A polarized light microscopy method for accurate and reliable grading of collagen organization in cartilage repair.

OBJECTIVES: Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. DESIGN: This PLM score uses an ordinal scale of 0-5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. RESULTS: The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. CONCLUSION: This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Structural characteristics of the collagen network in human normal, degraded and repair articular cartilages observed in polarized light and scanning electron microscopies.

OBJECTIVE: This study characterizes collagen organization (CO) in human normal (n = 6), degraded (n = 6) and repair (n = 22) cartilages, using polarized light (PLM) and scanning electron (SEM) microscopies. DESIGN: CO was assessed using a recently developed PLM-CO score (Changoor et al. Osteoarthritis Cartilage 2011;19:126-35), and zonal proportions measured. SEM images were captured from locations matched to PLM. Fibre orientations were assessed in SEM and compared to those observed in PLM. CO was also assessed in individual SEM images and combined to generate a SEM-CO score for overall CO analogous to PLM-CO. Fibre diameters were measured in SEM. RESULTS: PLM-CO and SEM-CO scores were correlated, r = 0.786 (P < 0.00001, n = 32), after excluding two outliers. Orientation observed in PLM was validated by SEM since PLM/SEM correspondence occurred in 91.6% of samples. Proportions of the deep (DZ), transitional (TZ) and superficial (SZ) zones averaged 74.0 ± 9.1%, 18.6 ± 7.0%, and 7.3 ± 1.2% in normal, and 45.6 ± 10.7%, 47.2 ± 10.1% and 9.5 ± 3.4% in degraded cartilage, respectively. Fibre diameters in normal cartilage increased with depth from the articular surface [55.8 ± 9.4 nm (SZ), 87.5 ± 1.8 nm (TZ) and 108.2 ± 1.8 nm (DZ)]. Fibre diameters were smaller in repair biopsies [60.4 ± 0.7 nm (SZ), 63.2 ± 0.6 nm (TZ) and 67.2 ± 0.8 nm (DZ)]. Degraded cartilage had wider fibre diameter ranges and bimodal distributions, possibly reflecting new collagen synthesis and remodelling or collagen fibre unravelling. Repair tissues revealed the potential of microfracture-based repair procedures to produce zonal CO resembling native articular cartilage structure. Values are reported as mean ± 95% confidence interval. CONCLUSION: This detailed assessment of collagen architecture could benefit the development of cartilage repair strategies intended to recreate functional collagen architecture.

A novel feeding behavior index integrating several components of the feeding behavior of finishing pigs.

Describing the feeding behavior of pigs is difficult given the large day-to-day variations observed for a given animal. The objectives of this study were to create an index that integrates the information from several components of feeding behavior in order to account for intra-animal variation within a day and between days, and to evaluate the capability of this index to study the impact of relevant nutritional factors affecting feeding behavior. Feed intake information from 160 pigs during the last 28 d of the growing phase from three studies was used. For each pig, the sum of the absolute values of the deviation areas between the regression line of the relative cumulative feed intake and the observed cumulative feed intake was used to calculate the weekly index measuring the irregularity of feed intake (IIFI). Spearman's correlations of IIFI with the number of daily meals (r = -0.42; P < 0.001), meal duration (r = 0.38; P < 0.01), and feed intake per meal (r = 0.41; P < 0.01) indicate that pigs with high IIFI have fewer meals of longer duration and higher feed intake compared to pigs with low IIFI. This shows that IIFI captures information from several components of feeding behavior. Analysis of variance showed no effect of treatment on feeding behavior for datasets 1 and 2. However, the correlation between IIFI and dietary levels of CP (r = 0.34) indicates that diets high in CP were associated with pigs having more irregular meals. In dataset 3, pig feeding behavior was more regular in control diets than in pigs fed fibrous diets (IIFI; 164 vs. 197, respectively; P < 0.05). Additionally, IIFI was smaller in pigs fed canola by-product diets than in pigs fed wheat by-product diets, indicating that the source of fiber may also influence the feeding behavior of pigs. In most cases, IIFI was more effective at identifying differences in feeding behavior between dietary treatments than conventional feeding behavior variables. These results show the ability of IIFI to integrate information from several conventional components of the feeding behavior of pigs and its potential to successfully evaluate the effect of nutritional factors on feeding behavior.

Precision livestock farming: real-time estimation of daily protein deposition in growing-finishing pigs.

Precision feeding using real-time models to estimate daily tailored diets can potentially increase nutrient utilization efficiency. However, to improve the estimation of amino acid requirements for growing-finishing pigs, it is necessary to accurately estimate the real-time body protein (BP) mass. The aim of this study was to predict individual BP over time in order to obtain individual daily protein content of the gain (i.e., protein deposition/daily gain, PD/DG) to be integrated into a real-time model used for precision feeding. Two databases were used in this study: one for the development of the equations for the model and the other for model evaluation. For the equations, data from 79 barrows (25 to 144 kg BW) were used to estimate the parameters for a Gompertz function and a mixed linear-quadratic regression. Individual BP predictions obtained by dual X-ray absorptiometry were regressed as a function of BW. Individual pig BP estimates were obtained by linear-quadratic regression using the MIXED procedure of SAS, considering pig measurements repeated in time. Individual Gompertz curves were obtained using the NLMIXED procedure of SAS. Both procedures generate an average or a general model, which was assessed for accuracy with the database used to generate the equations. Coefficients of concordance and determination were both 0.99, and the RMSE was 0.21 kg for the linear-quadratic regression. The Gompertz curve coefficients of concordance and determination were both 0.99, and the RMSE was 0.36 kg. In sequence, the linear-quadratic regression and Gompertz curve were evaluated in an independent data set (488 observations; 21 to 126 kg BW). The linear-quadratic regression to predict BP mass was accurate (mean absolute percentage error (MAPE) = 2.5%; bias = 0.03); the Gompertz model performed worse (MAPE = 3.9%; bias = 0.04) than the linear-quadratic regression. When using the derivative of these equations to predict PD/DG, the linear-quadratic regression was more accurate (MAPE = 4.8%, bias = 0.17%) compared to the Gompertz (MAPE = 10.6%, bias = -0.99%) mainly due to the linear decrease in PD/DG in the observed data. Further analysis using individual pig data showed that the goodness of fit of PD/DG curve depends on the individual shape of the growth curve, with either the Gompertz or the linear-quadratic regression being more accurate for specific individuals. Therefore, both approaches are provided to allow end users to select the model that best fits their needs. The proposed update of the empirical component of the original model, using either linear-quadratic regression or the Gompertz function, is able to predict BP in real-time with good accuracy.

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.

In order to promote an efficient humoral immune response, germinal center B cells modify both the antigen recognition and effector domains by programmed genetic alterations of their antibody genes. To do so, B cells use the enzyme activation-induced deaminase (AID), which transforms deoxycytidine into deoxyuridine at the immunoglobulin genes, triggering mutagenic DNA repair. Data accumulated during the past decade have significantly advanced our understanding of how AID activity is regulated and preferentially targeted to the immunoglobulin genes. There is also a better understanding of the ways by which AID-catalyzed uracil is recognized and the ensuing downstream processing underpinning the mechanisms of somatic hypermutation and class switch recombination. Here, we critically review these advances in the context of their relevance for the humoral immune response. A detailed understanding of these molecular mechanisms is paramount to uncover the basis of B cell intrinsic immunodeficiency, as well as to suggest tools and strategies that might allow boosting antibody gene diversification in the context of immunizations or infections that require the elicitation of rare or highly mutated antibody variants.

In vivo measures of mammary development in gestating gilts.

Potential links between measures of udder morphology obtained in live pregnant gilts and mammary gland development and composition measured in mammary tissue collected at slaughter were studied. Thirty-three gilts were used. In vivo measures of gland morphology using a tape or ultrasound imaging (parenchymal area measured by ultrasound [AREA]) were obtained on d 108 ± 1 of gestation. Gilts were then slaughtered on d 110 ± 1 of gestation to collect mammary glands for dissection and compositional analyses. The various tape measures were the distance between each teat on one side of the udder (DIST-TEAT), the distance between each teat pair (DIST-PAIR), the length of the udder (sum of all DIST-TEAT), the distance between the base of the teat and the ventral midline section of the udder (MID), and the distance between the base of the teat and the exterior junction of the udder with the abdomen (EXT). The variables MID, DIST-TEAT, DIST-PAIR, and length had very poor correlations with parenchymal weight, extraparenchymal weight, or any of the measured compositional variables. On the other hand, both AREA and EXT were correlated ( < 0.01) with the weight of parenchymal tissue, total parenchymal protein, total DNA, and total RNA. The ultrasound measure AREA and the tape measure EXT were also correlated with each other ( < 0.05). These measures could, therefore, be helpful to estimate mammary development in studies where animals cannot be slaughtered. The tape measure EXT seemed to better reflect the volume of the gland than MID, and it provided as reliable an estimate of parenchymal weight as the measure of parenchymal area using ultrasound while being much easier and cheaper to obtain.

Comparative study on the relations between backfat thickness in late-pregnant gilts, mammary development and piglet growth.

The potential relation between body condition of gilts in late-pregnancy and litter BW gain as well as mammary development was studied using 2 sets of data. Gilts either from a commercial herd (Part 1, n = 182) or from a series of trials looking at mammary development (Part 2, n = 172) were separated in 3 groups according to backfat thickness (BF) on d 110 of gestation. Group categorization was similar for Parts 1 and 2 of the study and was: low (LOW), 13.6 ± 1.6 mm (mean ± SD); medium (MED), 17.6 ± 1.0 mm (mean ± SD); and high BF (HIGH), 21.8 ± 1.8 mm (mean ± SD) for Part 1, and LOW, 14.2 ± 1.3 mm (mean ± SD); MED, 18.1 ± 1.0 mm (mean ± SD), and HIGH 23.4 ± 2.6 mm (mean ± SD) for Part 2. The effects of BF group on piglet BW gain (Part 1) or on various mammary gland characteristics (Part 2) were determined using ANOVA. Litters from HIGH sows tended to have a greater lactation BW gain than those from LOW sows (P < 0.10). Sows with HIGH BF had more mammary parenchymal tissue and more total protein and total DNA than MED and LOW sows (P < 0.05), which led to greater total protein and total DNA contents (P < 0.05). There were strong positive correlations (P < 0.0001) between parenchymal weight and total protein, total DNA, and total RNA. Results suggest that it is beneficial for primiparous sows to have greater BF (i.e., 20 to 26 mm) at the end of gestation to achieve optimal mammary development and greater litter BW gain in the subsequent lactation.

Effect of feed texture, meal frequency and pre-slaughter fasting on carcass and meat quality, and urinary cortisol in pigs.

Carcass and meat quality traits, and urinary cortisol variation was studied in 96 barrows assigned to the following treatments: feed texture (FT; mash vs. pellets), meal frequency (MF; 2 vs. 5 meals per day) and fasting time (F; 4, 14 and 24h) according to a 2×2×3 factorial design. Pigs fed mash, receiving feed five times a day and fasted for 24h before slaughter had lower carcass dressing yield (P<0.001). A higher (P<0.05) bruise score was found on carcasses from pigs fasted for 14 and 24h and fed either pelleted or mashed feed five times per day. The pH(u) value in the Longissimus muscle increased (P<0.05) with increasing fasting time, whereas in the Adductor muscle it was higher (P<0.05) in pigs fed with pellets in two meals per day and fasted for 24h. Urinary cortisol tended to be higher in pigs fasted for 14h compared to those fasted for 4 (P=0.10) and 24h (P=0.06). The results of this study show a significant influence of pellet feeding on carcass yield in fasted pigs, while the effects of pre-slaughter fasting time on meat quality traits were limited.

[Responses of ligamentous fibroblasts to mechanical stimulation]. / Réponses des fibroblastes ligamentaires à la stimulation mécanique.

Mechanical stimulation, as provided by physiotherapy or controlled motion is essentially the only factor able to improve anterior cruciate ligament (ACL) healing. We investigate the cellular effects of such stimulus. Two types of stimulations are applied on canine ACL fibroblasts: repetitive stretch of an elastomeric adhesion substrate and a laminar flow of culture media over the culture surface. Cell orientation, proliferation rate, synthesis and type of collagen as well as proteoglycans (PG) synthesis and hydrodynamic characteristics have been studied. According to our results, the fibroblasts tend to align perpendicularly to the deformation axis of their substrate, and along a laminar flow. The shear stress induced by the laminar flow does not modify significantly proliferation rate nor extracellular matrix synthesis. Substrate stretching however, increases proliferation rate, collagen synthesis, mostly type III, and PG synthesis, principally of small sizes. The characteristics of fibroblasts submitted to repeated deformation match those of fibroblasts from ligament scar tissues. Their orientation perpendicular to substratum deformation differs from the one usually encountered in the undamaged tissue: aligned on the ligament axis.

Shelf life of pork from five different quality classes.

A total of 117 loins were selected on the cutting line at 24h post-mortem to study the long term shelf life (35 days, 4 degrees C) of vacuum packaged pork from five different quality classes (PSE: pale, soft, exudative; PFN: pale, firm, non-exudative; RSE: red, soft, exudative; RFN: red, firm, non-exudative; and DFD: dark, firm, dry). The microbial load at 0 d was not significantly different (P>0.05) among the pork quality classes, indicating that the initial microflora was influenced by the dressing conditions at the plant, not by the meat quality class. But after 35 d of storage, total aerobic mesophilic and presumptive lactic acid bacteria counts were higher (P<0.05) in DFD pork due to its higher ultimate pH. RSE was the second quality class most susceptible to spoilage, whereas PFN, RFN and PSE pork had similar microbial loads. Further research is needed to elucidate the causes of the shorter shelf life in RSE pork.