Collagen Particles Research Articles

Simultaneous Use of Microfibrillar Collagen Hemostat and Blood Saving Devices in a Canine Kidney Perfusion Model

Intraoperative autotransfusion and topical microfibrillar collagen hemostats have been increasingly used, often simultaneously, in various surgical procedures to minimize intraoperative blood loss and thus reduce the inherent risks of homologous blood transfusion. As moderate amounts of small particles have been shown to pass through filtering devices during intraoperative autotransfusion, concern has been raised over the amount of heterologous collagen fibrils transfused and their effect on the host. We found that 2% of microfibrillar collagen hemostat particles pass through the 20-microns millipore filter contained in the tested autotransfusion device (William Harvey H-4700 cardiotomy reservoir). Using a canine kidney perfusion model, we found multifocal perivascular inflammatory reactions within the renal parenchyma five days after transfusion of filtered autologous blood containing minute amounts of microfibrillar collagen hemostat. The findings demonstrate a strong inflammatory foreign body response to heterologous collagen particles trapped in the microcirculation of the perfused kidneys. It is concluded that despite using filters with a pore size of 20 microns, using intraoperative autotransfusion and microfibrillar collagen hemostats simultaneously creates a potential risk because adverse reactions can be elicited especially within the microvasculature of tissues containing end-arterial circulation.

Effect of antimicrobial protection on tolerance to hepatic chemoembolization with a fibrous collagen carrier.

The effect of antimicrobial prophylaxis on tolerance to hepatic embolization and chemoembolization with collagen particles was examined in porcine liver. Embolization in the 36 animals consisted of hepatic artery injection of 3 mL of collagen (10 mg/mL) alone or combined with one to three drugs (cisplatin, mitomycin, and doxorubicin). Embolization was performed with or without antimicrobial protection, and the animals were killed at 5 days for gross and histologic evaluation of liver and for bacterial culture. When present, liver damage consisted of focal to whole-lobe necrosis. Liver damage was correlated with positive bacterial culture (P less than .002) and was reduced by antimicrobial protection. Vessel recanalization was inhibited by addition of drug to collagen (P less than .001) and by lack of antimicrobial protection (P less than .001). Perivascular necrosis was directly related to vessel size (P = .0001). Antimicrobial prophylaxis is recommended to enhance hepatic chemoembolization safety.

Attachment of Bacteroides gingivalis to collagenous substrata.

The ability of Bacteroides gingivalis 381 to attach to hydroxyapatite (HA) beads, treated with either human type I or type IV collagen, or to particles of bovine bone collagen was studied. All preparations were blocked with human albumin prior to being incubated with 3H-thymidine-labeled B. gingivalis 381 cells. The presence of collagen on HA surfaces (C-HA) significantly promoted attachment of the organism. HA treated with Type IV collagen bound B. gingivalis cells more effectively than did HA treated with type I collagen. Attachment of two additional strains of B. gingivalis to HA was also promoted by collagen. Binding to type I or type IV C-HA occurred rapidly, and equilibrium was attained within 45 min. B. gingivalis 381 cells also bound to particles of bovine bone collagen, and this appeared to be biphasic. Heating the bacteria abolished their ability to bind to C-HA. Attachment of B. gingivalis 381 cells to HA treated with type I collagen was strongly inhibited by the presence of soluble type I or type IV collagen, or gelatin, but not by the presence of human albumin, salivary proline-rich protein 1, or saliva. Human serum, fibronectin, fibrinogen, certain protease inhibitors, and some peptides were also inhibitory. 3H-fibronectin bound to bovine bone collagen particles and blocked the attachment of 14C-B. gingivalis cells. Mild trypsin treatment of the fibronectin-collagen complex restored its ability to promote 14C-B. gingivalis attachment concomitant with the loss of 3H-fibronectin. We suggest that elevated levels of proteases in the gingival sulcus, such as are associated with poor oral hygiene and gingivitis, might remove fibronectin and expose collagen molecules in the basement membrane, thereby promoting the attachment of B. gingivalis cells and facilitating their invasion into gingival tissues.

High-flow-rate arteriovenous malformation model for simulated therapeutic embolization.

A laboratory model of an arteriovenous malformation has been developed. Embolizations of the model were successfully performed under fluoroscopic guidance with collagen particles and cyanoacrylate liquid polymer. The model is inexpensive, easy to construct, and reduces the need for laboratory animals.

Particle Electrophoresis of Compact Bone Tissue

Fresh bovine bone, obtained from the diaphysis of a femur and a tibia, was used to determine the isoelectric point of bone particles. Particle electrophoresis equipment with a flat cell was used with a four electrode configuration. The results indicated that bone has an isoelectric point near pH 5 and that the overall electrophoretic mobility of the bone particles was similar to that of collagen particles obtained from human Achilles tendon.

Ultrastructural study on the release reaction of the platelet. Release II.

The ultrastructural changes of the platelets stimulated by release inducers II such as thrombin, collagen, and latex particles were studied to compare the differences in morphology of release I and II. The characteristics of platelets in response to thrombin were the decrease in number of alpha-granules and marked dilation of the open canalicular system containing fibrin preceded by the disappearance of very dense granules. In the platelets in response to collagen or latex, the release of alpha-granules was initiated after taking the particles into the open canalicular system. In all of the cases, the amount of microfibrils was seen in the cytoplasm, while contraction wave of the microtubules was rare in the cytoplasm during the release and uptake of particles. Accordingly, the release of very dense granules seems to be followed by that of alpha-granules. It is suggested that the microfibrils play a more important role than the microtubules in the release II as well as release I. Furthermore, the contents of alpha-granules may be discharged via the open canalicular system on the release II by thrombin, collagen or latex particles.

Studies on the interaction between collagen and a plasma kallikrein-like activity. Evidence for a surface-active enzyme system.

This study has demonstrated that collagen particles, after exposure to platelet-poor human plasma and subsequent washing, generate a kinin-like agent when incubated with prekinin substrate. The binding of kinin-generating activity to collagen in the plasma collagen incubation mixture occurs rapidly, whereas the loss of this activity in the incubation mixture occurs relatively slowly. The Hageman factor appeared to be necessary for the surface-bound kinin-generating activity, as this activity was absent in collagen exposed to Hageman factor-deficient plasma. These studies have partially characterized the plasma-derived enzymatic activity bound to collagen. Incubation of collagen with plasma caused a concentration-dependent reduction in the kinin-producing activity which was generated by the addition of ellagic acid, a known activator of plasma kallikrein. The kinin-inducing activity bound to collagen is inhibited by soybean trypsin inhibitor, Trasylol, serum C1 inactivator and the plasma alpha(2)-macroglobulin, but not by lima bean trypsin inhibitor. An eluate prepared from plasma-treated collagen, when compared with purified plasma kallikrein, shared a similar inhibitor profile. Selective chemical blockage of the free carboxyl groups on the collagen molecule, or heat denaturation, inactivated the ability of the collagen to generate kinin-like activity after incubation with plasma. Removal of the collagen telopeptides or blockage of the free amino groups failed to affect the collagen-plasma interaction. The binding of partially purified plasma kallikrein to collagen was found to have similar structural and chemical requirements. These data indicate that there is a structural and chemical specificity for the activation and binding of plasma kallikrein-like activity by collagen. These studies suggest that a plasma kallikrein may function as a surface-bound enzyme system.

The Effect of Adenosine Monophosphate, Arcaine and Anti-Inflammatory Agents on Thrombosis and Platelet Function in Rabbits

SummaryThrombi were produced by exposing femoral veins of rabbits to sodium morrhuate followed by partial ligation. The incidence of occlusive thrombosis in 30 min was 95% in animals with or without the infusion of 50-60 ml isotonic saline/kg body weight in 1½–2½ hrs. Comparable infusion of 0.04 M adenosine monophosphate reduced the incidence to 53% (p < 0.01). Arcaine was ineffective even at a toxic concentration. Injection of up to 180 mg phenylbutazone, 25 mg sodium salicylate, or 8.5 mg acetylsalicylic acid (ASA) per kg did not affect the incidence of occlusive thrombosis, but from 15 to 100 mg ASA per kg reduced the incidence to 46% (p < 0.01). Suspensions of connective tissue or collagen particles caused somewhat greater release of platelet-bound 14C-serotonin in heparinized than in citrated, platelet-rich rabbit plasma, and ASA more readily inhibited connective tissue- or collagen-induced aggregation in the latter. With varying connective tissue concentrations, this drug caused a constant reduction in the amount of 14C-serotonin released from the platelets in citrated, platelet-rich rabbit plasma, whereas considerably greater inhibition was observed in many samples of citrated, platelet-rich human plasma.

Demonstration of a Plasmatic Cofactor Different from Fibrinogen Necessary for Platelet Release by ADP and Adrenaline

SummarySecondary wave production and release of platelet factor 4 were studied in washed platelet suspensions and citrate plasma. Induction of a secondary wave requires the presence of large aggregates and a plasmatic cofactor different from fibrinogen. Acetyl-salicylic acid inhibited the production of a second wave both in plasma and platelet suspensions. Plasma taken after the intake of acetyl-salicylic acid induced a second wave in platelets harvested before the intake. The possible production of thrombin or allied enzyme inside the platelet aggregates due to changed internal milieu is suggested as a possible cause of the release reaction induced by ADP and adrenaline. Fundamental differences existed between the release induced by ADP and adrenaline and that induced by collagen, kaolin and other particles.

The release of nucleotides, 5-hydroxytryptamine and enzymes from human blood platelets during aggregation.

1. Adenosine diphosphate (ADP) and adrenaline caused the aggregation of human platelets suspended in plasma containing citrate anticoagulant and stirred at 37 degrees C. The aggregation occurred in two phases and the second phase was associated with the appearance in the plasma of up to 30% of the ATP and 55% of the ADP present in the platelets. The concentration of ADP appearing in the plasma was up to 7 times the concentration added.2. Radioactivity was released by ADP and by adrenaline from platelets labelled with radioactive 5-hydroxytryptamine; this release was closely correlated with the second phase of aggregation and with the release of nucleotides.3. Acid phosphatase, beta-glucuronidase and adenylate kinase were released to a small extent during second phase aggregation by ADP or adrenaline; thrombin and collagen particles caused significantly greater release of beta-glucuronidase than of either acid phosphatase or of adenylate kinase.4. Morphological changes indicating degranulation of the platelets were observed during the second phase of aggregation produced by adrenaline and by ADP.5. The second phase of aggregation, degranulation of platelets, and the release of nucleotides, of labelled 5-hydroxytryptamine and of enzymes, were all inhibited by concentrations of amitriptyline which did not inhibit aggregation.

The Effect of Various Enzymes on the Ultrastructure, Aggregation, and Clot Retraction Ability of Rabbit Blood Platelets

SummaryThe effect of various enzymes on rabbit blood platelets was studied with the following results :1. The proteolytic enzymes trypsin and papain destroyed the ability of the platelets to aggregate with (1) ADP and (2) suspensions of collagen particles. The aggregation ability was not restored by incubation of the platelets in plasma. The platelets treated with these enzymes did not adhere to tendon tissue in vitro. 2. The clot retraction activity of the platelets treated with trypsin and papain was decreased. This effect was especially pronounced with the trypsinized platelets.3. In about 20% of the trypsinized platelets intracellular membrane systems appeared. Trypsin did not cause marked loss of platelet organelles and no obvious effect on the boundary membrane could be detected with the electron microscopical technique.4. No effect of hyaluronidase on the platelets could be found.5. Whereas no effect of lipase from wheat germ could be detected, phospho-lipase A caused pronounced ultrastructural changes within the platelets and the structure of the boundary membrane was altered. Corresponding to these findings release of substances from the platelets was observed by spectrophotometry (with absorption maximum at 260 ιημ) and release of ADP was demonstrated by a specific enzymatic test.6. Platelets treated with neuraminidase aggregated spontaneously when resuspended in citrated plasma or EDTA-plasma. This effect was probably not due to released ADP. It is concluded that besides protein, neuraminic acid (linked to protein) may be of fundamental importance for the aggregation properties of blood platelets.

The heterogeneity of collagen solutions and its effect on fibril formation.

The reconstitution of fibrils from the collagen in solutions extracted from developing connective tissue has attracted a great deal of attention because of the possibility that it is related to collagen fibrillogenesis in vivo. Study of the kinetics of this reconstitution (Gross, 1956; Gross & Kirk, 1958; Bensusan & Hoyt, 1958; Wood, 1958; Wood & Keech, 1960; Wood, 1960a, b; Bensusan, 1960; Bensusan & Scanu, 1960) has helped to elucidate the mechanism of the process. Wood & Keech (1960) found that fibrils form in collagen solutions that have been extracted from purified calf dermis by dilute acetic acid in two distinct, effectively consecutive steps. These occupy an initial lag period and a growth phase. Kinetic and electron-microscope data were satisfactorily explained by postulating that the first step consists of the aggregation of asymmetric collagen particles in solution to form nuclei and that during the second these nuclei grow into fibrils by the accretion of further collagen from solution. The rate of the growth during the second step and the diameter of the fibrils ultimately produced depend to a large extent on the nucleation process, and are related to the number and shape of the nuclei (Wood, 1960a). Nucleation and growth were regarded as closely similar aggregation reactions, the main difference between them being that nucleation is a homogeneous reaction whereas in growth both solid and liquid phases are involved. Certain observations, particularly some aspects of the effect of chondroitin sulphate on the rate of fibril formation (Wood, 1960b; Keech, 1961), could only be reconciled with some difficulty with this simple model; the results suggested that nucleation and growth might be fundamentally different processes. One possibility is that nuclei are the product of aggregation of only a small fraction of the total collagen in solution, the remaining collagen being unable to form nuclei but able to aggregate on nuclei to form fibrils. A similar type of heterogeneity of acidsoluble collagen had been suggested by Buzagh (1942). Boedtker & Doty (1956) found evidence from light-scattering data for the presence of a small amount of aggregated material in solutions of citrate-soluble collagen, which could only be removed by prolonged centrifuging. Fessler (1957, 1960a, b) observed that, under certain experimental conditions, collagen which was precipitated on warming solutions of neutral-salt-soluble collagen to 370 could be divided into two fractions. One of these ('fraction A') redissolved when the precipitate was kept at 0° for 48 hr.; the other ('fraction C') did not. In addition, a portion of the original collagen ('fraction B') was not precipitated. Except for these studies there appears to be little evidence from their physical properties that the collagen molecules in a solution of collagen are not identical. However, Jackson & Bentley (1960) have recently concluded that extracts of collagenous tissue contain collagen molecules of different ages. The work reported here was designed to test the hypothesis that collagen molecules in solution are heterogeneous with respect to their ability to aggregate into fibrils. A preliminary account of this work has been published (Wood, 1962).