In vitro Exposure to Inflammatory Mediators Affects the Differentiation of Mesenchymal Progenitors.

The increasing prevalence of joint disease, and in particular osteoarthritis (OA), calls for novel treatment strategies to prevent disease progression in addition to existing approaches focusing mainly on the relief of pain symptoms. The inherent properties of mesenchymal stem cells (MSCs) make them an attractive candidate for novel tissue repair strategies, as these progenitors have the potential to differentiate into chondrocytes needed to replace degraded cartilage and can exert a modulating effect on the inflammatory environment of the diseased joint. However, the inflammatory environment of the joint may affect the ability of these cells to functionally integrate into the host tissue and exert beneficial effects, as hinted by a lack of success seen in clinical trials. Identification of factors and cell signalling pathways that influence MSC function is therefore critical for ensuring their success in the clinic, and here the effects of inflammatory mediators on bone marrow-derived MSCs were evaluated. Human MSCs were cultured in the presence of inflammatory mediators typically associated with OA pathology (IL-1ß, IL-8, IL-10). While exposure to these factors did not produce marked effects on MSC proliferation, changes were observed when the mediators were added under differentiating conditions. Results collected over 21 days showed that exposure to IL-1ß significantly affected the differentiation response of these cells exposed to chondrogenic and osteogenic conditions, with gene expression analysis indicating changes in MAPK, Wnt and TLR signalling pathways, alongside an increased expression of pro-inflammatory cytokines and cartilage degrading enzymes. These results highlight the value of MSCs as a preclinical model to study OA and provide a basis to define the impact of factors driving OA pathology on the therapeutic potential of MSCs for novel OA treatments.

Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

Music in the endoscopy suite: a meta-analysis of randomized controlled studies.

BACKGROUND AND STUDY AIM: Prior studies have suggested that music therapy can provide stress relief and analgesia. In this meta-analysis we focused on the effects of music therapy on patients undergoing gastrointestinal endoscopic procedures. MATERIALS AND METHODS: A literature search using the PubMed and Cochrane Library databases and a manual search led to the inclusion of six randomized controlled trials that examined the effects of music therapy on patients undergoing gastrointestinal endoscopic procedures. After data extraction, four separate meta-analyses were performed: in the three studies that did not use pharmacotherapy (group A), anxiety levels were used as a measure of efficacy; in the three studies in which pharmacotherapy was used (group B), sedation and analgesia requirements and procedure duration times were analyzed. RESULTS: A total of 641 patients were included in the analysis. In group A, patients receiving music therapy exhibited lower anxiety levels (8.6% reduction, P = 0.004), compared with controls. In group B, patients receiving music therapy exhibited statistically significant reductions in analgesia requirements (29.7% reduction, P = 0.001) and procedure times (21% reduction, P = 0.002), and a reduction in sedation requirements that approached significance (15% reduction, P = 0.055), in comparison with controls. CONCLUSIONS: Music therapy is an effective tool for stress relief and analgesia in patients undergoing gastrointestinal endoscopic procedures.

Bone morphogenetic protein-2 stimulates adipogenic differentiation of mesenchymal precursor cells in synergy with BRL 49653 (rosiglitazone).

Bone morphogenetic proteins (BMPs) were discovered as potent bone-inducing molecules. Their effect on adipogenic differentiation is not well understood, both stimulation and inhibition of the process have been described. We show here that BMP-2 strongly stimulates adipogenic differentiation of murine 3T3-L1 preadipocytes if applied together with an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma). On its own, BMP-2 (500 ng/ml) did not stimulate adipogenesis as quantified by flow cytometry with the lipophilic dye Nile Red. However, the protein strongly potentiated adipogenesis stimulated by the thiazolidinedione BRL 49653 as well as glycerol-3-phosphate dehydrogenase activity and induction of mRNAs for the adipogenic markers PPARgamma and adipsin. We confirmed the synergistic action of BMP-2 and BRL 49653 with primary cultures of rat bone marrow stromal cells. Our data demonstrate that BMP-2 can act as a potent adipogenic agent if presented together with activators of PPARgamma.

Stem cell characteristics of human trabecular bone-derived cells.

Human trabecular bone-derived cells (HTBs) have been used for many years as osteoblast progenitors. In this study we tested whether HTBs have stem cell characteristics; that is, whether they are pluripotent and able to self-renew. We show that HTBs readily differentiate into osteoblasts, chondrocytes, and adipocytes if subjected to the appropriate differentiating conditions. Importantly, differentiation into these three lineages is maintained in single cell clones derived by limiting dilution, following expansion over more than 20 cumulative population doublings. We conclude that cultures of HTBs are equivalent to cultures of "mesenchymal stem cells" (MSCs) isolated from bone marrow.

Multi-lineage potential of human mesenchymal stem cells following clonal expansion.

Bone marrow contains mesenchymal cells that can be isolated and grown in vitro. Using appropriate treatment protocols such cultures can be induced to differentiate to yield osteoblasts, adipocytes, and chondrocytes. However, previous experiments had not addressed the question whether single pluripotent stem cells exist and can give rise to these different cell lineages or whether bone marrow mesenchymal cell preparations represent a mixture of committed precursors. We have used human adult bone marrow-derived mesenchymal cells obtained from iliac crest biopsies to demonstrate clonal outgrowth after limiting dilution and we show that some clones can be expanded over more than 20 cumulative population doublings and differentiated to osteoblasts, adipocytes, and chondrocytes. Our data provide direct experimental evidence that cultures of bone marrow-derived mesenchymal cells contain individual cells that fulfil two essential stem cell criteria: (i) extensive self-renewal capacity and (ii) multi-lineage potential.

Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine.

Cytocompatibility, degradation, mechanical property retention and ion release profiles for phosphate glass fibre reinforced composite rods.

Fibre reinforced composites have recently received much attention as potential bone fracture fixation applications. Bioresorbable composites based on poly lactic acid (PLA) and phosphate based glass fibre were investigated according to ion release, degradation, biocompatibility and mechanical retention profiles. The phosphate based glass fibres used in this study had the composition of 40P2O5-24MgO-16CaO-16Na2O-4Fe2O3 in mol% (P40). The degradation and ion release profiles for the composites showed similar trends with the amount of sodium and orthophosphate ions released being greater than the other cations and anions investigated. This was attributed to low Dietzal's field strength for the Na(+) in comparison with Mg(2+) and Ca(2+) and breakdown of longer chain polyphosphates into orthophosphate ions. P40 composites exhibited good biocompatibility to human mesenchymal stem cells (MSCs), which was suggested to be due to the low degradation rate of P40 fibres. After 63 days immersion in PBS at 37 °C, the P40 composite rods lost ~1.1% of mass. The wet flexural, shear and compressive strengths for P40 UD rods were ~70%, ~80% and ~50% of their initial dry values after 3 days of degradation, whereas the flexural modulus, shear and compressive strengths were ~70%, ~80%, and ~65% respectively. Subsequently, the mechanical properties remained stable for the duration of the study at 63 days. The initial decrease in mechanical properties was attributed to a combination of the plasticisation effect of water and degradation of the fibre-matrix interface, with the subsequent linear behaviour being attributed to the chemical durability of P40 fibres. P40 composite rods showed low degradation and ion release rates, good biocompatibility and maintained mechanical properties similar to cortical bone for the duration of the study. Therefore, P40 composite rods have huge potential as resorbable intramedullary nails or rods.

Adult mesenchymal stem cells: differentiation potential and therapeutic applications.

Adult mesenchymal stem cells (MSCs) are a population of multipotent cells found primarily in the bone marrow. They have long been known to be capable of osteogenic, adipogenic and chondrogenic differentiation and are currently the subject of a number of trials to assess their potential use in the clinic. Recently, the plasticity of these cells has come under close scrutiny as it has been suggested that they may have a differentiation potential beyond the mesenchymal lineage. Myogenic and in particular cardiomyogenic potential has been shown in vitro. MSCs have also been shown to have the ability to form neural cells both in vitro and in vivo, although the molecular mechanisms underlying these apparent transdifferentiation events are yet to be elucidated. We describe here the cellular characteristics and differentiation potential of MSCs, which represent a promising stem cell population for future applications in regenerative medicine.

A high-capacity screen for adipogenic differentiation.

Glycerol-3-phosphate dehydrogenase (GPDH) is highly expressed in mature adipocytes. Activity of this enzyme is therefore routinely measured to assess adipogenic differentiation in cell cultures. Existing protocols for GPDH assays require relatively large amounts of cells, and throughput is limited due to multiple steps needed for cell harvest and enzyme extraction. We present here a new protocol allowing GPDH determinations to be performed in a 96-well-plate format. From the start of cell culture to the final readout all steps are carried out using the same multiwell plate, with a minimum of handling required. Our method is suitable for setting up high-throughput assays of adipogenic differentiation.

Enhanced marrow adipogenesis and bone resorption in estrogen-deprived rats treated with the PPARgamma agonist BRL49653 (rosiglitazone).

Thiazolidinediones are insulin-sensitizing agents and in clinical use for the treatment of type II diabetes. Under specific experimental conditions, these molecules induce adipogenic differentiation of mesenchymal precursor cells at the expense of osteoblasts in vitro, suggesting possible negative effects on the skeleton. We measured effects of the thiazolidinedione BRL49653 on bone tissue of intact and estrogen-deprived skeletally mature adult female Wistar rats (6-9 months old). Weight gain and decreased plasma triglyceride levels confirmed the effectiveness of the treatment. However, no change in bone mass or fat marrow volume was observed in intact rats treated for 8 weeks with 5, 10, or 20 mg/kg of BRL49653. Study of marrow cultures established at necropsy revealed a higher responsiveness to adipogenic differentiation protocols of cultures established from the 10-mg/kg group compared to vehicle control. In a second study, the effects of thiazolidinedione treatment on the skeleton of estrogen-deprived rats were investigated. Application of 10 mg/kg of BRL49653 for 12 weeks resulted in enhanced bone loss (+31%; pQCT) and increased fat marrow volume (+117%; histomorphometry) compared to vehicle-treated OVX control. Interestingly, osteoblast number was comparable in both cases. Bone resorption parameters were significantly increased in the treatment group (+27% osteoclast number, +30% eroded surface). Enhanced bone loss due to treatment was consistently observed in the tibia, femur, and the lumbar spine. Our data indicate that thiazolidinediones may enhance bone loss induced by estrogen deprivation.

Computed tomography in Crohn's disease.

Computed tomography, barium examinations, and endoscopy play complementary roles in documenting the extent of disease in patients with Crohn's disease. Computed tomography is most often requested for patients with suspected or known intraabdominal or pelvic abscesses. In addition to documenting the presence and extent of abscess formation, other pathological processes in patients with Crohn's disease, may be demonstrated on computed tomography scans. These include thickening and/or contrast enhancement of inflamed bowel wall, inflammatory changes in the mesenteric and perirectal fat, and fistulous tract formation.

Chronic microscopic enterocolitis with severe hypokalemia responding to subtotal colectomy.

The authors present the first case report of a 50-year-old woman with a 33-year history of severe, chronic watery diarrhea and hypokalemia secondary to chronic active microscopic enterocolitis with patterns similar to lymphocytic colitis but with acute cryptitis and terminal ileum involvement microscopically. The progressive nature of her illness resulted in multiple hospital admissions secondary to hypokalemia with subsequent chronic renal failure. High continuous doses of oral potassium supplements failed to correct the hypokalemic episodes. After subtotal colon resection, the patient made a marked clinical improvement with normal serum potassium levels without receiving potassium supplementation.

H. pylori infection and genotyping in patients undergoing upper endoscopy at inner city hospitals.

Kings County Hospital (KCH), and St. John's Episcopal Hospital (SJH) are inner-city hospitals in New York City serving predominantly minority populations. Staten Island University Hospital (SIUH) serves a predominantly middle-class Caucasian population. We examined H. pylori (HP) infection in patients undergoing upper endoscopy at these hospitals. Two gastric biopsies were obtained from each patient. One biopsy was examined by histology or the rapid urease test for the presence of HP. The other was subjected to analysis by PCR to detect HP DNA and to identify putative HP virulence factors. Of 200 subjects, 54% were African-American, 10% were Hispanic, and 36% were Caucasian. HP infection rates in African-American, Hispanic, and Caucasian patients were 43%, 20%, and 11%, respectively. Many of the African-American patients are recent immigrants from the Caribbean Islands. In these patients, an inverse relationship was observed between HP infection and the number of years living in the United States. Higher levels of HP infection were observed in patients with duodenitis and peptic ulcer disease. With respect to HP virulence factors, the vacA s1b and m1 alleles, as well as the iceA2 allele were the predominant alleles expressed in HP-positive samples obtained from African-Americans. The cagA gene was detected in 81% of HP-positive samples. However, CagA positivity was not related to any specific gastrointestinal disorder. Our findings indicate that among several ethnic groups served by three hospitals, African-American patients have the highest rate of HP infection. Moreover, in AfricanAmerican patients undergoing endoscopy: (1) HP infection was inversely related to the number of years the patients have been living in the USA; (2) HP infection rates were higher in patients diagnosed with duodenitis and peptic ulcer disease versus other disorders; (3) expression of the CagA gene was not associated with any specific gastroduodenal disorder; and (4) there was little allelic heterogeneity with respect to VacA and IceA subtypes. These findings suggest that inner-city African-Americans are more likely to be infected with HP and suffer from more serious gastroduodenal disorders than other ethnic groups.