In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

Human bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.     

Studies of the mechanisms of bradykinin generation in hereditary angioedema plasma

BACKGROUND: Factor XII-dependent bradykinin formation is thought to be responsible for the swelling associated with the various forms of C1 inhibitor deficiency, and complement activation is augmented during attacks of swelling. OBJECTIVES: To further elucidate the interactions of the kinin-forming cascade that lead to complement activation during attacks of swelling and to determine whether fibrinolysis is augmented as well. METHODS: We compared spontaneous and kaolin-induced activation of normal plasma with the plasma of patients with hereditary angioedema. RESULTS: Hereditary angioedema plasma demonstrated augmented factor XII activation, production of factor XIIf, prekallikrein activation, and high-molecular-weight kininogen cleavage, and, as a result, bradykinin formation was markedly increased. Baseline levels of C4a and plasmin-alpha 2 antiplasmin complexes increased, and, on activation with kaolin, levels increased further. CONCLUSIONS: All parameters indicative of activation of the bradykinin-forming cascade are activated in hereditary angioedema plasma vs normal plasma. Production of factor XIIf, demonstrated for the first time in whole plasma, may be responsible for C1 activation based on C4a production. The factor XII-dependent fibrinolytic cascade is also activated.     

The effects of separation and reunion on the behavior of mother and infant squirrel monkeys

Mother and infant squirrel monkeys that lived together in a socially restricted environment were separated for a period of seven days after the infants had become relatively independent, and were then reunited. Behavioral observations before, during, and after separation indicated that (1) female infants became independent of their mothers earlier than males, (2) neither mothers nor infants were severely affected by separation, and (3) an increase in attachment occurred following reunion only when the mother had a limited history of maternal experience. These results suggest that (1) certain characteristics in the maternal behavior of the squirrel monkey facilitate readjustment in mothers and infants following maternal separation, (2) maternal experience can influence the mother-infant relationship following a period of separation, and (3) the independence of infants from their mothers may be a function of both sex and species.     

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test

The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal and Aqualox elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion. The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.     

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.     

Low-grade intravascular coagulation and reticuloendothelial function

The present study evaluated the influence of experimentally produced intravascular coagulation on reticuloendothelial (RE) stability. Intravascular coagulation was initiated by the intraperitoneal injection of bovine thrombin (500 U/100 g body wt) into male rats. RE function was evaluated by the vascular clearance of an 131I-labeled RE test colloid. Thrombin injection resulted in a transient (0.5-2 h) (P less than .05) depression of the phagocytic index (K) with maximal depression at 1 h postthrombin challenge. The phagocytic index was unaltered after injection of saline or heat-inactivated thrombin. Vascular clearance depression was primarily due to a 37% decrease (P less than .001) in hepatic Kupffer cell colloid clearance and was associated with increments in lung (82%) and marrow (100%) colloid localization with no splenic alterations. While intravascular coagulation was associated with decreased hepatic blood flow at 30 min and 120 min, sinusoidal flow was normal during maximum RE impairment at 60 min. The in vivo clearance depression was not reflected as an intrinsic Kupffer cellular deficit when evaluated in an in vitro system. The results indicate a transient RE dysfunction during intravascular coagulation, the mechanism of which remains to be elucidated.     

Improved detection of cytomegalovirus viremia in AIDS patients using shell vial and indirect immunoperoxidase methodologies.

One hundred twelve peripheral blood specimens were tested for the presence of cytomegalovirus (CMV) by the tube culture indirect immunoperoxidase (TC-IPA) procedure, the shell vial assay [shell vials were pre- and postinoculation treated with medium containing 2 of 10% fetal bovine serum (FBS) or 100 micrograms% cortisol] (SV-IFA), and conventional (MRC-5) tube cultures (TC-CPE). CMV was detected in 25 (22%) of the 112 specimens tested by at least one of these methods. The detection/isolation of CMV among the 25 positive specimens in shell vials maintained with 2% FBS, 100 micrograms% cortisol + 2% FBS, and 10% FBS was 36, 44, and 52%, respectively. Detection/isolation of the virus from blood by TC-IPA and TC-CPE was 52% and 76%, respectively. A significantly greater CMV detection rate occurred using TC-CPE compared to SV-IFA treated with medium supplemented with an FBS concentration of 2% (P = .0132), but not medium containing the higher serum supplement or the glucocorticoid (P greater than .05). Differences in the identification of a CMV viremia were observed by IPA, SV-IFA, and TC-CPE methodologies on a patient-to-patient basis, denoting the necessity of incorporating each methodology into the CMV screening panel. Demographic analysis of 82 AIDS patients showed a CMV viremia prevalence of 9% (2/28) in intravenous drug users, 57% (27/47) in homosexual patients, and 22% (2/9) in heterosexual and transfusion patients. Overnight (24 hr) storage of whole blood at 4 or 24 degrees C, respectively, reduced CMV recovery by 40% and 65%, when tested by TC-CPE.(ABSTRACT TRUNCATED AT 250 WORDS)