Collagen fibers as a temporary scaffold for replacement of ACL in goats.

ACL substitutes made of braided or plied purified collagen fibers and cross-linked with hexamethylenediisocyanate were implanted into a total of 14 adult goats to achieve resorption within 8 to 10 months. Two types of collagen fiber prostheses differing in degree of collagen purification were tested. The implants were harvested 2 to 11 months postimplantation, tested for mechanical strength, and evaluated by morphological methods. In the first group (n = 5), the less purified and less cross-linked collagen fiber ACL implant induced fast connective tissue ingrowth. At 6 months postimplantation, 40 to 60% of the collagen implant was resorbed. No studies on breaking strength were done in this group. In the second group, highly purified and more crosslinked ACL implants were less infiltrated by cells and were resorbed only by 10 to 20%. Still, the breaking strength was decreased to 10% of the original implant strength. In the second group, the fixation of the ACL implant in the bone tunnel with a bone wedge was insufficient (n = 6); however, additional fixation with metal screws was successful (n = 3). We conclude that cross-linked collagen fibers alone cannot be used as a safe ACL substitute as they quickly lose mechanical strength despite limited biodegradation.

Effects of electrocautery on midline laparotomy wound infection.

This study compared the healing of midline fascial incisions made with either scalpel or electrocautery and inoculated with Escherichia coli in 57 Sprague-Dawley rats. At 7 days, tensile strength was significantly less when incisions were made with electrocautery than with a scalpel. Additionally, would strength was inversely related to the concentration of the inoculum of E coli. The use of electrocautery was also associated with more frequent bacteremia at 48 hours and higher mortality at 7 days. Our results suggest that the technique used to incise the abdominal fascia influences subsequent wound healing, particularly in contaminated wounds.

Inert wound dressing is not desirable.

Four Yorkshire piglets were inflicted with a total of 92 split-thickness wounds 4.8 cm2 in area and 400 microns deep. The wounds were treated with eight dressing regimens under the same experimental design. The rate of reepithelialization of the wound was quantitated by a morphometric method. The magnitude of inflammatory reaction of the wound to the dressing was scored from histological slides. The results indicate a relationship between the rate of reepithelialization of split-thickness wounds and the inflammatory response of the wound to the dressing. Dressings, such as collagen sponge, polyethyleneglycol, Duoderm, and lanolin ointment, induce moderate to severe inflammatory changes when placed on the wounds. These wounds reepithelialize significantly faster than control, gauze-covered wounds. This contrasts with inert dressings, such as hydrated hydrogel membrane, Carbopol 934P, or Silvadene cream, which did not affect the rate of reepithelialization when compared with the healing of control wounds. Simultaneously, these dressings induced no or minimal inflammatory reaction in the wound tissue. Only when the inflammatory reaction to the wound dressing was excessive (methylcellulose) was the rate of reepithelialization of the wounds significantly inhibited in comparison with control wounds. We hypothesize that wound dressings, by inducing inflammatory reaction, enhance healing by activating cells, such as macrophages or fibroblasts, that produce growth factors and other mediators of the repair process.

Effect of electrocautery on wound healing in midline laparotomy incisions.

The effect of electrocautery on midline fascial wound healing was studied in 108 Sprague-Dawley rats. Midline wound tensile strength was significantly reduced in fascia incised with the coagulation current compared with the cutting current or scalpel. In addition, tissue necrosis and inflammation as well as adhesion formation between the incision and abdominal viscera were more extensive in animals with incisions made using coagulation current. The results of the study indicate that the use of electrocautery coagulation current is associated with increased tissue damage and a significant reduction in the tensile strength of healing wounds. The contribution of electrocautery to wound complications in patients needs further evaluation.

Occlusion of the vas deferens in dogs with a biocompatible hydrogel solution.

The surgically exposed vasa deferentia of 21 dogs were injected, under visual inspection, with Hypan N-90 acrylic hydrogel (50-150 microL). The hydrogel was deposited inside the vas lumen via a 22-gauge Teflon intercatheter. The solution gelled within 120 seconds. Semen was collected by manual collection and analyzed for volume, sperm count and viability. After occlusion with 150 microL of the polymer in the distal direction (direction of the testis), the volume of ejaculate (2.2 mL) did not change. Subsequent samples showed no viable or dead spermatozoa. The stained smears of the ejaculate showed the presence of cell debris, granulocytes and few epithelial cells. When the vas was injected with 50 microL of the polymer in either a distal or proximal direction, the occlusion effectiveness was 75% and 25%, respectively. In the proximal direction, granulomas were noticed in the vas wall where semen leaked through the injection port. Histology of successfully occluded vasa (with Hypan) showed no cellular reaction or fibrotic changes in the proximity of the polymer. For less than or equal to 20 weeks after vas occlusion, no evidence of abnormal morphology was found in the epididymal and testicular tissue. This highly biocompatible polymer solution, when gelled in contact with tissue fluid, offers safe and effective occlusion of the vas with the promise of reversibility.

STM imaging of molecular collagen and phospholipid membranes.

The application of STM to biological materials has been limited by poor conductivity, sample geometry and stability of biological materials. In this paper we describe an STM study of the monomeric helical forms of collagen, a stable, conductive and widely prevalent structural protein. We have also used STM to image artificial Langmuir DPE (dipalmitoyl phosphatidyl ethanolamine) phospholipid membranes. Both molecular collagen and the phospholipid membranes were dried in air on highly oriented pyrolytic graphite (HOPG). Our STM images of collagen dried on HOPG reveal strands 15 A in diameter with a periodicity of about 30 A which correlates with that known to occur in collagen. Spikes which periodically protrude from strands in our STM images of collagen appear to represent pyrrolidine ring structures in the amino acids proline and hydroxyproline. Thus, we report the first STM imaging of native biomolecules revealing intramolecular details and what appear to be specific amino acids. STM imaging of phospholipid membranes show a lattice pattern with densities spaced approximately 4.5 A apart. These are thought to represent individual phospholipid molecules in an artificial membrane formed on the HOPG. We believe STM and its related technologies will have great future utility in biomolecular studies.

An optimal morphometric method for quantitating wound epithelization.

A new, inexpensive method for quantitative evaluation of reepithelization of shallow split thickness wounds in piglets is described. Wounds, 2.2 X 2.2 cm and 0.4 mm depth are inflicted by an electro-keratome knife in domestic piglets. At a specific time after wounding, the wound area is excised and processed for histology. A computer simulation, based on a randomized systematic sectioning of an entire wound, was used to conclude that only eight sections from the 2.2 X 2.2 cm wound are needed for the final evaluation. The results showed that the above method allows for determination of the epithelization magnitude within +/- 5% at a 95% confidence limit. It was found that in 15 kg piglets 50% epithelization of the above wounds was achieved in 65 hr; however, there exists a great interindividual variability. The rate of epithelization is age dependent and significantly faster in 7 kg body weight piglets than in those weighing 40 kg. The epithelization rate was the same at both the wound edge and the center of the wound, stressing the importance of hair follicles as a source of epithelization.

Comparative study of four wound dressings on epithelization of partial-thickness wounds in pigs.

Sixteen shallow wounds were inflicted in each of five Yorkshire white female piglets, 18-20 kg body weight, by a 2-cm diameter, fast-rotating abrasive disc. The injury is similar to a second-degree burn. The wounds were dressed with one of four dressings: Duoderm (Squibb), Op-Site (Smith & Nephew) [corrected], and collagen sponge, covered with either occlusive or semiocclusive polyurethane film (Datascope Corp.). The last two dressings were moistened with saline before application. The rate of epithelization by planimetric quantitation after 3 to 5 days was the same regardless of the dressing used, although the epithelium layer was thicker in wounds treated with Duoderm. Wounds dressed by either of the collagen sponge materials showed a better appearance when visually scored. Wounds dressed with Duoderm or Op-Site were often macerated; Duoderm's paste-like material remained on the wound and was difficult to remove without inflicting discomfort to the patient. Duoderm, and also Op-Site, adhered much more strongly to the intact skin than either type of collagen sponge dressing.

Identification of the depth of burn injury by collagen stainability.

Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.

Reaction of vaginal tissue of rabbits to inserted sponges made of various materials.

Sponges of the same size made of collagen (CS), polyurethane (PU), polyvinylalcohol (PVA) and acetylcellulose (AC) were inserted for 10 days in the vaginas of 22 rabbits. Light and scanning electron microscopy of the vaginal wall and its mucosal lining showed signs of cytotoxicity only with PU and AC while CS and PVA picture did not differ from sham controls. In order to explain the reasons for the toxic effects, all sponges were extracted into aqueous or organic solvent media and analyzed by gas liquid chromatography. Only several minute peaks in organic solvents were found. Extracts of all sponges tested for cytotoxicity in fibroblast cultures showed significant inhibition of H3-thymidine uptake. Nevertheless, extract of collagen sponge was significantly less cytotoxic than the extracts of all other sponges.

Studies on vaginal malodor. I. Study in humans.

Forty-two percent of collagen sponges tested as an intravaginal barrier contraceptive method developed malodor when retained for 5 days. Only 4% developed odor when the sponge was removed within 24 hours after intercourse, rinsed, and reinserted. While sexually active volunteers found odor in 37% of the sponges, odor formed only in 4% of the sponges worn by sexually inactive users. No difference in the rate of odor formation was found when neutral pH (7.0) and acid pH (3.4) collagen sponges were tested, although we believe that a pH 3.4 is too acid and promotes odor formation. The optimal pH of the sponge should be 4.5 to 5.5. Malodor was efficiently extracted from sponges by washing in acid milieu of tap water and vinegar or 0.1 M acetate buffer, pH 4.0. Alkali extraction procedures were ineffective, and lukewarm water was slightly less effective than acid extraction of odor. At the time of malodor development, the high content of polyamines (putrescine, spermine, spermidine) in the ejaculate decreased to undetectable values. We conclude that the ejaculate is the major source of malodor formation in intravaginally worn collagen sponges. Removal, rinsing optimally in vinegar solution, and reinsertion within 24 hours after intercourse reduces the chance of malodor formation.

PIP: 42% of collagen sponges tested as an intravaginal barrier contraceptive method developed malodor when retained for 5 days. Only 4% developed odor when the sponge was removed within 24 hours after intercourse, rinsed, and reinserted. While sexually active volunteers found odor in 37% of sponges, odor formed only in 4% of sponges worn by sexually inactive persons. No difference in the rate of odor formation was found when neutral pH (7) and acid pH (3.4) collagen sponges were tested, although it is believed that a pH of 3.4 is too acidic and promotes odor formation. The optimal pH should be 4.5-5.5. Malodor was effectively extracted from sponges by washing in acid milieu of tapwater and vinegar or .1 M acetate buffer, pH 4. Alkali extraction procedures were ineffective, and lukewarm water was slightly less effective than acid extraction of odor. At the time maloder develops, the high concentration of polyamines (putrescine, spermine, and spermidine) in ejaculate decreased to undetectable levels. It is therefore concluded that the ejaculate is the major source of malodor formation in intravaginally worn sponges.

Identification of ejaculate derived propylamine found in collagen sponge contraceptives.

Propylamine is identified as one of the compounds present in odoriferous intravaginal contraceptive sponges from sexually active women. This compound also appears in samples of human ejaculate incubated at 37 degrees C for seven days. We have identified propylamine by gas liquid chromatography, mass spectroscopy and as N-propylbenzoylamide. Evidently the compound forms enzymatically from spermine and spermidine. We believe that this is the first time that propylamine has been identified as forming from human tissue.