Extracellular matrix synthesis and degradation in functionally distinct tendons

Tendon injury is common in humans and horses, and incidence increases with age. The high-strain energy storing equine superficial digital flexor (SDFT) is injured more frequently than the low-strain positional common digital extensor (CDET). However, previous work indicated that matrix turnover is greater in the CDET than in the SDFT. It was hypothesised that matrix turnover is programmed by the cells’ strain environment; therefore high-strain energy storing tendons would have a lower rate of matrix turnover than low-strain positional tendons and the rate of matrix turnover would decrease with increasing age. The rate of matrix turnover was investigated by measuring the potential of the cells to synthesise and degrade matrix proteins, measuring the half-life of the collagenous and non-collagenous matrix proteins and assessing collagen turnover at the protein level. In vitro cell phenotype was also assessed in 2D and 3D culture and the effect of load on cells within native tissue was determined. The results show that turnover of collagenous and non-collagenous matrix proteins is differentially regulated in the functionally distinct SDFT and CDET. CDET tenocytes show greater potential for collagen turnover, whereas SDFT tenocytes have a greater potential for proteoglycan turnover; differences that are also present at the protein level. The differences in cell phenotype identified in vivo were lost in 2D and 3D culture, but tendon organ culture resulted in the maintenance of tenocyte phenotype. The cells’ ability to turnover the matrix does not decrease with increasing age, but collagen within the SDFT appears to become more resistant to degradation with ageing. This results in the accumulation of partially degraded collagen within the SDFT which may have a detrimental effect on tendon mechanical properties. These findings will help to elucidate the mechanisms behind the development of age-related tendinopathy and will be of use when developing treatment regimes

The Cholecystectomy As A Day Case (CAAD) Score: A Validated Score of Preoperative Predictors of Successful Day-Case Cholecystectomy Using the CholeS Data Set

Background Day-case surgery is associated with significant patient and cost benefits. However, only 43% of cholecystectomy patients are discharged home the same day. One hypothesis is day-case cholecystectomy rates, defined as patients discharged the same day as their operation, may be improved by better assessment of patients using standard preoperative variables. Methods Data were extracted from a prospectively collected data set of cholecystectomy patients from 166 UK and Irish hospitals (CholeS). Cholecystectomies performed as elective procedures were divided into main (75%) and validation (25%) data sets. Preoperative predictors were identified, and a risk score of failed day case was devised using multivariate logistic regression. Receiver operating curve analysis was used to validate the score in the validation data set. Results Of the 7426 elective cholecystectomies performed, 49% of these were discharged home the same day. Same-day discharge following cholecystectomy was less likely with older patients (OR 0.18, 95% CI 0.15–0.23), higher ASA scores (OR 0.19, 95% CI 0.15–0.23), complicated cholelithiasis (OR 0.38, 95% CI 0.31 to 0.48), male gender (OR 0.66, 95% CI 0.58–0.74), previous acute gallstone-related admissions (OR 0.54, 95% CI 0.48–0.60) and preoperative endoscopic intervention (OR 0.40, 95% CI 0.34–0.47). The CAAD score was developed using these variables. When applied to the validation subgroup, a CAAD score of ≤5 was associated with 80.8% successful day-case cholecystectomy compared with 19.2% associated with a CAAD score >5 (p < 0.001). Conclusions The CAAD score which utilises data readily available from clinic letters and electronic sources can predict same-day discharges following cholecystectomy

Sorghum and pearl millet as food-feed-crops in India

This paper reviews the role of and demand for food-feed-crops — crops that are grown to provide both grain for human food and crop residues as fodder for ruminant livestock. The paper argues that the predicted increase in demand for livestock products will coincide with shrinking common property resources and an increasing scarcity of arable land and water resulting in an increase in the importance of food-feed-crops smallholder crop- ruminant livestock systems. As a consequence, the International Crop Research Institute for the Semi-Arid Tropics and the International Livestock Research Institute have implemented collaborative research on the genetic improvement of fodder value of food-feed-crops, with emphasis on sorghum and pearl millet, which are important crops for the rural poor. The research has demonstrated the existence of significant genetic variation for fodder quality traits and it has identified cultivars that provide superior stover quality and quantity without detriment to grain yield. Initial estimates of heritability of pertinent stover quality traits were in the order of 0.7 suggesting good opportunities for further improvement of stover quality by genetic enhancement. However, certain socio-economic conditions need to be met for large-scale uptake of new varieties and hybrids

Chondrocyte dedifferentiation down regulates mechano-responsiveness and hedgehog signalling associated with changes in primary cilia structure

Purpose: with the limited availability of human chondrocytes for tissue engineering applications, passaging is used to increase cell yield. However, this 2D expansion leads to a loss of chondrogenic phenotype associated with changes in actin organisation. Primary cilia are microtubule structures that act as a signalling hub controlling a variety of signalling pathways including mechanotransduction and hedgehog signalling, both of which are involved in differentiation and osteoarthritis. Primary cilia structure and function are known to be regulated by changes in actin tension. Thus the present study tests the hypothesis that chondrocyte dedifferentiation during monolayer expansion leads to alterations in mechano-responsiveness and hedgehog signalling mediated by changes in primary cilia structure.Methods: chondrocytes were isolated from the metacarpophalangeal joints of 18-24 month old steers. Primary chondrocytes at passage 0 (P0) were cultured in monolayer up to passage 5 (P5) using DMEM containing 10% FBS, 1.9 mM L-glutamine, 96 U/ml penicillin, 96 mg/ml streptomycin, 20 mM HEPES buffer, and 0.74 mM L-ascorbic acid.For mechano-responsiveness, primary (P0) and dedifferentiated (P5) chondrocytes were cultured on collagen I-coated Bioflex plates and subjected to cyclic tensile strain (10%, 0.33 Hz, 1 hour) via the Flexcell 4000T system, prior to analysis of collagen II, aggrecan, ADAMTS5, and MMP13 gene expression. For hedgehog signalling, P0 and P5 chondrocytes were treated with 1 μg/ml Indian hedgehog (Ihh) for 24 hours. Hedgehog pathway activation was then quantified by analysis of Gli1 and Ptch1 gene expression. Controls were maintained in parallel for both studies. Total RNA was isolated using RNeasy Kit (Qiagen). Quantitect Reverse Transcription Kit (Qiagen) was used to covert 1 μg RNA to cDNA and real-time PCR was performed using KAPA SYBR® FAST qPCR Kit (KAPA Biosystems).Cells at P0 to P5 were also fixed with 4% paraformaldehyde and labelled for acetylated α-tubulin and Ki67, with DAPI counterstaining. Confocal microscopy was used to determine the percentage of Ki67 positive cells as well as primary cilia prevalence and length.Results: at P0, cyclic tensile strain significantly upregulated aggrecan, type II collagen and ADAMTS5 gene expression and downregulated MMP13 expression (Fig. 1). This response to loading was completely lost in the P5 chondrocytes. When chondrocytes at P0 were treated with Ihh, pathway activation was confirmed by a 5-fold increase in Gli1 and Ptch1 gene expression (Fig. 2). However both Gli1 and Ptch1 responses to Ihh were significantly reduced at P5 with a complete suppression of Ptch1 up-regulation.Cell proliferation, as indicated by Ki67, remained less than 30% although there was a significant increase by passage 3 (Fig. 3A). The percentage of ciliated cells increased significantly after the first passage and remained constant at 40-50% with subsequent passaging (Fig. 3B). Chondrocyte primary cilia length increased continuously with passage, doubling in length from P0 to P5 (Fig. 3C and D).Conclusions: changes in primary cilia length affects cilia function including mechanotransduction and hedgehog signalling and may also affect other cilia signalling pathways. As hypothesized, the reduction in mechano-responsiveness and hedgehog signalling with extended 2D culture is due to the associated elongation of primary cilia. To further identify the underpinning mechanisms, ongoing studies are using super resolution microscopy to examine whether cilia elongation at P5 is associated with alterations in cilia expression of ARL13B, and putative mechanoreceptors polycystin 2 and TRPV4. The observed reductions in mechano-responsiveness and ligand-induced hedgehog signalling with passage have important implications for the success of cartilage tissue engineering. Future work will investigate whether modulation of cilia length can maintain or rescue mechano- and hedgehog-responsiveness during expansion of chondrocytes for cartilage tissue engineering