Expanding the Spectrum of EWSR1-NFATC2-rearranged Benign Tumors: A Common Genomic Abnormality in Vascular Malformation/Hemangioma and Simple Bone Cyst.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

Mechanism of TGF-ß3 promoting chondrogenesis in human fat stem cells.

Clinically deficient cartilage is difficult to regenerate, and the availability of chondrocytes is very limited. However, human adipose-derived stem cells (ADSCs) can be obtained easily and in sufficient quantities. Therefore, we will find a way of replacing chondrocytes with fat stem cells to solve the problem of seed cell origin. Previous studies have revealed that transforming growth factor-ß (TGF-ß) can promote chondrocyte differentiation and maturation. In this study, we found that TGF-ß3 in the transforming growth factor family can effectively promote the transformation process from fat stem cells to chondrocytes, thus promoting chondrogenesis. At the same time, we also further reviewed and considered the mechanism of this process. Through flow cytometry, immunohistochemical, fluorescent microscopy, qRCR, Wb etc., we found that TGF-ß3 mainly plays a role through wnt5a/ß-catenin, promoting human fat stem cell growing into the cartilage. This discovery is expected to provide new ideas in the field of cartilage regeneration.

The study of targeted blocking SDF-1/CXCR4 signaling pathway with three antagonists on MMPs, type II collagen, and aggrecan levels in articular cartilage of guinea pigs.

OBJECTIVE: To explore the possibility and mechanism of targeted blocking SDF-1/CXCR4 signaling pathway using three antagonists TN14003, T140, and AMD3100 in vivo, and to investigate the function of three antagonists in delay degeneration process of articular cartilage. METHODS: Ninety-six male Duncan-Hartley guinea pigs (6 months old) were divided into groups A, B, C, and D randomly. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group A, and TN14003 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group B, and T140 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group C, and AMD3100 with concentration of 180 µg/ml was pumped every day. Hartley guinea pigs in group D remained untreated as the blank control group. At 2, 4, 6, 8, 10, and 12 weeks of treatment, 5 to 8 animals in each group were randomly chosen for blood collection via cardiac puncture. SDF-1 content using enzyme-linked immunosorbent assay (ELISA). At 12 weeks, all guinea pigs were sacrificed by injecting pentobarbital sodium (30 mg/kg) into the peritoneal cavity. Cartilages from the tibial plateau in each group were harvested for PCR testing and western blot analysis. SPSS19.0 was used for data analysis. RESULTS: Result of ELISA: the serum levels of SDF-1 of groups A, B, and C decreased gradually with time. Significant drop of SDF-1 level was seen in group A while increased SDF-1 was shown in group D. At the same time, the serum levels of SDF-1 of the group A were significantly lower than that of group B; those of group B were significantly lower than that of group C, which was significantly lower than that of group D, and their difference is statistically significant (P < 0.05). Real time quantitative PCR result: The mRNA levels of MMPs in group A were significantly lower than group B, and those of group B were significantly lower than group C, which was significantly lower than group D, and there was statistically significant (P < 0.05). The mRNA levels of type II collagen, aggrecan in group A were significantly more than group B; those of group B were significantly more than group C, which was significantly more than group D, and the difference was statistically significant (P < 0.05). H&E staining result: cartilage of group C was more significantly degenerative than other groups. CONCLUSIONS: The three antagonists can target SDF-1/CXCR4 signaling pathway in vivo, reduce the expression and secretion of MMP-3, MMP-9, and MMP-13 in cartilage tissue, and reduce the degradation of collagen II and aggregating proteoglycan, thus delaying the degeneration of articular cartilage, of which TN14003 has the strongest regulatory effect. Targeted blockade of SDF-1/CXCR4 signaling pathway by TN14003 in vivo delays articular cartilage degeneration more effectively than T140 and AMD3100.

Prediction of progression of damage to articular cartilage 2 years after anterior cruciate ligament reconstruction: use of aggrecan and type II collagen biomarkers in a retrospective observational study.

BACKGROUND: We aimed to determine whether synovial fluid (SF) biomarkers can predict the progression of articular cartilage damage as determined by arthroscopic evaluation during and after anterior cruciate ligament (ACL) reconstruction. METHODS: Arthroscopic assessment of articular cartilage damage was performed twice in 62 patients, first during ACL reconstruction and then approximately 2 years later during implant removal for ligament fixation. SF levels of the collagenase-generated cleavage neoepitope of type II collagen (C2C) and proteoglycan glycosaminoglycans keratan sulfate (KS), chondroitin-4-sulfate (Δdi-C4S), and chondroitin-6-sulfate (Δdi-C6S) were measured at ACL reconstruction. Associations between baseline biomarker levels and subsequent progression of cartilage damage were determined using receiver operating characteristic analysis and multivariable logistic regression analysis. RESULTS: No radiographic changes were observed in any of the patients. Progression of high-grade cartilage damage, observed arthroscopically, was negatively correlated with levels of Δdi-C6S and KS, as well as the ratio of Δdi-C6S to Δdi-C4S (C6S/C4S). Logistic regression analysis revealed significant associations of Δdi-C6S (cut-off: 55.7 nmol/ml, odds ratio (OR) 0.231, 95% confidence interval (CI) 0.061-0.879), KS (cut-off: 10.6 µg/ml, OR 0.114, 95% CI 0.024-0.529), and C6S/C4S ratio (cut-off: 4.6, OR 0.060, 95% CI 0.005-0.737) with the progression of high-grade cartilage damage after adjusting for age, the duration from injury to first surgery, sex, and the number of high-grade lesions (grades III and IV) at baseline. CONCLUSIONS: The progression of high-grade cartilage damage was significantly associated with baseline levels of proteoglycan glycosaminoglycan biomarkers; namely, Δdi-C6S, KS, and C6S/C4S ratio.

Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis.

BACKGROUND: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals. METHODS: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts. RESULTS: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent. CONCLUSIONS: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.

Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers.

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.

OBJECTIVE: We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. METHODS: HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. RESULTS: HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. CONCLUSION: HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Study of differential properties of fibrochondrocytes and hyaline chondrocytes in growing rabbits.

We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future.

Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration.

Cartilage degeneration is the major cause of disability and poses several challenges to repair and regenerate. Conventional surgical treatments often induce fibrous tissues and compromise its function. Alternative tissue engineering strategies utilized scaffolds, factors and cells alone or in combination with some degree of success. This study reports the use of nanostructured biomimetic scaffold system in regulating the rat bone marrow stem cells (rBMSCs) differentiation into chondrogenic lineage in vitro. The biometric scaffold is essentially a micro-porous polycaprolactone (PCL) spiral structure decorated with sparsely spaced bioactive PCL nanofibers. The bioactivity stems from the use of two major components of hyaline cartilage extracellular matrix (ECM) namely chondroitin sulfate (CS) and hyaluronic acid (HYA). The PCL spiral structure was surface functionalized with PCL nanofibers encapsulated with CS (20% (w/w)) and HYA (0.2% (w/w)). In order to retain and sustain the release of CS and HYA nanofibers were cross-linked using carbodiimide chemistry. This study also evaluated the effect of nanofiber alignment on rBMSCs differentiation and evaluated the production of characteristic hyaline cartilage proteins namely collagen type II and aggrecan in vitro up to 28 days. Rat bone marrow derived stem cells cultured on the aligned nanofibers expressed significantly elevated levels of collagen type II and aggrecan secretions (western blots) as compared to scaffolds decorated with random fibers and tissue culture polystyrene (TCPS). This fiber alignment dependent expression of collagen type II and aggrecan secretion were further confirmed through immunofluorescence staining. This biomimetic and bioactive scaffold may serve as a serve as an efficient scaffold system for cartilage regeneration.

Chondrogenic potential of stem cells from human exfoliated deciduous teeth in vitro and in vivo.

OBJECTIVE: The aim of this study was to investigate the chondrogenic potential of stem cells from human exfoliated teeth (SHED). MATERIALS AND METHODS: SHED cultures were isolated from human exfoliated deciduous teeth. Colony-forming capacity, odonto/osteogenic and adipogenic potential were measured. SHED were cultured for 2 weeks in chondrogenic differentiation medium containing dexamethasone, insulin, ascorbate phosphate, TGF-ß3 and bFGF. Toluidine blue staining and safranin O staining were used for chondrogenesis analysis. The related markers, type II collagen and aggrecan, were also investigated using immunohistochemistry. SHED were seeded onto the ß-TCP scaffolds and transplanted into the subcutaneous space on the back of nude mice. The transplants were recovered at 2, 4 and 8 weeks post-transplantation for analysis. RESULTS: SHED showed colony-forming capacity, odonto/osteogenic and adipogenic differentiation capacity. Chondrogenic differentiation was confirmed by toluidine blue staining, safranin O staining, type II collagen and aggrecan immunostaining. After in vivo transplantation, SHED recombined with ß-TCP scaffolds were able to generate new cartilage-like tissues. CONCLUSIONS: The findings demonstrate the chondrogenic differentiation capacity of SHED both in vitro and in vivo models, suggesting the potential of SHED in cartilage tissue engineering.

Fibronectin-aggrecan complex as a marker for cartilage degradation in non-arthritic hips.

PURPOSE: To report hip synovial fluid cytokine concentrations in hips with and without radiographic arthritis. METHODS: Patients with no arthritis (Tonnis grade 0) and patients with Tonnis grade 2 or greater hip osteoarthritis (OA) were identified from patients undergoing either hip arthroscopy or arthroplasty. Synovial fluid was collected at the time of portal establishment for those undergoing hip arthroscopy and prior to arthrotomy for the arthroplasty group. Analytes included fibronectin-aggrecan complex (FAC) as well as a standard 12 cytokine array. Variables recorded were Tonnis grade, centre-edge angle of Wiberg, as well as labrum and cartilage pathology for the hip arthroscopy cohort. A priori power analysis was conducted, and a Mann-Whitney U test and regression analyses were used with an alpha value of 0.05 set as significant. RESULTS: Thirty-four patients were included (17 arthroplasty, 17 arthroscopy). FAC was the only analyte to show a significant difference between those with and without OA (p < 0.001). FAC had significantly higher concentration in those without radiographic evidence of OA undergoing microfracture versus those not receiving microfracture (p < 0.05). CONCLUSION: There was a significantly higher FAC concentration in patients without radiographic OA. Additionally, those undergoing microfracture had increased levels of FAC. As FAC is a cartilage breakdown product, no significant amounts may be present in those with OA. In contrast, those undergoing microfracture have focal area(s) of cartilage breakdown. These data suggest that FAC may be useful in predicting cartilage pathology in those patients with hip pain but without radiographic evidence of arthritis.

Histological and molecular characterization of the femoral attachment of the human ligamentum capitis femoris.

The ligamentum capitis femoris (LCF) has increased in clinical significance through the development of hip arthroscopy. The histological pathologies and molecular composition of the femoral attachment of the LCF and the degeneration caused by LCF disruption were investigated in the human hip joint. Twenty-four LCFs were retrieved at surgery for femoral neck fracture (age range: 63-87 years). In the "intact" (i.e., intact throughout its length, n = 12) group, the attachment consisted of rich fibrocartilage. Fibrocartilage cells were present in the midsubstance. In contrast, the construction of the attachment in the "disrupted" (i.e., ligament no longer attached to the femoral head, n = 12) group had disappeared. The attachment in the disrupted group was not labeled for type II collagen or aggrecan, while that in the intact group was labeled for types I, II and III collagen, chondroitin 4-sulfate, chondroitin 6-sulfate, aggrecan, and versican. The percentage of single-stranded DNA-positive chondrocytes was significantly higher in the disrupted group than in the intact group. We conclude that the femoral attachment of the LCF has a characteristic fibrocartilaginous structure that is likely to adjust to the mechanical load, and suggest that its degeneration is advanced by disruption and should be regarded as a clinical pathology.

Fibroblast growth factor 2 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint.

BACKGROUND: Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space. Fibroblast growth factor 2 (FGF-2) is frequently applied in chondrogenic differentiation assays. Therefore, we hypothesized that FGF-2 might involved in the pathogenesis of SC. METHODS: SC synovium and loose bodies (LBs) specimens were observed by histological and immunohistochemical methods. Real-time PCR was conducted for comparing genes expressions in SC and normal synovium. SC synoviocytes were stimulated by FGF-2 in the presence or absence of its antagonist long pentraxin-3 (PTX3) for 6 days. Real-time PCR and alkaline phosphatase (ALP) activity were performed to examine the effects exerted by FGF-2 and PTX3. RESULTS: SC synovium, no matter facing the articular cavity or covering LB, was characterized by increased quantity of synoviocytes and blood vessels. FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs, and the wall of blood vessels. Expressions of chondrogenic genes (Sox9 and Wnt-4), osteogenic genes (Foxc2), FGF-2, and VEGF-A mRNA were significantly higher in SC synovium than that of the control group. The stimulation of FGF-2 on SC synoviocytes increased ALP activity and expressions of chondrogenic genes (Sox9, Col2α1, and Aggrecan), osteogenic genes (Foxc2, osteocalcin, and Col1α1), and VEGF-A, but PTX3 inhibited these effects. CONCLUSION: FGF-2 was responsible for the formation of cartilaginous loose bodies and involved in the pathogenesis of SC.

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-ß3 and bone morphogenetic protein-6 in a normal healthy impacted third molar.

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-ß3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-ß3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-ß3 and 220% by BMP-6. The synergetic effect of TGF-ß3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-ß3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

Arguments for an increasing differentiation towards fibrocartilaginous components in midportion Achilles tendinopathy.

PURPOSE: The objective of this study was to investigate the fibrocartilaginous differentiation occurring in midportion Achilles tendinopathy. METHODS: Tendon samples were retrospectively collected from 23 patients, who had undergone surgery for midportion Achilles tendinopathy resistant to conservative treatment. Based on histological scores, the biopts were subdivided into three categories: a light, moderate and severe histopathological stage. Throughout these stages, immunohistochemical staining was performed against biglycan, aggrecan and collagen type II, components characteristic for fibrocartilage. Staining of these components was evaluated using a semi-quantitative scoring method. RESULTS: The immunohistochemical scores of biglycan and aggrecan were statistically significant between the histopathological stages (P < 0.001). The immunohistochemical scores were positively correlated with the increasing histopathological stages [Spearman's correlation coefficient = 0.93 for biglycan and 0.78 for aggrecan (P < 0.001)]. Staining for collagen type II remained negative throughout these stages. CONCLUSION: Immunohistochemical staining of the fibrocartilaginous components biglycan and aggrecan showed a progressive increase, correlated with a further evolved histopathological stage. This observation gave arguments for an increased differentiation towards fibrocartilaginous components at protein level in midportion Achilles tendinopathy.

Up-regulation of interleukin-6 and vascular endothelial growth factor-A in the synovial fluid of temporomandibular joints affected by synovial chondromatosis.

Our aim was to explore important inflammatory mediators for synovial chondromatosis in the temporomandibular joints (TMJs) by analysing synovial fluid. Samples were collected from 10 patients with unilateral synovial chondromatosis of the TMJ. Control samples were obtained from 11 subjects with no symptoms in the TMJ. Concentrations of aggrecan, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A were measured in the samples of synovial fluid, and the results in the two groups compared. The tissues from the affected TMJ were examined histologically and immunohistochemically. Of the proteins evaluated, the concentrations of aggrecan, IL-6, and VEGF-A were significantly higher in the group with synovial chondromatosis. The immunohistochemical analysis showed that the synovial cells around the osteocartilaginous nodules were vigorously expressing VEGF-A. IL-6 and VEGF-A are thought to have important roles in the pathology of synovial chondromatosis of the TMJ.

Tri-lineage potential of intraoral tissue-derived mesenchymal stromal cells.

The purpose of this study was to analyse the potential of intraoral tissues as a source of mesenchymal stromal and progenitor cells (MSPCs) for usage in future cell-based therapy models. Cells were isolated from four different tissues harvested during oral surgery intervention: (1) bone explants from the posterior maxilla, (2) bone explants from the oblique line, (3) from the mandibular periosteum, and (4) from the dental pulp. Donor sites and tissues were evaluated in terms of their accessibility, donor-site morbidity and average time period until appearance of MSPC colonies. Cell characterization was performed by flow cytometry and evaluation of in vitro osteogenic, adipogenic and chondrogenic differentiation potential. Adherent cell colonies were isolated from tissues from all sites after 4-8 days. The cells showed characteristics of MSPCs, so they were expanded up to clinical scales and demonstrated multipotency. The lowest donor-site morbidity was observed in the posterior maxilla harvests, while the highest donor-site morbidity was associated with harvests from mandibular sites. All sites seem to be potential sources of mesenchymal stromal and progenitor cells for tissue engineering approaches. Therefore, harvest morbidity and patient acceptance should affect the choice of the appropriate site.

Osmolarity regulates chondrogenic differentiation potential of synovial fluid derived mesenchymal progenitor cells.

Cartilage is one of few tissues where adult stem/progenitor cells have not been putatively identified. Recent studies have provided strong evidence that a sub-population of mesenchymal progenitor cells (MPCs) derived from the synovial fluid may be able to affect some degree of cartilage repair both in vivo and in vitro/ex vivo, however this does not appear to be the case in patients with arthritis. Previously, it has been found that synovial fluid osmolarity is decreased in patients with osteoarthritis (OA) or Rheumatoid arthritis (RA) and these changes in osmolarity have been linked to changes in chondrocyte gene regulation. However, it is yet unknown if changes in osmolarity regulate the gene expression in synovial fluid MPCs (sfMPCs), and by extension, chondrogenesis of this cell population. In the present study we have collected synovial fluid samples from normal, OA and RA knee joints, quantified the osmolarity of the fluid and modified the culture/differentiation media to span a range of osmolarities (264-375 mOsm). Chondrogenesis was measured with Alcian blue staining of cultures in addition to quantitative PCR (qPCR) using probes to Sox9, ACAN and Col2A1. Overall, sfMPCs from arthritic joints demonstrated decreased chondrogenic potential compared to sfMPCs isolated from normal synovial fluid. Furthermore, the sfMPCs retained increased chondrogenic potential if differentiated under the same osmolarity conditions for which they were initially derived within. In conclusion, it does appear the synovial fluid osmolarity regulates the chondrogenic potential of sfMPCs, however, further study is required to elucidate the mechanism by which the changes in osmolarity are sensed by the cells and regulate chondrogenic gene expression.

Protection of articular cartilage by intra-articular injection of NEL-like molecule 1 in temporomandibular joint osteoarthritis.

Several studies have proven the ability of NEL-like molecule-1 (Nell-1) to induce chondrogenesis and make it as a potential candidate for articular cartilage repair. In the current study, the chondroprotective effect of Nell-1 on osteoarthritis (OA) of the temporomandibular joint (TMJ) was investigated by intra-articular injection. Bilateral partial discectomy was performed in 24 rabbits to induce TMJOA. Four weeks later, the right TMJ was treated with Nell-1 as the experimental group, whereas the left was treated with physiologic saline as the control group. Twelve rabbits each time were randomly killed at 12 and 24 weeks after injection, respectively. Histologic observation and metabolic analysis by reverse transcription-polymerase chain reaction were used for evaluation. All TMJs appeared as OA-like histologic changes after intra-articular injection. However, the degree of osteoarthritis in the experimental group was less severe than that in the control group during the experimental time. The expression of type II collagen and aggrecan messenger RNA was significantly higher than the control group at 12 weeks after injection. However, no difference in the expression of aggrecanase or interleukin 1 messenger RNA was observed. The results suggest that intra-articular injection of Nell-1 may be a good alternative for the treatment of cartilage degeneration in OA.