aversions, or parent-reported sleep problems. ConclusionsA subset of children with autism spectrum disorder exhibits higher urine glycosaminoglycan excretion. Further research is needed to explore the molecular basis of this finding.

Decellularized biological scaffold and stem cells from autologous human adipose tissue for cartilage tissue engineering.

Here, the in vitro engineering of a cartilage-like tissue by using decellularized extracellular matrix scaffold (hECM) seeded with human adipose stem cells (hASCs) which can both be isolated from the human waste adipose tissue is described. Cell-free, highly fibrous and porous hECM was produced using a protocol containing physical (homogenization, centrifugation, molding) and chemical (crosslinking) treatments, characterized by SEM, histochemistry, immunohistochemistry and in vitro cell interaction study. A construct of hECM seeded with hASCs was cultured in chondrogenic medium (with TGF-β3 and BMP-6) for 42 days. SEM and histology showed that the biological scaffold was highly porous and had a compact structure suitable for handling and subsequent cell culture stages. Cells successfully integrated into the scaffold and had good cellular viability and continuity to proliferate. Constructs showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins, Collagen type II and Aggrecan. Dimethylmethylene blue dye binding assay demonstrated that the GAG content of the constructs was in tendency to increase with time confirming chondrogenic differentiation of hASCs. The results support that human waste adipose tissue is an important source for decellularized hECM as well as stem cells, and adipose hECM scaffold provides a suitable environment for chondrogenic differentiation of hASCs.

Link N as a Therapeutic Agent for Early Intervertebral Disc Degenertion

Purpose Although the disc has limited endogenous repair activity, induced repair of disc tissue may be possible by the intradiscal injection of growth factors to stimulate the production of disc matrix. We previously demonstrated that Link N (DHLSDNYTLDHDRAIH), a naturally occurring peptide generated by the N-terminal proteolytic fragmentation of link protein during tissue turnover, can act as a growth factor in the disc. It can stimulate matrix production in vitro, in vivo and in intact ex vivo human intervertebral discs (IVDs). We have recently discovered that AF cells have the ability to proteolytically process Link N resulting in a fragment spanning amino acid residues one-eight (US Patent # 61870394) – short Link N (sLink N). Our in vitro data indicates that the biologically active sequence is preserved within this fragment and, thus, sLink N could represent a potential stable growth factor able to stimulate disc repair. Separately, we developed a long-term organ culture model with vertebral bone. The purpose of the present study was to evaluate the effect of sLink N and compare its efficacy to Link N in this novel organ culture model of early disc degeneration. Method Caudal IVDs from the tails of 20–24 months old steers were isolated with adjacent vertebral bone. After seven days of preconditioning in culture, degeneration was induced in IVDs by a single injection of 50 μg trypsin into the NP. Seven days after induced-degeneration, the trypsin-treated discs were injected with either sLink N or Link N (100 μg/disc, n = six discs/group). Four of the trypsin-treated degenerate discs were injected with PBS alone to serve as a control for degeneration while four discs served as non-degeneration controls. At two, four and eight weeks post treatment, two discs from each treatment and control groups were processed for biochemical analyses. Proteoglycan (predominantly aggrecan) synthesis in the NP was monitored as sulfated glycosaminoglycan using dimethylmethylene blue dye-binding assay, and Western blotting was performed to determine the expression of aggrecan and type two collagen in the tissue. Results Without intervention, at all time points, the GAG content in degenerate discs dropped to ~50% of that in non-degenerate controls. In contrast, sLink N, significantly increased the GAG and collagen content of the discs compared with that in degeneration control discs. Conclusion The results revealed that sLink N has the ability to restore tissue content in an early state of disc degeneration. These results have implications in relation to using either sLink N in regenerating a functional NP in the degenerated IVD or in retarding disc degeneration. Separately, we have developed and validated a novel long-term IVD organ culture model that retains vertebral bone and easy to prepare. Our model is ideal for testing potential drugs and alternate-based therapies, in addition to investigating the long-term effects of loading paradigms on disc degeneration and repair. Disclosures: None

Parthenolide inhibits pro-inflammatory cytokine production and exhibits protective effects on progression of collagen-induced arthritis in a rat model

Objectives: Progressive destruction of synovial joint cartilage and bone occurs in pathological conditions such as rheumatoid arthritis (RA) because of the overproduction of pro-inflammatory cytokines and activation of nuclear factor kappa B (NF-κB). Through the screening of NF-κB inhibitors by a luciferase reporter gene assay, we identified parthenolide (PAR) as the most potent NF-κB inhibitor, among several PAR analogue compounds. This study was undertaken to determine whether PAR inhibits pro-inflammatory cytokine production, cartilage degradation, and inflammatory arthritis.Method: The mRNA levels of pro-inflammatory cytokines were examined by real-time polymerase chain reaction (PCR). Proteoglycan content and release were determined by measuring glycosaminoglycan (GAG) levels using the dimethylmethylene blue (DMMB) dye-binding assay. The potential role of PAR in treatment of arthritis was studied using a collagen-induced arthritis (CIA) model.Results: We established that PAR, as a prototype compound, suppressed lipopolysaccharide (LPS)- and tumour necrosis factor (TNF)-α-induced increases in matrix metalloproteinase (MMP)-1, MMP-3, inducible nitric oxide synthase (iNOS), and interleukin (IL)-1β mRNA in chondrocytes. In addition, PAR prevented proteoglycan degradation triggered by pro-inflammatory cytokines. PAR treatment at the onset of CIA symptoms significantly reduced synovitis, inflammation, and pannus formation scores. Reduced synovial inflammation after PAR treatment was also reflected in significantly less bone erosion and cartilage damage.Conclusions: These data indicate a protective effect of PAR on the catabolic insults of pro-inflammatory cytokines on chondrocyte metabolism and GAG release in vitro and in CIA. PAR had anti-inflammatory and structure-modifying effects on experimental arthritis, suggesting that PAR may be useful as a potential alternative or adjunct therapy for inflammatory arthritis.

Link N and Mesenchymal Stem Cells Can Induce Regeneration of the Early Degenerate Intervertebral Disc

Link N is a naturally occurring peptide that can stimulate proteoglycan synthesis in intervertebral disc (IVD) cells. IVD repair can also potentially be enhanced by mesenchymal stem cell (MSC) supplementation to maximize extracellular matrix (ECM) production. In a previous study, we have shown that Link N can inhibit osteogenesis and increase the chondrogenesis of MSCs in vitro. The aim of the present study was to determine the potential of MSCs and Link N alone or in combination with regard to tissue repair in the degenerate disc. Bovine IVDs with trypsin-induced degeneration were treated with MSCs, Link N, or a combination of MSCs and Link N. Trypsin-treated discs were also injected with phosphate-buffered saline to serve as a degeneration control. The ECM proteins and proteoglycans were extracted from the inner nucleus pulposus (NP) of the discs, and sulfated glycosaminoglycans (GAGs) were analyzed by the dimethyl methylene blue dye-binding assay. The expression of type II collagen was analyzed by western blot. To track the MSCs after injection, MSCs were labeled with PKH67 and observed under confocal microscopy after the 2 week culture period. The GAG content significantly increased compared with the degeneration control when degenerate discs were treated with MSCs, Link N, or a combination of both Link N and MSCs. Histological analysis revealed that the newly synthesized proteoglycan was able to diffuse throughout the ECM and restore tissue content even in areas remote from the cells. The quantity of extractable type II collagen was also increased when the degenerate discs were treated with MSCs and Link N, either alone or together. MSCs survived, integrated in the tissue, and were found distributed throughout the NP after the 2 week culture period. MSCs and Link N can restore GAG content in degenerate discs, when administered separately or together. Treatment with MSCs and Link N can also increase the expression of type II collagen. The results support the concept that biological repair of disc degeneration is feasible, and that the administration of either MSCs or Link N has therapeutic potential in early stages of the disease.

The Effects of Link N and Bone Marrow-Derived Mesenchymal Stem Cells on the Regeneration of Intervertebral Disc

Introduction Link N is a naturally occurring peptide that can stimulate regeneration of the intervertebral disc (IVD). IVD repair can also potentially be enhanced by mesenchymal stem cell (MSC) supplementation to maximize extracellular matrix production. In a previous study, we have shown that Link N can inhibit osteogenesis and increase the chondrogenesis of MSCs in vitro. The aim of the present study is to determine the effects of injecting MSCs and Link N alone or together on the repair of degenerated discs. Materials and Methods The largest first four caudal discs with intact cartilaginous endplates were isolated from the tails of 24- to 30-month-old bovines. After a 3-day preconditioning period, the discs were injected with 100 μg trypsin in 75 μL PBS, to induce degeneration. They were then cultured for another 4 days, followed by the injection of MSCs (105), Link N (75 μg), or a combination of MSCs and Link N, in a final volume of 75 μL PBS. Seven of the trypsin-treated discs were injected with PBS alone to serve as a degeneration control. After the discs were cultured for another 14 days, the extracellular matrix (ECM) proteins and proteoglycans were extracted from the inner nucleus pulposus of the discs by guanidinium chloride. Sulfated glycosaminoglycans (GAGs) were analyzed in tissue extracts ( n = 7/group) by the dimethyl methylene blue (DMMB) dye-binding assay. The expression of type II collagen was analyzed by Western blot. To track the MSCs after injection, MSCs were labeled with PKH67 and observed under confocal microscopy when the discs were dissected. Results After injection, MSCs were distributed throughout the discs and retained their morphology. When the trypsin-treated discs were injected with PBS only, the final GAG concentration in the ECM was 15.12 µg/mg wet weight tissue. When the degenerated discs were injected with MSCs only, the GAG concentration increased to 25.66 µg/mg wet weight tissue. When the degenerated discs were injected with Link N alone or both Link N and MSCs, the GAG concentrations increased to 29.63 and 32.66 µg/mg wet weight tissue, respectively. Compared with the degeneration control, the expression of type II collagen also increased when the degenerated discs were injected with MSCs and Link N, either alone or together. Conclusion MSCs and Link N can both increase GAG production in degenerated discs, with Link N showing a greater effect than MSCs when administered separately. However, an additive effect was found when MSCs and Link N were administered together. The injection of MSCs and Link N can also increase the expression of type II collagen in the degenerated discs. Thus, the administration of MSCs and Link N has therapeutic potential in intervertebral disc repair. Disclosure of Interest None declared

HIF-1α regulates configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors

Purpose: Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in hypoxic conditions for cell survival in various tissues. In chondrocytes, previous studies showed that HIF-1α was necessary for chondrocyte survival in endochondral ossification and for interzone formation in joint development; however, the underlying molecular mechanisms remain unclear. Moreover, physiological and pathological functions of HIF-1α in articular cartilage are yet to be revealed. Here we have investigated HIF-1α functions and the underlying mechanisms of the configuration and maintenance of articular cartilage by HIF-1α. Methods: We generated tissue-specific knockout mice of HIF-1α by mating Sox9-Cre knock-in mice (Sox9-Cre) or tamoxifen-inducible Col2a1-Cre transgenic mice (Col2a1-CreERT2), with mice homozygous for a floxed Hif1a allele (Hif1afl/fl). For analyses of cartilage configuration, we examined Sox9-Cre;Hif1afl/fl mice at E15.5, E18.5 and P1. To analyze articular cartilage maintenance and osteoarthritis (OA) progression after maturation, we injected tamoxifen into 7-week-old Col2a1-CreERT2;Hif1afl/fl mice, and created a surgical OA model by inducing instability in the knee joints one week later. OA severity was quantified by the OARSI histopathology grade 8 weeks after surgery. For histological analyses of these knockout mice, we performed Safranin O staining, TUNEL staining, and immunofluorescence. Quantitative gene expression in limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/fl neonates was determined based on real-time RT-PCR analyses using samples obtained directly from their limb cartilages. To assess the catabolic ability of HIF-1α, we measured the concentration of aggrecan released from the 3-week-old mouse femoral heads cultured with or without CoCl2, a potent enhancer of HIF-1α protein stability, utilizing dimethylmethylene blue dye-binding assay. Mouse primary articular chondrocytes were used for further in vitro functional analyses. Results: Sox9-Cre;Hif1afl/fl embryos and neonates showed dwarfism with severe limb shortening throughout the embryonic stages and died in the perinatal period. This phenotype was consistent with previous reports using Prx1-Cre and Col2a1-Cre mice. Notably, the knee joints of Sox9-Cre;Hif1afl/fl embryos showed a widespread epiphysis with a massive cavity continuing from the joint surface forming a trumpet shape, in which the columnar architecture was completely abolished and the cell numbers were vastly reduced(Fig.1). Safranin O staining showed a decrease in proteoglycan content in comparison with the control Hif1afl/fl embryo. TUNEL staining accentuated ectopic apoptosis broadly across the limb cartilage, and immunofluorescence showed enhanced expression of Mmp13 surrounding the defective area. When we created the experimental OA model one week after the tamoxifen injection into Col2a1-CreERT2;Hif1afl/fl mice, the OA development in the knee joints was markedly accelerated as compared to the control Hif1afl/fl joints. Apoptosis and Mmp13 expression were upregulated by the conditional knockout of HIF-1α after maturation in articular cartilage, as well as in the limb cartilage. To reveal altered gene expression by HIF-1α deficiency, we obtained RNA samples directly from the limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/f embryo. Real-time RT-PCR using these samples revealed increases of catabolic factors including Mmp13 and Mmp9, and decreases of anabolic factors including Col2a1 and Sox9 by the conditional knockout of HIF-1α. When we deleted HIF-1α in primary articular chondrocytes from Hif1afl/fl mice by adenoviral vector expressing Cre recombinase, expressions of the catabolic and the anabolic genes were changed in ways similar to those in the in vivo analyses, under both normoxic and hypoxic (1% O2 concentration) conditions. Similar results were also obtained under both O2 conditions by HIF-1α silencing in primary articular chondrocytes from WT mice using siRNA. Furthermore, in the organ culture of mouse femoral heads, stabilizing HIF-1α protein by CoCl2 treatment markedly decreased aggrecan release into the medium. Conclusions: HIF-1α regulates the configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors. Elucidation of the molecular network related to HIF-1α may lead to cartilage regeneration and OA treatment.

Identification of fibroblast growth factor-18 as a molecule to protect and regenerate articular cartilage

Purpose: Aiming at the disease-modifying treatment of osteoarthritis (OA), we sought to identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions by gene expression profile analyses. The mechanisms of the identified molecule underlying the protection and regeneration were further investigated. Methods: To identify genes that have both protective and regenerative effects on adult articular cartilage, we performed two sets of microarray analyses. First, to select genes that work for maintenance of articular cartilage, we compared gene expression profiles between adult articular (AA) and adult growth plate (AG) cartilages in 10-week-old rats. Second, to find genes that function for regeneration of articular cartilage, we compared the profiles between infant superficial (IS) and infant deep (ID) layers of epiphyseal cartilage in 6-day-old rats. For genes which were up-regulated ≥10-fold both in AA than in AG and in IS than in ID, we performed real-time RT-PCR for the confirmation. In vivo expression was examined by immunohistochemistry of articular and growth plate cartilage of 14-week-old rats. The therapeutic effect of intra-articular injection of sprifermin (recombinant human fibroblast growth factor 18 (FGF18)) was examined in the experimental OA model by surgical induction of instability in the knee joints of adult rats. To further learn the underlying mechanism, the protective ability of articular cartilage was assessed by measuring the amount of sulfated glycosaminoglycan (sGAG) released into the medium in the ex vivo culture of bilateral femoral heads of 3-week-old mice using dimethylmethylene blue dye-binding assay. Proliferation and migration were analyzed in the cultures of mouse articular chondrocytes using Cell Counting Kit-8 and Oris Cell Migration assay systems, respectively. Expression levels of catabolism-related factors (Mmp9, Mmp13, Adamts4, Adamts5, Timp1, Timp2, and Timp3) and anabolism-related factors (Col2a1 and aggrecan) in the cultures of mouse femoral heads and mouse articular chondrocytes were analyzed by real-time RT-PCR. Results: Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The ratios of the 16 genes were confirmed to be ≥10 fold by real-time RT-PCR analysis. Among them three genes were expressed more strongly in AA than in IS. In these three genes, FGF18 was the extracellular and secreted factor of which the AA/AG ratio was the highest in the microarray analysis. Immunohistochemistry showed that FGF18 was strongly expressed in the articular cartilage chondrocytes of adult rats but was hardly detected in the growth plate cartilage. In the rat surgical OA model, a once-weekly injection of sprifermin given 3 weeks post-surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with a significant effect at 10 μg/week of sprifermin. As an underlying mechanism, sprifermin suppressed the sGAG release into the culture medium in the ex vivo culture of mouse femoral heads. Furthermore, sprifermin accelerated proliferation and migration of cultured mouse articular chondrocytes. Among catabolic and anabolic factors, sprifermin decreased Adamts4 and increased Timp1 expressions in the cultures of mouse femoral heads and murine articular chondrocytes; however it decreased Col2a1 and aggrecan expressions in both cultures. Conclusions: The present gene expression profiling analysis identified sprifermin as a molecule to protect and regenerate adult articular cartilage, causing prevention of cartilage degeneration by the once-weekly intra-articular injection in a rat model. This effect may be due to inhibition of cartilage catabolism, and acceleration of proliferation and migration of articular chondrocytes, providing a disease-modifying OA treatment.

Biomarkers in dogs surgically treated for ruptured cranial cruciate ligaments

Thirty-one dogs with cranial cruciate ligament rupture (CCLR) were randomly treated with tibial plateau levelling osteotomy (TPLO; TPLO group) or proximal tibial osteotomy (PTO; PTO group). Synovial fluid was collected...

Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro

The meniscus has limited ability to repair itself following injury. However, tissue engineering provides new means of meniscus repair. Myoblasts, which possess the potential of multi-directional differentiation, may be ideal seed cells in meniscus tissue engineering. Myoblasts from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated myoblasts from canines and induced chondrogenesis in order to establish a new experimental model of seed cells. Myoblasts were isolated and harvested from Beagle canines. To induce chondrogenesis, cartilage-derived morphogenetic protein-2 (CDMP-2) at different concentrations (10, 20, 50 and 100 ng/ml), transforming growth factor-β1 (TGF-β1; 10, 20, 30 and 50 ng/ml), and different concentrations of CDMP-2 (10, 20, 50 and 100 ng/ml) together with TGF-β1 (20 ng/ml) were added to the cultured pellets. After 21 days of in vitro culture, chondrogenic differentiation was evaluated by histological and immunohistochemical techniques. The degree of gene expression was measured by quantitative RT-PCR. Based on the histological staining of glycosaminoglycan, using the toluidine blue dye-binding method, we found that CDMP-2 initiated chondrogenic differentiation of myoblasts, as did TGF-β1. Furthermore, CDMP-2 conferred a stronger stimulatory effect than TGF-β1. The combination of CDMP-2 and TGF-β1 synergistically induced chondrogenesis of myoblasts. This synergistic chondrogenic effect of CDMP-2 together with TGF-β1 was further confirmed by quantification of glycosaminoglycan using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific proteins collagen I and II. Canine myoblasts can be induced into chondrocytes by CDMP-2 and TGF-β1 in vitro, suggesting that myoblasts are suitable as seed cells for meniscus tissue engineering.

477 NEW ALGINATE-CHITOSAN HYDROGEL BEADS WITH ANTI-INFLAMMATORY AND ANABOLIC EFFECTS ON HUMAN CHONDROCYTES

Purpose: Proper selection of cell source is a persisting problem for cartilage tissue engineering because it is difficult to attain sufficient number of chondrocytes from sparsely cellular cartilage tissue. Chondrocytes can be increased in number by cell culture however they readily dedifferentiate towards fibroblast like and lose their chondrocytic phenotype. Previously we have shown that co-culture of these cultured passaged dedifferentiated chondrocytes with small numbers of primary chondrocytes induces redifferentiation in dedifferentiated cells. Here we show that the age affects ability of primary chondrocytes to induce redifferentiation in passaged cells. Methods: Cell preparation: Human fetal (17 weeks), human adult articular (18–60 yrs.) and bovine articular cartilage (young 6–9 months, old 24–36 months) were harvested and chondrocytes were isolated by sequential enzymatic digestion. Co-culture: the passaged dedifferentiated cells were mixed with primary chondrocytes at ratio of 4:1 and cocultured on MillicellTM filter inserts which allow for 3D culture in 20% FBS containing DMEM-HG. Analysis: After 4 weeks the newly formed tissue was characterized and compared with bovine and human primary chondrocyte cultures. Histological qualitative assessment was done by Toluidine Blue staining. Proteoglycan accumulation was quantitatively determined by dimethylmethylene blue dye-binding assay. Values were normalized to total DNA content determined by Hoechst 33258 dye binding assay. Gene expression was quantified by qPCR. Statistics: Results were expressed as mean ± SD of 3 independent experiments and significance was determined by one-way ANOVA. Results: To determine the effect of age on the ability of chondrocytes to accumulate proteoglycans and induce redifferentiation chondrocytes from older and younger cows were compared with each other after 4 weeks of in vitro culture (Fig. 1). After 4 weeks when visualized under microscope after Toluidine blue staining more young cells appeared to be alive and surrounded by newly deposited cartilaginous matrix. Dimethylmethylene blue assay showed that the proteoglycan content of tissue formed by co-cultured of young primary chondrocytes and old passaged cells at 4 weeks was significantly higher than tissue formed by the co-culture with old primary chondrocytes. qPCR showed higher gene expression of type II collagen, aggrecan, and SOX9. passged cells alone failed to produce cartilaginous matrix regardless of age or species difference. Conclusion: We conclude that young primary chondrocytes produce factors which affect the cartilaginous matrix deposition capabilities of dedifferentiated cells. Further studies on human fetal chondrocytes are in progress. Acknowledgements: Supported by CIHR.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

075 OSTEOARTHRITIS PATIENTS' PERCEPTIONS REGARDING APPROPRIATENESS FOR TOTAL JOINT REPLACEMENT SURGERY

stem cell line (GFP-hES2) was cultured in DMEM/F-12 containing 20% serum replacement, 10% mouse embryonic fibroblast conditioned media and β-Mercaptoethanol on gelatin-coated plates seeded with irradiated mouse feeder cells. Chondrogenic differentiation: We developed a novel two step protocol for chondrogenic differentiation of GFP-hES2. 1. Mesoderm differentiation: Embryoid bodies were developed in lo-cluster tissue culture plates in the presence of BMP4, bFGF, and Act-A. 2. Chondrogenesis: The second step was co-culture of the mesodermal cells with primary bovine chondrocytes (bP0). bP0 and GFP-hES2 were mixed at a ratio of 1:4 and co-cultured on MillicellTM membrane inserts, which allow for 3D growth, in DMEM supplemented with 20%FBS. After 4 weeks the newly formed tissue was characterized and compared with tissue formed by bP0 and hES2 alone. Analysis: FACS analysis of the GFP-hES2 cells was carried out using selected antibodies. Histological qualitative assessment of the newly formed tissue was done by using light microscopy and toluidine blue staining. Proteoglycan accumulation was semi-quantitatively determined by dimethylmethylene blue dye-binding assay. Values were normalized to total DNA content determined by Hoechst 33258 dye binding assay. Gene expression was quantified by qPCR. Statistics: Results were expressed as mean ± SD of 3 independent experiments and significance determined by one-way ANOVA. Results: FACS analysis after the first stage of culture at day 5 showed that only 5-7% of cells remained positive for ckit, an embryonic stem cell marker, indicating a shift towards more differentiated state. Furthermore, 50-60% of the cells were KDR and PDGF double positive, indicating that these cells attained mesodermal features. At this stage cells were co-cultured with bP0. After 4 weeks GFP-hES2 appeared to be present and surrounded by newly deposited cartilaginous matrix when visualized under fluorescent microscopy. Toluidine blue staining and dimethylmethylene blue assay showed that the proteoglycan content of tissue formed by co-cultured embryonic stem cells at 4 weeks was significantly higher than tissue formed by the bovine chondrocytes alone. qPCR showed higher gene expression of type II collagen, aggrecan, and SOX9. GFP-hES2 cultured alone failed to accumulate any cartilaginous matrix. Conclusions: We conclude that bovine primary chondrocytes produce factors which promote chondrogenic differentiation of partially differentiated embryonic stem cells. Understanding the mechanism regulating this differentiation may aid in developing a source of cells suitable to use for cartilage repair. Acknowledgement: Supported by CIHR.

Membrane-Based Cultures Generate Scaffold-Free Neocartilage In Vitro: Influence of Growth Factors

Scaffold-free cultures provide promising potential in chondrogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a novel scaffold-free membrane-based culture system, in which hMSCs were cultivated on a cellulose acetate membrane filter at medium-gas interface, was evaluated for chondrogenesis under the addition of growth factors. Chondrogenic differentiation of hMSCs has been described in scaffold-free pellet cultures with good results. In our study membrane-based cultures (1 x 10(6) hMSCs) were produced, maintained at the medium-gas interface and cultured for 21 days. Results were compared with findings from standard pellet cultures (2.5 x 10(5) hMSCs). The effects of the following growth factors were examined: human transforming growth factor-beta(3) (TGF-beta(3)) +/- insulin-like growth factor-1 or +/- human fibroblast growth factor 2. After 3 weeks of culture, chondrogenesis was assessed by Safranin-O staining, immunohistochemistry, a dimethylmethylene blue dye binding assay for glycosaminoglycans, and quantitative real-time polymerase chain reaction for cartilage-specific proteins. Membrane-based cultures containing growth factors formed hemispherical structures with a large surface area (65 mm(2)). When removed from the membrane they showed a histologically smooth cartilage-like surface. Membrane-based cultures stained positive for Safranin-O and collagen type II and contained a high content of glycosaminoglycans. Expression of cartilage-specific markers like collagen type II, aggrecan, and SOX9 was observed under the addition of TGF-beta(3), whereas combinations of growth factors let to a significant increase of collagen type II expression. A markedly reduced expression of collagen type X was found in membrane-based cultures when only TGF-beta(3) was added. Pellet cultures showed similar results besides an increased expression of collagen type X and type II that were observed. Membrane-based cultures provide a differentiation system, comparable in chondrogenesis to pellet cultures, which is able to generate scaffold-free neocartilage. The key benefit factors of membrane-based cultures are a histologically smooth cartilage-like surface and reduced expression of collagen type X, both of which are suitable features for its future application in cartilage regeneration.

Osteogenic protein-1 with transforming growth factor-β1: potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro

BackgroundRecently, cells derived from synovial mesenchymal stem cells (MSCs) have been regarded as a potential source of cells to induce repair of articular cartilage. To investigate more effective methods for promoting chondrogenesis, we examined the effects of osteogenic protein (OP)-1 with or without transforming growth factor-β (TGFβ1) on chondrogenesis of human MSCs in vitro. MethodsMSCs were isolated from the synovial membrane of patients with rheumatoid arthritis undergoing knee replacement surgery. After expansion of the cells, pellet cultures were performed in chondrogenic medium with OP-1 100–200 ng/ml, TGFβ1 10 ng/ml, or both agents for 3 or 6 weeks. Chondro-genesis was evaluated histologically with safranin O staining, reverse transcription polymerase chain reaction for aggrecan and type II collagen mRNA, and quantification of glycosaminoglycan (GAG) content using a dimethylmethylene blue dye-binding assay. GAG content was normalized by DNA content measured using Hoechst 33258 dye. ResultsAt 3 weeks of culture, mRNAs for type II collagen and aggrecan were expressed by MSCs treated with either TGFβ1 or OP-1; however, substantial matrix production was not induced. At 6 weeks, OP-1 increased GAG accumulation dose-dependently in the presence or absence of TGFβ1, and the GAG content was the highest after combined treatment with 200 ng OP-1 and TGFβ1. Histological staining for safranin O was poor after treatment with OP-1 or TGFβ1 alone and slightly increased after combined treatment with TGFβ1 and OP-1 at 3 weeks. At 6 weeks, OP-1 increased the intensity of staining dose-dependently in the presence or absence of TGFβ1. However, the histological appearance of the cells treated with OP-1 alone was similar to that of hypertrophic chondrocytes, which was different from that of cells with combined treatment with OP-1 and TGFβ1. ConclusionsA high dose of OP-1 was useful for enhancing chondrogenesis from synovium-derived MSCs in combined treatment with TGFβ1.

Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis

To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system. 6 healthy horses, 6 months to 5 years of age. Bone marrow-derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm(2) and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye-binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs. Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification. FGF-2 treatment of bone marrow-derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair.

Effects of Insulin-Like Growth Factor I on Transforming Growth Factor β1 Induced Chondrogenesis of Synovium-Derived Mesenchymal Stem Cells Cultured in a Polyglycolic Acid Scaffold

The aim of this study was to demonstrate the induction of chondrogenesis by transforming growth factor (TGF)-β₁ from synovium-derived mesenchymal stem cells in a three-dimensional polyglycolic acid (PGA) scaffold, and to evaluate the effects of insulin-like growth factor (IGF)-I on TGF-β₁-induced chondrogenesis. Adult human synovial membranes were obtained from the knees of patients with osteoarthritis or rheumatoid arthritis. Cells were expanded in monolayers, seeded onto a PGA scaffold, and cultured for 4 or 8 weeks in chondrogenic medium containing TGF-β₁ with or without IGF-I. As a control, the cells were cultured in chondrogenic medium without TGF-β₁. The glycosaminoglycan content was quantified using dimethylmethylene blue dye-binding assay, and the DNA content was measured fluorometrically. Histological examination was also performed using safranin-O staining. The expression of mRNA for aggrecan and collagen type II was confirmed by RT-PCR. After 4 weeks of cultivation with TGF-β₁, the cells differentiated to a chondrocytic phenotype, and these chondrogeneses were more potent when cultured for 8 weeks. The combination of IGF-I and TGF-β₁ produced higher amounts of glycosaminoglycan than TGF-β₁ alone at 8 weeks. In conclusions, chondrogenesis from human synovium-derived mesenchymal cells was identified, and IGF-I plays a role in maintaining the extracellular matrix in combination with TGF-β₁.

Proinflammatory Cytokine Activities, Matrix Metalloproteinase‐3 Activity, and Sulfated Glycosaminoglycan Content in Synovial Fluid of Dogs with Naturally Acquired Cranial Cruciate Ligament Rupture

To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. Prospective clinical study. Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.

Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes

Mesenchymal stem cells (MSCs) are able to differentiate into many types of cells including chondrocytes. Transforming growth factor β1 (TGF-β1) is very important in the regulation of chondrogenesis. Since cartilage-derived morphogenetic protein-1 (CDMP-1) belongs to the TGF-β superfamily, we tested whether CDMP-1 plays any role in the regulation of the differentiation of MSCs into chondrocytes using a high density pellet culture system. Based on the histological staining of glycosaminoglycan using toluidine blue dye-binding method we found that CDMP-1 could initiate chondrogenic differentiation of MSCs as did TGF-β1. However, CDMP-1 was less stimulatory than TGF-β1. The combination of CDMP-1 and TGF-β1 synergically induced chondrogenesis of MSCs. This synergic chondrogenic effect of CDMP-1 together with TGF-β1 was further confirmed by quantification of GAG using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific protein collagen II. This study may provide an improved induction approach using MSCs for repairing damaged cartilage.

Effects of growth factors on temporomandibular joint disc cells

The effects of growth factors on cartilaginous tissues are well documented. An exception is the temporomandibular joint (TMJ) disc, where data for growth factor effects on proliferation and biosynthesis are very limited. The purpose of this study was to quantify proliferation of and synthesis by TMJ disc cells cultured in monolayer with either platelet derived growth factor-AB (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-I (IGF), at either a low (10 ng/ml) or high (100 ng/ml) concentration. Proliferation was assessed with a DNA quantitation technique, collagen synthesis was measured via a hydroxyproline assay, and GAG synthesis was determined with a dimethylmethylene blue dye binding assay at 14 days. Overall, the most beneficial growth factor was bFGF, which was most potent in increasing proliferation and GAG synthesis, and also effective in promoting collagen synthesis. At the high concentration, bFGF resulted in 96% more cells than the control and 30 to 45% more cells than PDGF and IGF. PDGF and bFGF were the most potent upregulators of GAG synthesis, producing 2–3 times more GAG than the control. IGF had no significant effect on GAG production, although at its higher concentration it increased collagen production by 4.5 times over the control. Collagen synthesis was promoted by bFGF at its lower concentration, with levels 4.2 times higher than the control, whereas PDGF had no significant effect on collagen production. In general, higher concentrations increased proliferation, whereas lower concentrations favoured biosynthesis.