Method Of Producing Collagenic Material For The Closure Of Lesions

Abstract

The production of collagenic material for the closure of lesions in enhanced, and thus the healing of a lesion accelerated by applying histamine to the lesion and then radiating the tissue cells bounding the lesion with electromagnetic radiation. The electromagnetic radiation being in the non-ionizing range of frequency, and having an intensity less than one milliwatt.

Description

This invention relates to a method of producing collagenic material for the closure of lesions.

It has already been noted in an article by C. Romero-Sierra, Susan Halter and J.A. Tanner entitled "Effect of an Electromagnetic Field on the Sciatic Nerve of the Rat," in "The Nervous System and Electric Currents," Vol. 2. Edited by Norman L. Wulfsohn and Anthony Sances, Jr., Plenum Press, 1971, page 84, lines 9-16, that away from the center of an electromagnetic field in rat specimens subjected to electromagnetic radiation for a short period of time only, collagen appeared in tests to have increased in amount and collagen-like fibrils, different from those found in rat specimens which were not subjected to electromagnetic radiation, appeared protruding from the Schwann cell cytoplasm into the endoneural space. The tests were carried out on the exposed sciatic nerve of a right leg of the specimen rats.

Whilst these test showed that collagen increased in amount using small dosages of electromagnetic energy of low intensity, the increase in collagen was insufficient to indicate the use of electromagnetic radiation in a practical manner for the closure of lesions.

It is an object of the present invention to provide a method of producing collagenic material which can be used in a practical manner for the closure of lesions.

According to the present invention there is provided a method of producing collagenic material for the closure of lesions, comprising applying histamine to the lesion, and radiating tissue cells bounding the lesion with electromagnetic radiation, the electromagnetic radiation being in the non ionizing range of frequency, and having an intensity less than one milliwatt, so that the reproduction of collagenic material by the tissue cells is enhanced.

In the accompanying drawings which illustrate by way of example, comparative tests which have been carried out, with embodiments of the present invention.

FIG. 1 is a diagrammatic view of an adult rat having a lesion coated with histamine and subjected to electromagnetic radiation to produce collagenic material.

FIG. 2 is a magnified transverse view (450X) of the incision area of a control rat wherein the incision has been treated with saline only,

FIG. 3 is a magnified transverse view (450X) of a control rat wherein the incision has been treated with saline and radiated with an electromagnetic field,

FIG. 4 is a magnified (450X) transverse view of the incision area of a rat treated with histamine only, and

FIG. 5 is a magnified (450X) transverse view of the incision area of a rat treated with histamine and radiated with an electromagnetic field.

In FIG. 1 there is shown an adult rat 1 which has been anesthetized with ether and Nembutal. The back of the rat has been shaved and a horizontal lesion or incision 2 made with scissors (not shown) in the area just below the scapulae.

A rod electrode 4, is connected by a cable 6 to an electromagnetic energy generator 8 for producing a 27 MHz field of electromagnetic radiation. The generator 8 is connected by a cable 10 to a power source 12.

In the tests according to the present invention rats 1 after being anesthetized were given the incision 2, and histamine was applied to the incisions 2. The incisions 2 were then radiated with electromagnetic energy from the electrode 4 with the electrode 4 placed lengthwise along the incision 2 and held proximal thereto. These tests were compared with tests wherein the incisions 2 in other rats 1 were treated with saline and not radiated, and others treated with histamine and not radiated.

THE TESTS

Twelve adult rats 1 weighing from 200 to 300 grams were used in this study. The rats were divided into four groups: control rats treated only with saline applied locally to the incision 2; test rats 1 treated with two drops of saline to the incision 2 and 15 minutes exposure to a VHF (27MHz) field with the electrode 4 held 0 - 5 mm from the area; control rats 1 treated with two drops of histamine diphosphate (1.0 mg per ml); applied to the incision 2 and test rats 1 treated with two drops of histamine disphosphate (1.0 mg. per ml) applied to the incision 2 and 15 minutes exposure to a VHF (27MHz) field with the electrode held 0-5 mm above the incision 2.

After anesthetizing the rats with ether and Nembutal, the backs of test and control rats 1 were shaved and a 1 to 1.5 cm horizontal incision 2 was made with scissors in the area just below the scapulae. Any blood that appeared in the wound area near the incision 2 was removed with gauze and the point of a 20 guage needle was used to adjust the edges of the incision 2 and to appose them. After application of the saline or histamine to the incision 2, the edges of both test and control wounds near the incisions 2 were carefully apposed and held tightly together. For the test rats 1, the electrode was held at a distance of 0 - 5 mm above the incision 2. After 15 minutes both control and test rats 1 were returned to individual cages for 24 hours. An attempt was made to perform each operation in as uniform a way as possible and to limit each operational procedure to a maximum of 25 minutes.

After 24 hours the wounds from the incisions 2 were carefully examined and the rats 1 sacrificed with ether. An area of skin 1 .times. 11/2 in. surrounding the incision 2 was excised and placed in Bouin's fluid. Paraffin sections were cut and stained with haemotoxylin and eosin, and van Gieson's stain.

RESULTS -- GROSS OBSERVATIONS

CONTROL GROUP No. 1 WITH SALINE

In all rats 1 a small amount of blood appeared between the two apposed edges of the incisions 2 during the operation. The edges of the incision 2 did not appear to to adhere well and one of the rats 1 shook the edges loose at the end of the 15 minutes. After 24 hours the edges did not appear to be well apposed in any of the incisions 2 and the healing process was incomplete.

TEST GROUP NO. 1 WITH SALINE AND ELECTROMAGNETIC FIELD

Before application of the electrode 4, the edges of the incisions 2 of the test rats were similar to the incision 2 of the control rats 1 treated with saline. However, after exposure to the field, the edges of the incisions 2 appeared to be more adherent and more easily applied to each other. After 15 minutes exposure, the incisions 2 appeared cemented. Over a period of 24 hours, healing was grossly completed and in two of the rats 1 the edges of the incisions 2 were very neatly apposed.

CONTROL GROUP NO. 2 WITH HISTAMINE

The edges of the incisions 2 treated with histamine appeared to be much more adhesive and pliable than those treated with saline. After 15 minutes of tightly apposing the sides, the edges seemed to adhere better than those of the incisions 2 treated with saline. After 24 hours the edges were not as well apposed as they were originally and gaps could be seen.

TEST GROUP NO. 2 WITH HISTAMINE AND ELECTROMAGNETIC FIELD

The edges of these incisions 2 were adhesive like those of the control group with histamine and after application of the electromagnetic field became even more manageable. After 15 minutes exposure only a thin hair-line incision 2 could be seen. After 24 hours, the incisions 2 appeared to be healing very well compared to all the other groups, including the control group No. 2 with histamine.

RESULTS -- LIGHT MICROSCOPE OBSERVATIONS AFTER 24 HOURS

In comparing Test Group No. 1 rats 1 treated with saline and electromagnetic field to the controls treated only with saline, it was found that the incisional gap appeared smaller on light microscope examination. There was less white blood cell infiltrate in the incision area and there appeared to be more of fibrin base to the incision 2. There were more fibroblasts and macrophages in the incisional gap of the test rats 1 than in the wound area of the controls. Active phagocytosis by macrophages could be seen in the test rat 1 incisions 2 but only occasionally was it observed in the control groups (See FIGS. 2 and 3).

In FIG. 2 there is shown a magnified (450X) transverse view of the incision area of a control rat 1 receiving only saline (control group 1 with saline). The relatively large infiltration of cells predominantly polymorphonuclear leukocytes, and their relative lack of fibrin base should be noted.

In FIG. 3 there is shown a magnified (450X) transverse view of the incision area showing the predominance of mononuclear cells in the infiltrate and the density of the fibrin base for test group 1 treated with saline and radiated with an electromagnetic field.

The blood vessels in the area around the incisions 2 of both Test Group No. 2 and Control Group No. 2 rats 1 treated with histamine appeared more dilated than those of the rats 1 treated with saline alone or with saline and the electromagnetic field. There was more fibrin in the base of the incision area of the controls treated with histamine than in the controls treated only with saline but this was less than that observed in the incisional area of the test rats 1 treated with both histamine and the electromagnetic field. There were not as many white blood cells in the incisional gap 2 of the controls treated with histamine as there were in the controls treated with saline. There were still fewer in the incisions 2 of the test rats 1 treated with histamine and electromagnetic field. In addition, there was a greater number of fibroblasts in the incisions 2 of the controls treated with histamine than in the saline control.

The Test Group No. 2 rats 1 treated with histamine and the electromagnetic field had a comparable number of fibroblasts to the histamine controls. (See FIGS. 3 and 4).

In FIG. 4 there is shown a magnified (450X) transverse view of the incision area of a rat 1 of test group No. 2 treated with histamine and not radiated with an electromagnetic field. The incision area contains approximately equal numbers of polymorphonuclear cells and mononuclear cells.

In FIG. 5 there is shown a magnified (450X) transverse view of the incision area of a rat 1 of test group No. 2 treated with histamine and radiated with an electromagnetic field contains a large amount of fibrin and only a few scattered mononuclear cells.

In order to understand the effects which an electromagnetic field has on wound healing, the exact sequence of events involved in normal wound healing must be known and understood. Although wound healing has been an area of considerable interest for hundreds of years, the actual sequence of events has only been known over the last 15 to 20 years. There is still much which needs to be learned about the functional events involved.

Ordman, L.J. and T. Gillman (1966) "Studies in the healing of cutaneous wounds," 1. "The Healing of incisions through the skin of pigs" Arch. Surg. 93 (6): 857-882, studied the normal sequence of events involved in the healing of cutaneous wounds in young pigs. They found that in the first day polymorphonuclear leukocytes were present throughout the length and depth of the incision and continuous with the blood and fibrin clot which formed the surface of the incision. Epithelial sheets were beginning to bridge the gap. By the second day mononuclear cells had replaced the polymorphonuclear cells and were phagocytizing cells and debris in the incisional area. On the third day vertically oriented spindle-shaped mononuclear cells and argyrophilic fibrils were first seen together with round cells in the incision. Vertically oriented fibroblasts were seen on the fourth day and mitoses were at maximal incidence within the intra-incisional new connective tissue.

The following table prepared from the above tests shows that it was found that there was less of an infiltration of polymorphonuclear white blood cells into the incisions of both test groups as compared to the controls. In contrast, there were many more fibroblasts and macrophages in the incisions of the test group than in the controls. This would suggest that the test wounds were an entire day ahead of the control groups in the healing process if one compares these findings to the sequence of events in normal wound healing as described by the above Ordmann and Gillman reference. In addition, the greater amount of fibrin in the test incisions would possibly provide a stronger supporting structure for the future healing process. ##SPC1##

It is shown that histamine is essential for normal wound healing. Although its function in the healing process is not entirely understood, it is known that histamine causes dilatation of the capillaries in the body and an increase in the permeability of the fine vessels on the venous side of the capillary bed. It would not be possible to say by what exact mechanism histamine is acting in the present study to influence wound healing. One specific advantage provided by the treatment with histamine according to the present invention was the increased manageability of the wound edges. Since malapposition of incised skin is one reason for delayed wound healing, the ability to avoid this may provide an additional advantage in treating wounds according to the present invention with an electromagnetic field and histamine was superior to the other incisions including the test wound treated only with an electromagnetic field and saline and indicates another advantage of the present invention.

Whilst the present invention has been described in the tests in relation to the production of collagenic material for the closure of lesions in rats, it is also useful for production of collagenic material for the closure of lesions in other living bodies, in particular in humans.

Claims

We claim:

1. A method of producing collagenic material for the closure of lesions, comprising applying histamine to the lesion, and radiating tissue cells bounding the lesion with electromagnetic radiation, the electromagnetic radiation being in the non-ionizing range of frequency, having an intensity less than one milliwatt so that the reproduction of collagenic material by the tissue cells is enhanced.

2. A method according to claim 1, wherein the histamine is histamine diphosphate.

3. A method according to claim 1, wherein the tissue cells are radiated with a 27 MHz electromagnetic radiation from an electrode held proximal to the tissue cells for 15 minutes.

4. A method according to claim 1, wherein the electromagnetic field is radiated from an electrode, the electrode is a rod electrode, and the electrode is placed lengthwise along the lesion.