Increasing Collagen to Bioink Drives Mesenchymal Stromal Cells-Chondrogenesis from Hyaline to Calcified Layers.

The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.

Behaviour of human dental pulp stem cell in high glucose condition: impact on proliferation and osteogenic differentiation.

OBJECTIVE: The aim of this study is to investigate the changes of human dental pulp stem cell (hDPSC) viability, proliferation and osteogenic differentiation in high glucose condition. DESIGN: After 21 days of culture in low (5.5 mM) and high (20 mM) glucose medium, hDPSC viability and proliferation were assessed with respectively the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst assays. To investigate the influence of glucose on osteogenic differentiation hDPSCs were cultured for 28 days in low or high glucose medium with osteoinductive cocktail. Mineralization was examined by alizarin red staining/quantification and the expression of osteogenic-related genes [Runt-related transcription factor 2 (RUNX2), Osteocalcin (OCN), Collagen 1A1 (COL1A1)] analyzed by RT-qPCR. RESULTS: We observed no significant difference (p >  0.05) on hDPSC proliferation or cell viability between low or high glucose groups. We did not highlight a significant difference after alizarin red staining and quantification between hDPSCs cultured with high or low glucose concentration in the culture medium. In the same manner, high glucose concentration did not appear to modify osteogenic gene expression: there was no significant difference in osteogenic-related gene expression between high or low glucose groups. CONCLUSION: Proliferation, viability, and osteogenic differentiation of hDPSCs were not changed by high glucose environment.

Respective stemness and chondrogenic potential of mesenchymal stem cells isolated from human bone marrow, synovial membrane, and synovial fluid.

BACKGROUND: MSCs isolated from bone marrow (BM-MSCs) have well-established chondrogenic potential, but MSCs derived from the synovial membrane (SM-MSCs) and synovial fluid (SF-MSCs) are thought to possess superior chondrogenicity. This study aimed to compare the in vitro immunophenotype and trilineage and chondrogenic potential of BM-MSCs to SM-MSCs and SF-MSCs. METHODS: MSCs were isolated from bone marrow (BM-MSCs), synovial membrane (SM-MSCs), and synovial fluid (SF-MSCs) extracted from the hips (BM) and knees (SM and SF) of advanced OA patients undergoing arthroplasty. Flow cytometric analysis was used at P2 to evaluate cell stemness. The trilinear differentiation test was performed at P2. At P3, MSC-seeded collagen sponges were cultured in chondrogenic medium for 28 days. Chondrogenic gene expression was quantified by qRT-PCR. Finally, the implants were stained to assess the deposition of proteoglycans and type II collagen. RESULTS: Despite variability, the immunophenotyping of BM-MSCs, SM-MSCs, and SF-MSCs was quite similar. All cell types were positive for the expression of stem cell markers and negative for exclusion markers. Additionally, chondrogenic differentiation and hypertrophy were more pronounced in BM-MSCs (ACAN, SOX9, COL2B, and COL10A) than in SF-MSCs, with SM-MSCs having intermediate characteristics. Concerning matrix synthesis, the three cell types were equipotent in terms of GAG content, while BM-MSC ECM synthesis of type II collagen was superior. CONCLUSIONS: Chondrogenic MSCs are easily collected from SM and SF in advanced human OA, but in vitro chondrogenesis that is superior to age-matched BM-MSCs should not be expected. However, due to intra-articular priming, SF-MSCs did not overexpress hypertrophic gene.

PLGA-Based Nanoparticles: a Safe and Suitable Delivery Platform for Osteoarticular Pathologies.

PURPOSE: Despite the promising applications of PLGA based particles, studies examining the fate and consequences of these particles after intra-articular administration in the joint are scanty. This study was carried out to evaluate the neutrality of the unloaded delivery system on different articular cell types. To facilitate tracking, we have thus developed a fluorescent core of particles, combined to a hyaluronate shell for cell recognition. METHODS: Fluorescence pictures were taken at time intervals to assess the internalization and the corresponding inflammatory response was monitored by RT-qPCR and biochemical measurements. After NPs pre-treatment, mesenchymal stem cells (MSCs) were cultured into chondrogenic, adipogenic or osteogenic differentiation media, to investigate if NPs exposure interferes with differentiation ability. Finally, intra-articular injections were performed in healthy rat knees and joint's structure analysed by histological studies. RESULTS: Particles were detected in cytoplasm 8 h after exposure. Internalization led to a slight and reversible increase of inflammatory markers, but lower than in inflammatory conditions. We have confirmed particles exposure minimal neutrality on MSCs pluripotency. Histological exams of joint after intra-articular injections do not demonstrate any side effects of NPs. CONCLUSIONS: Our findings suggest that such a delivery platform is well tolerated locally and could be used to deliver active molecules to the joint.

Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.

Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5-20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOF mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGFß1 and patient-to-patient differential expression. Novel targets of TGFß1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. BIOLOGICAL SIGNIFICANCE: The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFß1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it.

Increasing the bioactivity of elastomeric poly(ε-caprolactone) scaffolds for use in tissue engineering.

BACKGROUND: Biodegradable polymers used in tissue engineering applications, such as poly(ε-caprolactone) (PCL), are hydrophobic leading to a lack of favorable cell signalization and finally to a poor cell adhesion, proliferation and differentiation. To overcome this problem, scaffolds undergo generally a surface modification. OBJECTIVE: Our laboratory has demonstrated that the grafting of poly(sodium styrene sulfonate) (pNaSS) onto titanium or poly(ethylene terephthalate) surfaces, leads to a more specific protein adsorption and a better control of cell proliferation. The objective of this work is to develop, through a straightforward way, bioactive elastomeric PCL scaffolds by grafting pNaSS. METHODS: Porous elastomeric PCL scaffolds were developed using a particulate-leaching process. pNaSS was grafted into the scaffold by a "grafting from" technique. In vitro tests were carried out to assess cell adhesion and protein expression. RESULTS: pNaSS was grafted homogeneously onto PCL scaffolds without degrading the biodegradable polymer or the porous structure. The in vitro studies have shown that pNaSS grafted onto PCL improves the cell response with a better expression of collagen, fibronectin and integrin α1. CONCLUSIONS: The grafting of pNaSS onto biomaterial surfaces is a versatile method that can provide a new generation of biodegradable scaffolds which could be "biointegrable".

Ambivalent properties of hyaluronate and hylan during post-traumatic OA in the rat knee.

AIM: To determine whether viscosupplementation with intra-articular (i.a.) low- or high-molecular-weight hyaluronate (HA) injections influenced both chondral and synovial lesions in rats with surgically-induced OA knee. METHODS: On D0, rats underwent anterior cruciate ligament transection (ACLX) and were divided in 4 groups: sham group, ACLX-saline control group, ACLX-hyaluronate group, ACLX-hylan group. IA injections were performed on D7, D14 and D21. Histological grading of chondral and synovial lesions were performed on D28. Concomitant immunostainings of Caspase3a and Hsp70 were also performed. RESULTS: Articular damages were significantly reduced in both HAs-treated knee joints. In contrast, a significant increase of histological score of synovial inflammation was noted in both ACLX + HAs groups. Apoptotic events significantly decreased as anti-apoptotic Hsp70 expression increased significantly in both HAs groups. CONCLUSION: HAs may exert, independently of its molecular weight, ambivalent properties on articular structures, simultaneously exerting chondroprotective properties and promoting long-term subacute synovitis.

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix.

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5ß1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5ß1 integrin in vitro. TGFß1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.

Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators?

INTRODUCTION: We have taken advantage of the large screening capacity of a multiplex immunoassay to better define the respective contribution of articular versus systemic cytokines in experimental arthritis. METHODS: We performed a follow up (from 7 hours to 14 days) multiplex analysis of 24 cytokines in synovial fluid and sera of rats developing Antigen-Induced Arthritis (AIA) and confronted their protein level changes with molecular, biochemical, histological and clinical events occurring in the course of the disease. RESULTS: The time-scheduled findings in arthritic joints correlated with time-dependent changes of cytokine amounts in joint effusions but not with their blood levels. From seven hours after sensitization, high levels of chemokines (MCP-1, MIP1α, GRO/KC, RANTES, eotaxin) were found in synovial fluid of arthritic knees whereas perivascular infiltration occurred in the synovium; local release of inflammatory cytokines (IFNγ, IL-1ß, IL-6) preceded the spreading of inflammation and resulted in progressive degradation of cartilage and bone. Finally a local overexpression of several cytokines/adipocytokines poorly described in arthritis (IL-13, IL-18, leptin) was observed. CONCLUSIONS: Distinct panels of cytokines were found in arthritic fluid during AIA, and the expected effect of mediators correlated well with changes occurring in joint tissues. Moreover, multiplex analysis could be helpful to identify new pathogenic mediators and to elucidate the mechanisms supporting the efficacy of putative targeted therapies.

Alternative for anti-TNF antibodies for arthritis treatment.

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-α antibodies has shown its efficacy in rheumatoid arthritis (RA) and is now widely used. Nevertheless, some patients currently treated with anti-TNF-α remain refractory or become nonresponder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. In this study, we investigated in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α triplex-forming oligonucleotide (TFO), as judged from effects on two rat arthritis models. The inhibitory activity of this TFO on articular cells (synoviocytes and chondrocytes) was verified and compared to that of small interfering RNA (siRNA) in vitro. The use of the anti-TNF-α TFO as a preventive and local treatment in both acute and chronic arthritis models significantly reduced disease development. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-α TFO modulates arthritis in vivo, thus providing proof-of-concept that it could be used as therapeutic tool for TNF-α-dependent inflammatory disorders.

Evaluation of a rat knee mono-arthritis using microPET.

AIM: Assessing the activity of synovitis, which is characterized by an increase in cell metabolism, is important for the prediction of future articular destruction in clinical and preclinical studies. To evaluate the correlation between ¹8F-FDG accumulation and arthritis pathology during its establishment, we used microPET to evaluted ¹8F-FDG accumulation in vivo during rat Mycobacterium wall-induced knee arthritis. METHODS: ¹8F-FDG PET images of arthritic rats were acquired on days 1, 2, 3 and 7 after arthritis induction. The subjects (n=2/time) were subsequently subjected to macro-autoradiography, and ¹8F-FDG accumulation was compared with histological findings. RESULTS: ¹8F-FDG PET images depicted swollen joints, and ¹8F-FDG accumulation increased with the progression of arthritis. Histologically, increased ¹8F-FDG accumulation correlated with the pannus rather than the infiltration of inflammatory cells around the joints. CONCLUSION: ¹8F-FDG accumulation in arthritis reflects proliferating pannus and inflammatory activity enhanced by inflammatory cytokines. ¹8F-FDG microPET should be effective for quantifying the inflammatory activity of arthritis and/or its therapeutic response.

Evaluation of intra-articular delivery of hyaluronic acid functionalized biopolymeric nanoparticles in healthy rat knees.

The aim of this study is to evaluate the toxicity of nanoparticles of poly(D,L-lactic acid) (PLA) or poly(D,L-lactic-co-glycolic acid) (PLGA) covered by chemically esterified amphiphilic hyaluronate (HA) which will be used for intra-articular injection as a drug carrier for the treatment of arthritis (RA) and/or osteoarthritis (OA). PLA and PLGA are FDA approved polymers that are already used for the preparation of nano or microparticles. HA is a natural polysaccharide already present in the articulations known to interact with the CD44 receptors of the cells (especially chondrocytes). Therefore, we can envisage that the HA covering can improve the interactions between the cells and the nanoparticles, leading to better targeting or biodistribution. The knee of healthy male rats was injected one to two times weekly, with various concentrations of nanoparticles encapsulating Dextran-FITC. The synovial membranes and the patellae were collected aseptically and histologically analyzed to assess the effects and localization of the nanocapsules in the knee joint. We did not observe significant modifications in the synovial membranes (weak hyperplasia) or patellae integrity after local administration of nanodevices into the rats. While we found some nanoparticles in the synovial membrane, none were detected in the patellae. Moreover, the histological observations for patellae were confirmed by radiosulfate intake, which depicted no decrease in proteoglycans biosynthesis in nanoparticles treated animals. Concerning the safety towards synovial membranes, we also had a look at the inflammatory response after injections of nanoparticles covered by amphiphilic HA or polyvinyl alcohol (PVA) by monitoring the mRNA expression levels of some specific early cytokines (IL-1ß and TNF-α). Once again, no differences were observed between the control rats and the rats treated with nanoparticles. Considering these preliminary results obtained in healthy rats, we can establish that neither the amphiphilic HA-covered PLGA nanoparticles nor their degradation products induce major modifications of articular tissues functions, while injected into the knee of healthy rats. These results should be confirmed in OA or RA rat models, in order to confirm that nanoparticles do not worsen already altered (degenerative or inflamed) articular tissues. Once confirmed, such tuneable nanoparticles could be proposed as a safe drug delivery system for the treatment of articular disease, allowing a wide range of encapsulating molecules.

Local induction of heat shock protein 70 (Hsp70) by proteasome inhibition confers chondroprotection during surgically induced osteoarthritis in the rat knee.

AIM: to determine if chondrocytic Hsp70 induction, via intra-articular injections of a reversible proteasome inhibitor (MG132), can protect articular chondrocytes from cellular death in experimental rat OA knee induced surgically by anterior cruciate ligament transection (ACLT). MATERIALS AND METHODS: ACLT was performed on D0. Histological lesions in naive (sham) controls (ACLT+saline) and treated (ACLT+MG132) rats were assessed according to Mankin's score. Repeated intra-articular injections (1.5 muM MG132 or saline were performed on D1, D7, D14 and D21. Rats were sacrificed sequentially on D7, D14 and D28. Detection of active caspase-3 and protein expression of Hsp70 was also determined on D7, D14 and D28 by immunostaining methods. RESULTS: MG132 significantly reduced OA lesions on D28 in the MG132 treated group. The expression of Hsp70 increased 11-fold in the MG132-treated group versus 2-3-fold in ACLT-control rats on D28. Concomitantly, cells expressing caspase-3 increased 4-fold in ACLT model and decreased 2-fold with MG132 treatment. CONCLUSIONS: Intra-articular induction of Hsp70 by MG132 could be a safe and interesting tool in chondrocytes protection from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

Short hairpin RNA-mediated inhibition of HSV-1 gene expression and function during HSV-1 infection in Vero cells.

AIM: To evaluate the efficiency of 3 short hairpin RNA (shRNA) interfering with the herpes simplex virus type 1 (HSV-1) gene coding glycoprotein D (gD) for inhibiting the gD expression and virus replication in vitro. METHODS: Vero cells were selected for an in vitro model of infection. Three shRNA sequences (shRNAgD1, -gD2, and -gD3) targeting specifically the gD gene of HSV-1 were selected for evaluating the antiviral effects. The antiviral effects of shRNA in the cells infected with HSV-1 were evaluated by cytopathic effect (CPE) observations and plaque assays. The transcription level of viral RNA and the gD expression were studied by RT-PCR, Western blotting, and flow cytometry. RESULTS: With the 3 shRNA at a final concentration of 120 nmol/L, a significant inhibition of CPE in the HSV-1-infected cells was observed. The ED50 of shRNA-gD1, gD2, and gD3 were 48.74+/-2.57, 57.13+/-3.24, and 114.64+/-5.12 nmol/L, respectively. The gD gene decreased significantly after viral infection in the Vero cells pretreated with shRNA compared to the virus group. The expressions of the gD protein, determined by Western blotting and flow cytometry, were also drastically decreased in shRNA-transfected cells. CONCLUSION: Exogenous shRNA molecules can suppress the HSV-1 gD expression. They are inhibitors of HSV replication during infection in Vero cells.

Hyaluronate-covered nanoparticles for the therapeutic targeting of cartilage.

Hyaluronic acid (HA) has a high affinity for the CD44 receptor present at the surface of articular cells, particularly of chondrocytes. HA-covered polylactide nanoparticles containing bioactive compounds such as HA and chondroitin sulfate (CS) were thus prepared in order to achieve a controlled delivery targeted to cartilage cells after injection near articular alterations/erosions. Such nanoparticles (diameter = 700 nm) were prepared by double emulsion/solvent evaporation, using amphiphilic derivatives of HA, as stabilizer of the secondary emulsion. These nanoparticles were incubated with articular cells, and several tests were carried out. First, they proved that the nanospheres provoked no decrease in cell viability, even after 72 h of contact. Second, a confocal microscopy analysis on fluorescent HA-covered particles showed that they were captured by articular cells, while with those covered with poly(vinyl alcohol), the uptake was far lower. Third, a scattering electron microscopy analysis proved that the HA-coated nanoparticles were localized in the cell intracytoplasmic area.

Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis.

Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

Bovine chondrocyte behaviour in three-dimensional type I collagen gel in terms of gel contraction, proliferation and gene expression.

This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2 x 10(6) chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.

In vivo follow-up of rat tumor models with 2-deoxy-2-[F-18]fluoro-D-glucose/dual-head coincidence gamma camera imaging.

UNLABELLED: Before studying the impact of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) imaging with a dual-head coincidence gamma camera (DHC) for the follow-up of animal tumor models, we wanted to optimize this technique. METHODS: Three different animal tumor models (osteosarcoma, melanoma, and breast cancer) were studied after FDG injection. Dynamic and dual time point FDG/DHC imaging were studied from one hour to five hours postinjection. In vitro tumor cell FDG uptake was assessed in eight different tumor cell lines. In one model (osteosarcoma), tumor growth, lung metastasis emergence, and survival were assessed by classical clinical follow-up and compared to FDG imaging in a control group (n = 6) and in a group treated by endostatin liposome complexes (n = 6). RESULTS: Images obtained five hours after injection were more reliable for tumor growth follow-up than standard images (one hour). In vitro tumor cell FDG uptake confirmed in vivo imaging studies. In eight different tumor cell lines the FDG uptake was higher after five hours incubation than after one hour (p < 0.002). With FDG follow-up, we found that FDG uptake was strongly correlated with survival and that lung metastasis larger than 5 mm could be detected. CONCLUSION: Using the optimization proposed above, DHC/FDG functional imaging seems to be a powerful tool to study rat tumor models and to help develop novel cancer therapies.

Effect of proteoglycan depletion on T2 mapping in rat patellar cartilage.

PURPOSE: To evaluate experimentally the sensitivity of T2 mapping with magnetic resonance (MR) imaging at 8.5 T in depicting variations in proteoglycan content and concurrent extracellular matrix of rat patellar cartilage. MATERIALS AND METHODS: The study was performed in 36 immature (age, 5 weeks) and 36 mature (age, 10 weeks) Wistar rats. Maintenance and care of the rats were conducted in accordance with National Institutes of Health guidelines. Fifty-six rats underwent T2 mapping in 28 right patellae degraded with hyaluronidase for 1 and 6 hours and in 28 undegraded age-matched patellae that served as controls. After MR mapping, the rats were sacrificed, and the patellae were studied histologically to evaluate proteoglycan and collagen content and collagen network organization in cartilage. Biochemical analysis was performed in 88 patellae to quantify sulfated glycosaminoglycan and hydroxyproline content. Effects of age and/or degree of degradation were evaluated after rank transformation of continuous data by using rank analysis of variance (ANOVA). Associations between continuous variables were assessed with the Spearman rank correlation coefficient. RESULTS: Results of histologic analysis showed proteoglycan loss after hyaluronidase degradation without alteration of collagen network. No significant variation in hydroxyproline sulfate content was observed with depletion of proteoglycan. Proteoglycan losses of 19% and 13%, found after 1-hour degradation in immature and mature groups, respectively, were associated with significantly increased global T2 values (ANOVA, P < .001). Six-hour degradation resulted in more severe proteoglycan losses of 45% and 53% in immature and mature groups, respectively, inducing significant increases in global T2 values in immature and mature groups (ANOVA, P < .001). Variations in T2 values between superficial and deep cartilage zones were not affected by proteoglycan depletion. CONCLUSION: In rat patellar cartilage, T2 mapping permits detection of slight or severe proteoglycan depletion and concurrent changes of extracellular matrix when age-matched samples are compared.