Autologous Mesenchymal Stromal Cells Immobilized in Plasma-Based Hydrogel for the Repair of Articular Cartilage Defects in a Large Animal Model.

The treatment of cartilage defects in trauma injuries and degenerative diseases represents a challenge for orthopedists. Advanced mesenchymal stromal cell (MSC)-based therapies are currently of interest for the repair of damaged cartilage. However, an approved system for MSC delivery and maintenance in the defect is still missing. This study aimed to evaluate the effect of autologous porcine bone marrow MSCs anchored in a commercially available polyglycolic acid-hyaluronan scaffold (Chondrotissue®) using autologous blood plasma-based hydrogel in the repair of osteochondral defects in a large animal model. The osteochondral defects were induced in twenty-four minipigs with terminated skeletal growth. Eight animals were left untreated, eight were treated with Chondrotissue® and eight received Chondrotissue® loaded with MSCs. The animals were terminated 90 days after surgery. Macroscopically, the untreated defects were filled with newly formed tissue to a greater extent than in the other groups. The histological evaluations showed that the defects treated with Chondrotissue® and Chondrotissue® loaded with pBMSCs contained a higher amount of hyaline cartilage and a lower amount of connective tissue, while untreated defects contained a higher amount of connective tissue and a lower amount of hyaline cartilage. In addition, undifferentiated connective tissue was observed at the edges of defects receiving Chondrotissue® loaded with MSCs, which may indicate the extracellular matrix production by transplanted MSCs. The immunological analysis of the blood samples revealed no immune response activation by MSCs application. This study demonstrated the successful and safe immobilization of MSCs in commercially available scaffolds and defect sites for cartilage defect repair.

Absence of maternal cell contamination in mesenchymal stromal cell cultures derived from equine umbilical cord tissue.

This study aimed to determine whether maternal cell contamination exists in cells derived from equine umbilical cord tissue, a perspective material for cell-based therapies in veterinary medicine. Potential maternal cell contamination was analyzed at DNA level via a set of 16 microsatellite markers in cells originating from the cord tissue of 22 foals. In these cells no maternal cell contamination was detected at a sensitivity level of 0.01%. Our results suggest that equine umbilical cord tissue-derived cells are entirely of fetal origin.

Effect of protein supplement source on porcine oocyte maturation and subsequent embryonic development after parthenogenetic activation.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine.

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.

Relations between genetic distance of parental pig breeds and heterozygosity of their F1 crosses measured by genetic markers.

The aim of this paper is to examine the extent to which increment of heterozygosity in F1 crosses can be predicted from genetic distance of parental breeds. For this purpose, 38 polymorphic marker loci (blood groups, allotypes, polymorphic proteins and enzymes) were tested in 1115 purebred animals (Duroc, Hampshire and Czech Meat Pig as sire breeds; Landrace, Large White and Black Pied Prestice as dam breeds) and in 1428 crossbred animals of the resulting nine crossbred groups. The number of animals in each genetic group ranged from 75 to 230. On the basis of the allele frequencies of the scored loci, three measures of genetic diversity (heterozygosity, standardized heterozygosity, effective number of alleles) were calculated in all 15 genetic groups. Furthermore, two measures of genetic distance (Nei's standard genetic distance and Gregorius' absolute genetic distance) were calculated between the parental populations. High correlations (Pearson product-moment correlation 0.62 to 0.73; Spearman rank correlation 0.58 to 0.85) were found between the increment of heterozygosity in the crosses (in relation to the mean of the heterozygosities of parental populations) and the genetic distance between the parental populations.

Increase in fibrinogenemia in the postoperative period of open-heart surgery.

Postoperative thromboses, with no effective prevention currently available, are a serious complication of open-heart surgery. Conventionally, anticoagulants (heparin, coumarine derivatives) are used after prosthetic heart valvular surgery (PHVS), and antiplatelet drugs (ASA) are administered after coronary artery by-pass graft operation (CABG)). Postoperative thrombophilia may be enhanced by an increase in plasma fibrinogen (FBG) levels, from 2.6 +/- 0.4 to 6.0 +/- 1.8 g/l, p < 0.01, as observed following open-heart surgery in CABG pts (n = 19), and from 2.8 +/- 0.5 to 4.2 +/- 1.0 g/l, p < 0.01) in PHVS pts (n = 12) during postoperative days 6 to 10. This increase in FBG in both groups of pts on different antithrombotic regimens significantly correlated with an increase in others plasma proteins alpha-1-antitrypsin A1AT (r = 0.43, p < 0.01), coeruloplasmin, CRPL (r = 0.53, p < 0.01), orosomucoid, ORM (r = 0.37, p < 0.02), and with prolongation of euglobulin clot lysis time (r = 0.42, p < 0.01) during the 21-day postoperative period. A negative correlation was demonstrated between FBG and the plasma levels of transferrin, TRF (r = -0.389, p < 0.02). Whereas FBG, ORM, A1AT, and CRPL are referred to as "positive" acute phase proteins, TRF is a "negative" acute phase protein. It follows from multivariate factor analysis that the reactive increase in these acute phase proteins (incl.FBG) is due to one "common" stimulating factor during the postoperative period. The "common" stimulating factor also raised thrombocyte count.(ABSTRACT TRUNCATED AT 250 WORDS)

[Levels of C-reactive protein in serum--comparison of 2 methods used for quantitative determination]. / Koncentrace C-reaktivního proteinu v séru--srovnáni dvou metod pouzívaných pro jeho kvantitativní stanovení.

The authors compared the results of two methods used for the quantitative assessment of C-reactive protein: the method of radial immunodiffusion and the microturbidimetric method. The testing was based on examination of 68 sera from patients. The samples comprised sera with a CRP concentration under 230 mg/l. There was a statistically significant correlation between the results of the two compared methods (r = 0.949, p = 0.0001).

[Quantitative determination of C-reactive protein using the microturbidimetry method]. / Kvantitativní stanovení C-reaktivního proteinu mikroturbidimetrickou metodou.

C-reactive protein (CRP) is the protein of the acute stage responding sensitively to changes which take place in the organism in some pathological conditions. The authors were concerned with its quantitative estimation by a microturbidimetric method the advantage of which is greater rapidity and sensitivity, as compared with hitherto used methods for the assessment of the CRP level in our laboratories.

Plasma free N-terminal fibronectin 30-kDa domain as a marker of endothelial dysfunction in type 1 diabetes mellitus.

The plasma free N-terminal fibronectin 30-kDa domain was measured in 44 type 1 diabetic patients and in 20 healthy subjects. A significantly raised mean concentration of a free N-terminal fibronectin 30-kDa domain was found in plasma of diabetic patients with proliferative retinopathy as compared with healthy persons (P less than 0.001). A positive correlation was observed between free N-terminal fibronectin 30-kDa domain and von Willebrand factor in plasma of all examined subjects (r = 0.62, P less than 0.01). A similar correlation was present between 30-kDa domain and albuminuria (r = 0.56, P less than 0.01). However, no relationship was found between fibronectin 30-kDa domain and control of diabetes as assessed by fructosamine concentration. The free N-terminal fibronectin 30-kDa domain may be used as a marker of actual endothelial cell dysfunction in diabetes.

Changes in haemostasis and proteins of the acute phase following cardiac surgery.

After cardiac surgery performed with extracorporeal circulation (ECC) involving heart valve prostheses (PHVS, n 12) and after a bypass of the coronary arteries by a venous graft (CABG, n 19), the authors investigated the dynamics of changes of haemostasis on the 1st, 3rd, 6th, 10th and 21st day after operation. As anti-thrombotic treatment after PHVS anticoagulants were used, after CABG thrombocyte inhibitors. On the 1st day after operation in both groups thrombocytes decline, while after the 10th day their numbers increase. From the 6th day there is in both groups a rise of fibrinogen and other proteins of the acute phase (alpha-1-antitrypsin, orosomucoid and ceruloplasmin, while there was a drop of transferrin. On the 3rd and 6th day after operation fibrinolysis activators decline (euglobulin fibrinolysis). These findings suggest an increased risk of thrombophilia during the postoperative period and are probably associated with the release of interleukin-1 after Ecc and the stress of cardiac surgery. In patients with CABG on the 1st day a major drop of thrombocytes occurs, on the 6th day an elevated fibrinogen value was recorded and on the 10th day a reduced fibrinolytic activity, as compared with patients with PHVS. These changes will be, however, associated rather with a greater development of general atherosclerosis in patients with CABG, which leads to a further alteration of haemostasis, rather than with the applied antithrombotic treatment.