Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

Effects of grape seed extract on properties of type I collagen scaffolds

To obtain a material with potential for use in tissue engineering, anionic collagen was obtained from porcine serosa (S) and bovine tendon (T) by alkaline hydrolysis for 72h. Part of this collagen was mixed with water to obtain 4 % (weight/weight) collagen suspension and part was solubilized in acetic acid pH 3.5 to obtain 1.5% (w/w) gel. The suspensions were mixed with their respective gels (2:1) (suspension: gel) and grape seed extract, whose main product is proanthocyanidin, was added at concentrations of 0.03% and 0.5%, thus obtaining the scaffolds SC (serosa collagen suspension and gel), TC (tendon collagen suspension and gel), SCP003 (SC with 0.03% extract), TCP003 (TC with 0.03% extract), SCP05 (SC with 0.5% extract added) and TCP05 (TC with 0.5% extract). The materials were analyzed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and characterized by phosphate buffered saline absorption assay and in vitro biological stability assay. By DSC it is observed that the addition of 0.5% of extract increases the denaturation temperature (Td) of collagen, indicating that at this concentration the extract acts as polymer crosslinking agent. SEM shows disorganized cross-section pores in all scaffolds, not exceeding 130 μm. Absorption and degradation assays indicated that the addition of 0.5% extract increases the absorption of phosphate buffered saline (PBS) by the scaffolds and decreases the degradation percentage by collagenase. These results suggests that the scaffolds can be used for different applications, e.g. as hemostatic agent.

Abstract 157: A Highly Reproducible, Clinically Relevant Large Animal Model of Myocardial Ischemia and Heart Failure

Background: Heart failure (HF) is the leading cause of mortality in United States. Animal models used to test novel HF therapeutics are inadequate as pathological features of human HF are not replicated. Available large-animal swine myocardial ischemia (MI) models mimic human HF; however, do not achieve ejection fractions (EF) less than 40%. Objective: We sought to develop a reproducible swine MI model with EF below 35%. Methods: Yucatan miniature pigs (n=18) were anesthetized, catheterized and collagen suspension (COL) of microfibrillar hemostat (INSTAT MCH, Ethicon) mixed in contrast and saline solution was injected into the left anterior descending coronary artery (LAD) under fluoroscopic guidance. COL injections started distally and filled side branches during catheter retraction. Complete transient LAD occlusion was confirmed at 2hrs by coronary angiography. Cardiac function was evaluated at 3mos using a 1.5T MRI System (Siemens Magnetom Avanto). LV volumes at end systole (LVESD) and diastole (LVEDD), EF, wall thickness (WT) in area at risk (AAR) and normal zones (NZ) were calculated. Uninjected Yucatan pigs (n=3) served as controls. Results: Twelve of 18 pigs survived COL infarction. At 3mos, EF was reduced in COL injected pigs compared to control (27.0±2.3 vs. 61.6±1.3%, respectively, P<0.001). LVESV and LVEDV were 4- and 2-fold higher and WT in AAR and NZ were reduced 6- and 2-fold, respectively in COL injected compared to controls. Conclusions: A reproducible large animal model of MI was produced by catheter based LAD collagen delivery resulting in EFs below 35% and progression to HF. By closely resembles human HF, it a highly useful for testing potential HF therapies.

Influence of Acidity in Wet-Chemical Synthesis on the Joint of Nano-Sized β-Tricalcium Phosphate Particles with Collagen Fibrils in Their Composites

β-tricalcium phosphate (β-TCP)/collagen composites are in the limelight for their biomedical applications. It is believed that joint status of β-TCP particles with collagen fibrils plays key roles in both osteoconductivity and biodegradability of composites. In this work, the influence of acidity during synthesis on the joint status between nano-sized β-TCP particles and collagen fibrils is investigated. The composites are characterized by X-ray diffractometer and Field Emission Scanning Electron Microscope. The results show that the joint status of nano-sized β-TCP particles with collagen fibrils in the composites depends on the acidity in collagen suspensions. A desired joint status with obvious disassembled collagen fibril, good particle dispersion and strong boding between the particles and the fibrils could be obtained when acidity of the collagen suspension is pH 2.

Hereditary abnormality of platelet aggregation attributable to nucleotide storage pool deficiency

An abnormality of platelet aggregation has been detected in six family members with mild bleeding tendencies. In citrated platelet-rich plasma, primary aggregation induced by ADP or epinephrine and agglutination in response to ristocetin were present but second wave aggregation and aggregation in response to collagen suspension were absent or greatly reduced. Sodium arachidonate-induced aggregation was normal although aggregation in response to prostaglandin G2 was reduced and depended entirely on the presence of plasma or ADP. Further tests indicated that the platelets produced prostaglandins but did not release ATP in response to thrombin or sodium arachidonate. Platelets from the patients were found to contain reduced amounts of ADP and 5- hydroxytryptamine and to be unable to retain radioactivity during prolonged incubation at 37 degree C with radiolabeled 5- hydroxytryptamine. Although electron microscopy revealed an absence of very dense bodies, the platelets appeared otherwise normal. The findings are discussed in relation to previous studies of nucleotide storage pool deficiency and the light they shed on platelet physiology in general.

Hereditary abnormality of platelet aggregation attributable to nucleotide storage pool deficiency

An abnormality of platelet aggregation has been detected in six family members with mild bleeding tendencies. In citrated platelet-rich plasma, primary aggregation induced by ADP or epinephrine and agglutination in response to ristocetin were present but second wave aggregation and aggregation in response to collagen suspension were absent or greatly reduced. Sodium arachidonate-induced aggregation was normal although aggregation in response to prostaglandin G2 was reduced and depended entirely on the presence of plasma or ADP. Further tests indicated that the platelets produced prostaglandins but did not release ATP in response to thrombin or sodium arachidonate. Platelets from the patients were found to contain reduced amounts of ADP and 5- hydroxytryptamine and to be unable to retain radioactivity during prolonged incubation at 37 degree C with radiolabeled 5- hydroxytryptamine. Although electron microscopy revealed an absence of very dense bodies, the platelets appeared otherwise normal. The findings are discussed in relation to previous studies of nucleotide storage pool deficiency and the light they shed on platelet physiology in general.

Platelet-Derived Growth Factor Production During the Platelet Release Reaction Independent of The Endoperoxide Pathway.

Ross et al (P.N.A.S. 71:1207, 1974) showed that a platelet-derived growth factor (PGF) promotes the proliferation of arterial smooth muscle cells (SMC) and thus may be important in atherogenesis. Although PGF is found in plasma when platelets are exposed to thrombin or collagen it has not been conclusively established that the material is released from platelet granules. Inhibitors of the release reaction and of platelet cyclo-oxygenase were used to examine the relation between release of 14C-serotonin (14C-5HT) from rabbit platelets and the appearance of PGF in the suspending fluid. Washed platelets (2 × 106/mm3) were suspended in Tyrode solution containing albumin and apyrase at 37°C and treated with collagen suspension, thrombin or ADP. After 5 min. the platelets were removed and the supernate assayed for 14C-5HT, malondialdehyde (MDA) and PGF. Incorporation of 3H-thymidine into cultured rabbit aorta SMC DNA was used as an index of PGF concentration. There was a direct relation between the concentration of collagen or thrombin and the amount of PGF in the supernate. Indomethacin (20μM) or sulfinpyrazone (ImM) reduced 14C-5HT release in response to collagen from 45-65% to 5-8%, completely blocked MDA formation and reduced the amount of PGF in the supernate to 33-44% of control values. These inhirbitors reduced release caused by thrombin (5U/ml) from 90-95% to 85-88% and, although they completely blocked MDA formation, did not decrease PGF. PGF was not made available by aggregation of platelets with ADP.(Release does not occur with rabbit platelets exposed to ADP). Thus, the appearance of PGF does not depend on primary aggregation but is associated with the release reaction and is independent of the endoperoxide pathway.

Experimental Modification of the Platelet Release Reaction Induced by Collagen

Collagen-induced release of platelet constituents can be divided into two kinetically-distinct phases, only one of which is associated with platelet aggregation, and the aggregation-independent release is less susceptible to inhibition by pharmacological agents (Drummond and Gordon, 1974). Minor variations in experimental conditions alter the release reaction profile. Collagen was incubated with platelet rich plasma (PRP) for one minute at 37°, under conditions in which no aggregation occurred ( + 3nM EDTA or in absence of stirring). The reaction was ‘terminated’ by addition of ice-cold EDTA-saline and samples were then centrifuged (14,800 g) under the conditions described in the table.No significant release was detected under any of the above conditions if saline were substituted for collagen suspension.These results indicate that the storage temperature and time after centrifugation are as important as the centrifugation conditions themselves, and suggest reasons for discrepancies reported in previous studies of collagen induced release kinetics.Drummond, A. H. and Gordon, J. L. (1974). Brit. J. Pharmacol. 52, 130 P.

Blood Coagulation and Platelet-function Parameters before and during Parenteral Administration of Medroxyprogesterone Acetate as a Contraceptive Agent

SummaryThe effects of medroxyprogesterone acetate on blood coagulation and platelet collagen aggregation were studied in nineteen women. After parenteral administration of the compound for 4 and 7 months, the activated partial thromboplastin time showed a significant decrease. The percentage of the prothrombin time test rose after 4 months, but not significantly after 7 months ; factor II decreased after 7 months of treatment. The reaction time during which no aggregation occurred after addition of a collagen suspension to a stirred platelet-rich plasma, was shorter after both 4 and 7 months of treatment. Other coagulation and platelet-function parameters were not altered. Although a trend toward hypercoagulability was detected, the changes were not the same as those reported for combined oral contraceptives.