Thomas A. Chvapil

Identification of the depth of burn injury by collagen stainability.

Heat-denatured collagen in burned skin stains red instead of blue in Massons 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.

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.

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.

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.

Reaction of vaginal tissue of rabbit and of cheek pouch of hamster to inserted collagen sponges treated with either zinc or copper

Abstract Resilient collagen sponges either untreated (CS) or treated with zinc (Zn-CS) or copper (Cu-CS) were inserted intravaginally into 41 adult New Zealand white rabbits. Ultimately, a distal vaginal ligature proved the most effective method of keeping the CS in place. A group of sham-operated control animals had ligated vaginas without sponges. After 10 days, the animals with ligated vaginas were killed. Vaginal secretions and vaginal and uterine tissues were analyzed for zinc and copper content. The morphology of vaginal tissue and of the various CS was examined grossly and microscopically. The CS caused minimal but definite irritation to the vagina, seen mostly in localized lysis of epithelial cells, the presence of a few leukocytes in the submucosal layer, and cellular infiltration of the CS. The morphology of vaginas with Zn-CS inserted did not differ from those of sham-operated animals. No signs of inflammation in vaginal tissue and no infiltration of leukocytes appeared. Conversely, Cu-CS produced striking inflammatory changes with diffuse necrosis of epithelial lining, leukocytic infiltration of the lamina propria, and extensive cellular infiltration of the sponge. The semiquantitative rating of the morphology of the Cu-CS was 14, hence unacceptable, while that of CS was six and that of Zn-CS was only two. A similar set of sponges was inserted for three days in the cheek pouches of golden hamsters. Mucosal cell linings were not affected by CS or Zn-CS. However, Cu-CS produced marked inflammation, ulceration of epithelial cells, and leukocytic infiltration of subepithelial connective tissue and of inserted sponges. We conclude that Zn-CS did not produce a topical toxic effect in either rabbit vaginas or hamster cheek pouches. Cu-CS proved unacceptable because of the toxic effects that these caused in both models.

STM imaging of molecular collagen and phospholipid membranes.

The application of STM to biological materiais 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 15Å in diameter with a periodicity of about 30Å 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 ∼4–5Å 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.

Laboratory and clinical testing of an intravaginal contraceptive collagen sponge.

Abstract The efficacy of collagen sponge (CS) used as intravaginal contraception barrier depends largely on the continuous retention of CS in the upper vault of the vagina, prompt absorption of ejaculate into CS and inactivation of sperm by acidity of the sponge. All three factors were tested in in vitro studies, and retention was also evaluated in human volunteers. Retention seems to depend on adequate resilience of the CS. This can be controlled by the degree of crosslinking of collagen fibers. With adequate resilience the expulsion rate, originally 33%, was reduced to 2.5%. Our tests were conducted by 30 volunteers who tested eight types of CS in a total of 213 insertions. The rate of fluid absorption by the CS could be significantly increased by treating the sponge with a surfactant, such as Triton X-100 (0.1%). It was found that prewetted sponge absorbs fluid instantaneously; thus, modification of CS with surfactant is not necessary. CSs were equilibrated to various pHs (4 to 7). Extracts from these were applied to fresh human spermatozoa to test the effects on sperm motility. At pH 5.0 the motility stopped after 4 min. while at pH 7.0 the cells were still active after 30 min. observation. This is one of the reasons why CSs used as contraceptive should have acidity around 5.0. The various methods of insertion and removal of CS were tested by five volunteers for 5 consecutive days. Both digital and applicator insertion were found acceptable. Removal of CS was facilitated by a loop or strand of umbilical tape fixed to the CS.

Ultrastructure of the Vaginal Tissue of Rabbits Treated With Collagen Sponge Alone and Medicated with Zinc and Copper Salts and Copper Wire

We tested the reaction of rabbit vaginal epithelium to inserted collagen sponges which have been proposed for human use as intravaginal contraceptive barriers. Some collagen sponges were medicated with zinc and copper sulfate and oher (untreated) sponges were wrapped loosely with copper wire. Scanning and transmission electron microscopy was performed on vaginal tissue after 10days of intravaginal contact with the sponges. The characteristic longitudinal rugate arrangement of polygonal, flat epithelial cells with a dense, even covering of microvilli seen in intact vaginas was not changed by the presence of a collagen sponge for 10days. In the presence of zinc sulfate in the sponge, the microvilli were in general shorter; however, the cells remained flat and continuous, forming visible, longitudinally oriented folds. Sponges containing copper sulfate induced marked changes in the epithelial lining of the rabbit vagina. Cells of differing sizes protruded above the plane of the surface. These cells had a hypertrophic appearance with narrow neck and widening at the top. Microvilli in these cells were very short and widely spaced, in contrast to the profuse microvillous projections in other vaginal samples. Copper wire induced definite abnormalities which were classified into three categories: (1) altered appearance similar to that caused by copper sulfate; (2) localized complete denudation of cells; (3) necrotic changes with penetration of the continuity of epithelial lining and excessive leukocytic infiltration of the lesion at the site of direct contact of the vaginal mucosa with the wire.

Studies on vaginal malodor

SummaryVarious factors contribute to the formation of malodor in intravaginal collagen sponges (CS) worn for several days by sexually active persons. To study this problem we developed an in vitro model: CS was infiltrated with semen and incubated at various pHs or in the presence of various drugs, at 37° and 95% O2-5% CO2 in humid atmosphere. The odor was measured by subjective olfactometry as well as quantitated and characterized by gas liquid chromatography. Based on previous findings concerning the disappearance of polyamines from the semen in sponges with malodor and present detection of maximal malodor formation at pH 7.4 and absence of malodor with heat-inactivated semen we propose that malodor forms through the enzymatic oxidation of polyamines or diamines possibly by diamine oxidase (DAO) of the semen. Direct assay of DAO showed that all factors inhibiting or stimulating the malodor affected the activity of DAO in the same direction. Thus, while copper ions were stimulatory to odor and DAO activity, zinc ions, acid environment (3.4 pH), exogenous spermine administration, isoproniazid and pargyline, both known inhibitors of MAOs and DAOs, reduced malodor and DAO activity.We found that more intensive malodor developed in sponges made of polyurethane, cellulose or spongin (sea sponge) than of collagen.Whole semen caused greater malodor formation than prostatic fluid and seminal plasma. Both washed spermatozoa and vaginal secretion did not induce any subjectively detectable offensive odor in sponges. Addition of a mixture of nonpathogenic microorganisms to the semen did not enhance the formation of subjectively detectable malodor. GLC showed characteristic differences between volatile compounds formed by ejaculate and bacteria. While most of the malodor formed by the semen-CS system was enzymatically related, some evidence indicates a portion of malodor to be nonenzymatic process.It is concluded that the most feasible method preventing the semen related vaginal malodor is to keep the CS acidic (pH 5) and to impregnate sponges with low concentrations of zinc sulfate (40 mg/sponge), unless the sponge is removed 24 h after the intercourse, washed and reinserted.