The role and applicability of hyaluronidase in clinical dentistry

Operative

Oral

Surgery

THE ROLE AND APPLICABILITY OF HYALURONIDASE DENTISTRY GEORGE H. HESKEL,

CAYTAIN~ liSAIF

IN CLINICAL

(UC)

Introduction N 1930 an investigation led to the discovery of what, became known as a I spreading factor. This spreading factor was subsequently identified as the enzyme,- hyaluronidase, a tissue permeability factor which promises to assume considerable importance in biochemistry, bacteriology, and many mediral fields. Developments in the field of enzyme chemistry in recent years have already associated hyaluronidase with such diverse fields as gynecology, rheumatology, oncology, bacteriology, internal medicine, pediatrics, orthopedics. dentistry, and others. Hyaluronidase opens a new approach to fundament,al problems in each of these areas. The prime objective of this article is to correlate the known properties 0-f hyaluronidase, and to apply them to clinical dentistry. Herein is an attempt at compilation of the findings of the more pertinent investigations which are of direct, interest, to and have active application in clinical dentistry. The work reported here was accomplished in the oral surgery section 01’ a dental internship at F’itzsimons Army Hospital, Denver, Colorado.* From a review of medical and dental literature, a great variety of possible dental Many of these uses were studied from the theoretical and uses was postulated. clinical aspect. There was limited time available for investigation; consequently, this present study includes neither a sufficient number nor variety of cases to be stat,istically valid. The cases in this study, however, showed promise in all areas and indicate nerd for further investigation and acceptance by the profession. An enzyme which increases tissue yermeabilit,y naturally suggests its role in the treatment of traumatic swelling and hematomas and its application in Presented as part of the requirement of the Oral Surgery Section of the Dental InternProgram at Fitzsimons Army Hospital, Denver, Colorado. The opinions expressed in this paper are those of the author, and endorsement by any of the Armed Forces Dental Services is not to be infrrrrd. *The author is indebted to Colonel H. R. Dierdorff for his permission to undertake thr clinical portion of this study and to Colonel Dierdorff and T&utenant Colonel R. J. T:llhot for their guidance and support throughout. ship

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local anesthesia and hypodermoclysis. These are obvious potentialities of the enzyme; others are somewhat more subtle. Several applications, at this juncture, have been merely hypothesized. In this article, the proposed dental application of hyaluronidase will include : (1) hypodermoclysis, (2) traumatic swelling, (3) hematomas, (4) local anesthesia, (5) topical anesthesia, (6) temporomandibular disturbances, and (7) miscellaneous references to other potential uses. The work thus initiated at Fitzsimons Army Hospital is being continued, with special emphasis toward the problem of temporomandibular joint disturbance, a condition which has long perplexed the dental profession. A brief, general review of the more pertinent literature will be presented to familiarize the clinician with the properties and current uses of hyaluronidase and to aid in a more critical appreciation of the problem at hand. Review of the Literature:

Physiologic and Pharmacologic Aspects

Duran-Reynals13 originally discovered that the spread of vaccinial infection in rabbit skin was enhanced by simultaneous injection of testicular extract. McClean30 confirmed and extended these investigations and showed that the action of the extract in spreading infection was by increasing tissue permeability. This spreading factor was then found in such diverse sources as certain bacteria, various tissues, some tumors, snake and spider venoms, and leech heads. While investigation of the spreading factor continued, Meyer and Palmer33 reported the presence of a mucopolysaccharide in vitreous humor of cattle eyes which they called hyaluronic acid. The correlation of these and similar investigations was made by Chain and Duthie,7 who described the phenomenon of the spreading factor as an enzymatic effect on a polysaccharide of connective tissue, that is, hyaluronic acid. Hyaluronic Acid.-Our knowledge of the mucopolysaccharides, of which hyaluronic acid is a member, is limited ; Meyer,32 however, has proposed the following classification : I. Mucopolysaccharides-polysaccharides amine as one component

which contain hexos-

A. Neutral mucopolysaccharides B. Acid polysaccharides 1. Simple-N-acetylhexosamine plus uranic and hexonic acids. (Example : hyaluronic acid) 2. Complex-Same as above plus sulfuric or phosphoric acid. (Example : heparin, chondroitin sulfate)

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Mucoids or mucoproteins-contains less than 4 per cent amino sugars. (Example : blood group A substance of gast,ric mucosa) Glycoproteins-contains less than (Example : serum albumin)

4 per (bent amino sugars.

Hyalnronic acid is a mucopolysaccharide which consists of equimola,r proportions of glucuronie acid and N-acetylglucosamine. Its properties are : it is of high molecular weight, it is viscous, soluble in water, precipitable in acetic acid, insoluble in fat solvents, relatively resistant to denaturation, is not antigenie, does not dialyze through a collodion membrane of great porosity, and (very important) it probably occurs in varying proportions in all connective t,iSsue.2G,44 Hyaluronic acid and chondroitin sulfuric acid are important constituents OE ground substance. Hyaluronic acid may be the intercellular ground sul)stance of connective Gssue, the amorphous, viscous, transparent, jellylike substance of connective tissue in which argyrophilic fibers? collagenous bundles. lymph, and blood vessels are imbedded and in which the fibroglia and cells are included. The hyaluronic acid may serve an architectural function or it may bind water in interstitial spaces.22 In any case, it is felt that it has a function in holding cells in their jellylike mat,rix and is a component of the lubricant and shock absorber in joints. Synovial fluid is possibly a function of the connective tissue synovial layer of the joint capsule. Hyaluronic acid provides viscosity t,o the connective tissues and their secretions which necessarily must be altered in order that substances may pass through, although met’abolites might usually move through, impelled by capillary force.15, 2fi Capillary permeability itself is quite possibly related to hyaluronic acid.’ The extraordinary wide latitude of the permeability of blood capillaries tends to preclude the possibility that this property is primarily that of living cells c*omposing the capillary walls. Many substances to which living cells are itnpermeable readily pass through the capillary wall.‘” The intercellular cement substance, rather than the endothelial cell itself, may be responsible f’or capillary permeability. There is a definite connection between the ground substance of connective tissue in general and the cement substance of blood and lymph vessels.‘;’ The c2ement between the endothelial cells is a sticky, argyrophilic matter. (‘OIItinuously secreted by the cells t,hemselves,” and the I)ermeahility 01: the Y~SPW lar system can be locally or generally increa.sed I)y preparations rich in s])rtB;ltlitig factors.15, I6

Hyaluronidase.-The term “spreading factor” is not synonymous with hyaluronida.se. Various substances, such as ascorbic a&l, azoproteins anti diazo compun(ls, urethane, hirudin, comtrlrrc*ial I)rptotlrs, ;1nt1 lrcaithins, protttotc in viva spreading illld imlnce the viscosity 01’ SoIiIc sul)slr:lff~s it1 yitro.lT>. 22.L!fi On the other hand, hyaluronidases from any sout’c’(b (venom. leech extracts, ba.cterial toxin, etc.) are spreading factors. ~la.mmalian testicau-

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lar tissue is the most potent source. In the animal body, hyaluronidase is quite possibly constituted as a group of related enzymes rather than as a single enzyme.15 Hyaluronidase, as the suffix implies, is an enzyme. It gives the usual tests for protein, contains inorganic phosphorus and organic sulfur; it is filterable, nondialyzable, heat labile, insoluble in fat solvents, soluble in water, It is inactivated, precipitated by acetic acid, and is of high molecular weight. after heating for one hour at 60° C., by pepsin and trypsin and by chloroform. Material available for clinical use is a vacuum-dried, powdered extract of bull testes which is free from pyrogens. It is stable indefinitely in the dry state at room temperature or refrigerated. It is soluble in aqueous solution, and this solubility has been enhanced by the pharmaceutical houses by compounding the extract with lactose. Ready solubility is very important, since the enzyme is inactivated by even gentle shaking. Reconstituted in physiologic saline, hyaluronidase is stable for a minimum of two weeks when refrigerated, provided that sterility is maintained.4”> 5o AS with most other enzymes, hyaluronidase has an optimum pH, temperature, and concentration of salts, The optimum pH is about 6.0, some.what more acid than the human pH, but a factor actually out of our control with in vivo experimentation. The presence of sodium chloride is of marked importance to hyaluronidase activity. Various investigators have found the optimum concentration to be between 0.15M NaC1 and 0.30M NaCI. Physiologic saline is a very convenient 0.154M NaCI. The Q10 has been reported as 1.75 to 2.0. That is, an increase in temperature of 10’ C. will increase the activity of the enzyme 1.75 to 2.0 times. The Qlo is applicable to a maximum of 40° C.‘2, 2(i Normal body temperature is again a convenient 37O C. Hyaluronidase acts on its polysaccharide substrate through a process of disaggregation and depolymerization, although the actual steps of this process are unknown. The breakdown occurs in at least two”” or threc4’ steps. This breakdown of the ground substance decreases the interfibrillar a.nd interAfter cellular viscosity and renders the connective tissue more permeable. breakdown, the nat,ural hyaluronic acid barrier is partly restored in twentyfour hours and completely restored in forty-eight hours.23 The enzyme may be slightly antigenic, but hypersensitivity reactions are rare to nonexistent.4, 8. In, 3Z,1-1 Toxicity is minimal; a dose of 200,000 times the maximum therapeutic dose must be injected to produce the first sign of Accidental injection directly into the blood stream is also quite toxicity.42 inhibitor in norma. harmless,j, r,slo due at least partly to a. hynlnronidasc bIood.5, 0, 35,17,22 Large dosrs of salicylates, cortisone, ACTH, estrogens, and antihistamines also either have an inhibitory action on hyaluronidase or render the substrate partly refractory to the action of the enzyme.“, ‘l

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A turbidity reducing unit is tlefiiletl as the itlrlol~llt of hywlar~o~li~~~~s~~ which, under a.ssay conditions, will reduce the turbidity caused by 0.2 mg. 01 potassium hyaluronate in acidified horse serum to that, caused by 0.1 mg:” One TRTT is eyuivalent to approximately 3.33 VRT’; that. is. 150 TRl- is eWi=llent to about 500 VRTT.2”, 50 150..TRT: or 500-VRIV vials are supplied by v;irious pherma.ceutical houses ; their potencies are about) equal. Throughout thv remainder of t,his article, the turbidity reducing unit iTI enzyme are needed to produce a given spreading effect in Srgrors pat,ients.5

Hypodermoclysis The use of hyaluronidase as an aid t,o hypodermoclysis is an est~ahlishetl hospital practice. The enzyme enhances the effectiveness of clysis solutions, atld has incressed a,bsorption of fluids from five to fourteen times.l”’ *‘, *’ The absorption of most of the commonly used solutions (saline, saline-glucose. Tissue derivatives, such as blood sodium lactate. Ringer’s, etc.) is facilitated. which inhibit the and possibly heparin, however, contain cert.ain inactivators artion of hyaluronidase, as mentioned previously. The dentist normally has little occasion to require clysis. In cases in which fluid administration is desirable and adequate veins are not available (as seen especially with infants and aged patients), or in the event of cmcrgencg and lack of skilled att.endants, hypodermoclysis rnay be llecessary. As a member of the healing arts in our present civilization, it behooves t,he dentist to he familiar with modern lifesaving methods: hence, the brief cliscussion of hypodermoclysis in this article. I’ery little hyaluronidase serves to break down tissue barriers. AZu effective clysis may be obtained by injecting 150 TRTi (one vial) of re’constituted hyaluronidase into the rubber tubing, as close to the needle as possible, at the start of clysis. This amount will facilitate absorption of 1,000 C.V. or more of solution.“” Up to 400 CC. per hour (in adults) can be administered with little of the swelling or discomfort usually associated with simple hypetlermoclysis without the enzyme.24

Traumatic Swellings General and oral surgeons are continually confronted with the problem of traumatic swelling and all its complications. When the swelling is due to surgical trauma, the postoperative problem can prove to be very disturbing to the patient and to the doctor.

468

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HEiYKEL

With a. surgical, physioIogic, or acci(lenI,itl iitsuIt to the tissue, swelling at the site 01’ insult to a gre;ltel* (~1’lesser tlegt~ec is to be exl)ected. It is frc(]~~~:~~tlythe result of mechanical illjury to t,he blood vessels and lymphatics in the area. The swelling is uncomfortable at best. lt is probably disfiguring to the patient, and adds to the problem of postoperative management. If the molar regions are involved, there will often develop an accompanying trismus. Pressures in any area interfere with lymph flow, tend to occlude vessels mechanically, and contribute further to general stasis.” With stasis and persistent edema, fibrosis and induration are noted along with a striking drop in natural resistance to infection.12 The resolution of these postoperative sequelae is a relative phenomenon, depending much on the “healing powers” and “resistance” of the patient. The problem is not entirely objective, since variations in individual thresholds of pain and relative discomfort complicate the postoperative picture.3 Various methods have been employed to combat t,he problem of postoperative swelling. Such diverse methods as local application of heat and cold, diathermy, heliotherapy, massage, physical therapy, radiotherapy, psychotherapy, application of packs and poultices, administration of antibiotics and chemotherapeutics, and many others have been attempted with variable and generally unsatisfactory results. The most widely used treatment, namely, local application of heat and cold, was recently investigated by P~ssoff.~~ He reported little difference in swelling or cytologic response caused by application of heat and cold or nothing during the first twenty-four hours, and no demonstrable difference in the rate of healing. Until Benzer3 suggested the use of hyaluronidase to increase tissue permeability and thus enhance dispersion of accumulated inflammatory exudate, nothing had ever been injected into a swelling in order to reduce it. His work was fundamental to dental application, and laid the foundation upon which much of the present report on traumatic swelling is built. After surgical interference, the postoperative sequelae usually regress after only a few days, and the surgeon has only to prescribe appropriate drugs for analgesia. When the swelling persists, stasis occurs, and induration begins, a different approach is indicated. Although the body has an efficient mechanism of inflammation to bring to an area of tissue insult the necessary reparative, protective, and healing fluids, it often does not have such an efficient mechanism for resolution of these fluids. Thus, it was felt that the use of hyaluronidase might be indicated to increase tissue permeability and to aid resolution of fluids which had potentially served their purpose. In order to appreciate the rationale for use and the clinical results, a basic understanding of present concepts of the inflammatory mechanism is acts upon the ground substance of connective necessary, since hyaluronidase tissue, the substance in which the phenomena of inflammation occur.15 Inflammation might be termed the basic lesion in pathology. It is the local defensive reaction that occurs when an injurious agent penetrates the

HY$LURONIDASE

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tissues. The inflammat,ion might be caused by bacterial, mechanical, physi(a:ll. or chemical agents; it is a chemical reaction, whatever the cause. The purpw of this defensive reaction is at least fourfold: to destroy or wall off t,he irritant! to neutralize toxic substances liberated? to dilute the irritant, and to remove necrotic debris. Stimulation of healing and regeneration of new cells by ltletabolic products is a.nother possible function of the inflammatory process.‘~’ The gross characteristics of inflammation ;\raewrll known. They are : Redness -increased amount of blood in the inflamed area. Heat, -greater amount. of blood and increased rate of flow. Swelling-increased amount of blood. mostly due to accumulatei~ serous exudate and cellular c~lernents. -irritation of sensory nerves by toxic substances and I’ilill by the tension due to the swelling. Blood leukocytes, small blood vessels. and sul)pol*ting connective t,issues When a noxious element is present ~111participate in the defensive reaction. in the tissue, the blood leukocytes collect along the small vessels of the area, giving rise to the phenomenon of margination. “Emigration” then denotes the passage of these leukocytes through the vascular walls, while “exudation’! describes the collection of fluid a.nd cells in the tissue spaces. The original (Nation of the small vessels which lcads to slowing of the blood and ma.rg:ination is caused by direct action of the irritants on the vessels; there is little This dilation is combined wit,h an increase ill cont,rol by the nervous system. permeability, leading to the emigration and exudation. This serous exudate is very similar to ~~lasl~~a. I“ibrinous exudates MT caused by coagulation, the quantity depending upotl the nature of the irritant. I’llcrease in the permeabilit,y ot site of injury. and structure of the tissue. a.fferent lymphatics adds to the collection of fluid in the extravascular spats. Traumatic swellings are basically composed of inflammatory exudate and are accompanied by obstruction of lymph charlnels.1 During t,he early stages of edema, the formation of exudate is rapid: in lateI* stages, the opposite is true, Thus, we see that traumatic swelling is a l)asic physiologic process thai serves a, definite purpose. It brings protective bodies to the injured arra. ii may contain opsonins or agglutinins, it may conta,in hacteriolysins or antitoxins, and it dilutes the irritant. Later it may stimula.tc repair. To rid itself of this accumulation of fluid, the body usually begins the pro+ MS of resorption. The inflammatory lesion at the surgical site should hegirr to resolve after fort,y-eight hours, barring secondary irlfcction or a general If it is not rrsorhe(1. the ~I’OWHS ol’ organization will systemic disturbance.:’ set in. 12rwley has describrtl a succession of l)hnscs 01‘ mnversiou from t~lem;~ to gelatinous groUllc1 SUl)StilllW to argyrol)hilic fil)c>l% to (~Ollil~!.rll. It is l’LIIS1 htlI, I)elievrd that ground sulAance in the atlult is a pwtluct. of the scvretioll (11‘ connective tissue cells and that progressive transformation of this substanpc. yields the various components of adult connective tissue.“, ”

470

GEORGE

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Hence, this ground substance, cement substance, or mesenchymal interfibrillar substance is basic. It is a precursor to other connective tissue elements and is the binder through which most substances reaching cells must pass.‘5 Conversely, any accumulation of extravascular elements must pass through the ground substance on its way back to the vascular system. The permeability of the connective tissue (or, more specifically, the ground substance) has a normal, constitutional state or tonus which can be increased by certain factors and decreased by others.2a I5 The permeability is increased during edema formation, but soon returns to a state of lesser permeability and the exudate is often destined to slow resorption or organization. This permeability may be promoted or maintained by breakdown of the hyaluronic acid barrier with hyaluronidase. The prophylactic use of hyaluronidase to promote or maintain the permeability of early edema formation and prevent postoperative swelling has been proposed.lO. N That the technique is clinically feasible has been established. However, many factors should be weighed before such prophylactic use. Benze+ 31 states that after surgical procedures one should not interfere with edema and swelling which ensue. Such interference aborts the protective physiologic process of inflammation. If the tissue injury is fresh and the tissue pathways are opened, it is possible that the tissue bleeding is still present and open tissue pathways will lead to a widespread dissemination of blood, resulting in an extreme ecchymosis4 If ecchymosis does not occur, it is quite probable that the enzyme has been inactivated by the blood factors and will have no effect upon subsequent swelling. Lymphatics are usually blocked within eight hours4” while about twenty-four hours are needed for consolidation of clotted capillaries.” It has also been found that there is establishment of a resistance to infection with a variety of bacterial preparations within twentyfour hours.14, I5 Thus, it seems that it is not advisable to use hyaluronidase too soon. Advocates of prophylactic injection of the enzyme have at least one justification for use that has not been presented as such. It is felt that a “ critical concentration’ ’ of an organism is necessary to establish an infection.5s I5 Thus, by dispersing the concentration, hyaluronidase indirectly exposes more organisms to antibodies, phagocytes, and/or antibiotics. Also, the enzyme ma.y depolymerize certain encapsulated organisms.5 In most clinical instances, however, the virulence of the organism is unknown. Therefore, the local use of hyaluronidase without antibiotic coverage for the organisms conceivably present is potentially dangerous.” The critical concentration theory lends further justification to the use of hyaluronidese to reduce swelling after a period of normad healing. There should be little tlanger in injecting the enzyme into an area of swelling even though there were potential or actual pathogcuic organisms. Tf the organism were walled off, as it most probably would be after a period of normal healing, the enzyme would have no effect upon the infection unless injected directly

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into it, becaqse the enzyme cannot penetrate a fibrinous barrier Ilut, ra2the1,, tliffuses ;~rtjll~ltJ it, following the Ilath of le~~sl wsist;~I~w.“‘~ “’ Oti the other withoul hantl, if an injection were inatle directly into an i\.r(‘ii of infr&on simultaneous antibiotic or chen~othel,al)eutic ~()\~(~ril~t~, the ol*g;nnistn WIII~ quite possibly be dispersed to the esteut that its concentration would IW 1~s than the critical concentration necessary to establish an infection. In the more practical case, where infection is suspected and a,ntibic)tic or chemotherapeutic coverage had been administ,rretl previous to hyaluronidasr administration, the enzyme would disperse the organisms so that they would be more exposed to antibacterial action. The potential capsule depolymerization action of the enzyme could also render the antihact,erial ~~OC~SS~S mart contbirretl cff active. There is some indication that injec4ion of hyaluronitlase, with an antibiotic in aqueous suspension, directly into the mass of the swelling is of definite advantage in treatment of infection.‘. “’ The antibiotic disperses rapidly and has local effect, besides being absorbed more rapidly for systemic The presence of a localized infection does not contraindicate t,he list’ effect. of the enzyme,““, 4’ although it would not be advisable to iqject into a localizctl infection if this can be avoided. Thus, with a critical evaluation and corrrlation of the physiology and pharmacology prescribed above. we can make tenable the utilization of hyaluronidase as a valuable adjunct in the treatment ctt certain cases of traumatic swelling. First, it might be expected that the judicious use of the enzyme after a reasonable period of normal healing might enhance diflusion of the collectiotl of inflammatory exudate away from the surgical site. Second, the induration that might be forming in the area due to a prolonged edema and the beginning of organization would be broken up dur tc; hydrolysis of the new hyaluronic acid and barrier mucopolysac.chari(les l)eing formed with fibrosis. Third, with the reduction of the hard swelling to a soft swelling, usually of less magnitude, pressures on the tissues arc’ reduced and the lymphatics ant1 capillaries might more readily regenerate and alleviate the stasis. Rith rrliet of stasis and pressure, there would be more rapid healing ant1 a greater suppl!. of nutrients to the involved tissue, .P’ourth, the possibility of infection would be dt~crrased and, if infrctiotl were already present. the resolut,ion of tha.t infection wonId be Illore rapid. Fifth. the efficacy of an antibiotic or ~henlotheri~l)eutic agent would IW enhanced.

Clinical Results.-In cases of traumatic swelling that were treated with hyaluronidase at, Fitzsimons Army Hospital, the clinical results closely paralleled the rationale for use. The most st.riking changes were subjective in nature. Although objective changes, as recorded by measurements, were not{4 in each case, subjective symptoms of tension or pressure in the area, pain, and restriction of motion were relieved to a marked degree. In general, the patiellds experienced almost complete suhjcctive relief within a thirty-minute perio(f afttlr injection.

472

GEORGE H. HENKEL

The method of utilization for hyaluronidase for relief in traumatic is simple. Either the intraoral or extraoral route can bc used.

swelling

Method.--The arJ1lilllJetlt;lriuln I’or the ittdcction is shown in Fig. 1. The mucous membrane over the swellin g is drictl with a sterile sponge and the area is painted with Metaphen. A few drops of local anesthetic agent are then deposited just, under the mucosa to prevent discomfort when a larger needle for injection of the enzyme is inserted. The enzyme is then reconstituted with physiologic saline. The usual dose is 150 to 300 TKU of hyaluronidase, reconstituted in 3 to 4 C.C. saline. This a.llows sufficient increase of interstitial pressure to enhance diffusion. Resistance of connective tissue bo penetration by fluid is not, overcome until a pressure of 8.5 cm. of water is reached.l”

Fig. I.-Basic

armamentarium

for

hyaluronidase

injection.

With localized fluid pressure present, the enzyme will spread and remove the hyaluronic acid barrier at sites more distant to t.he point of injection. When the fluid pressure is high, as in the case of localized edema, or when the enzyme is introduced into the tissues with a volume of fluid, the diffusion is enhanced. The rate of diffusion is proportionate to the amount of enzyme, and the extent is proportionat,e to the volume of solution.“” The reconstituted enzyme is placed in a Lok type syringe with a 22- to 2-I-gauge needle. The tip of the needle is then passed through the anesthetized mucous membrane into the center of the swelling (Fig. 2). The enzyme is then slowly deposited.

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,ZTtcr with&awal of the needle, sterile sponges are placed betwcell t1lC teeth and thcbcheek, and a folded towel is pressed by the patient on the outside pressure pack increases and oi’ the cheek (I+‘&. 3). This intraoral-extraoral maintains interstitial pressure. The pressure pack is held by the patient for thirty minutes.

Fig. 3.

Fig. 2. Fig. 2.-Intraoral injection Fig. 3.-Intraoral-egtraoral

Fig. $.---Method

of obtaining

of hyaluroni~lxse. pressure pack in position.

objective

measurements.

474

GEORGE

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Following this procedure, no discomfort was experienced by most patients. A slight warm to burning sensation was experienced by two patients, but this sensation passed within three minutes. It is not necessary to anesthetize the deep structures, since sensory nerve endings in the area to be injected are no problem. In fact, deep anesthesia may be detrimental in that the vasoconstrictor in the anesthetic could counteract the effect of the enzyme, For the purpose of the Fitzsimons study, two measurements were taken as an attempt at quantitative resulm. Two tattoo marks were placed on opposite cheeks at the point of greatest dimension of swelling with an indelible pencil and a measurement is taken across these points with a Snow type face-bow. The intermaxillary space, as measured between the incisal edges of the anterior teeth, was also recorded (Fig. 4). These objective measurements were taken before the injection and at various intervals post,injection. Case histories follow.

A. Fig. 5.-A, Postoperative swelling of swelling forty-five minutes after administration of swelling, accompanied by subjective relief.

B.

forty-eight hours’ of hyaluronidase.

duration. There

I?, Postoperative has been reduction

Case L-A 2%year-old man was seen forty-eight hours after removal of a vertically impacted R-16 and an impacted R-8. The patient had had hemorrhage into his mouth and There was a firm, localized swelling in the into the tissue for twelve hours postextraction. right premolar region, as well as a generalized soft swelling of the right cheek and neck. There was slight ecchymosis; the firm swelling was diagnosed as a hematoma. The patient’s intermaxillary opening was 30 mm. (Fig. 5, a).

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Hyaluronidaxe, 150 TRU reconstituted in 3 C.C. physiologic saline, was injected intrsThe patient noted a burning orally into the hard swelling after the usual preparation. sensation in his cheek which passed within three minutes. An intraoral-extraoral pressure pack was applied for thirty minutes. When the pressure pack was removed, it was noted that the swelling in the neck had decreased markedly (Fig. 5, B). Mrasurement with the face-bow apparat,us showed a 4 mm. dccrrascS i a lateral dimension. The intermaxillary space had increased 6 mm., to 36 mm. The swelling Huhsequent recover!; was which remained !vas soft and the patient \ras more comfortable. uneventful. Case 2.-A %-year-old woman was seen forty-eight hours after removal of impacted B-8 and R-16. She had a large, generalized swelling of the right body of the mandible. The skin over the area felt tight and had a shiny appearance. Intermaxillary space was restricted to 21 mm. and the patient stated that she was very uncomfortable (Fig. li? -4 ),

.4.

Fig. K-A, Postoperative swelling thirty minutes after the cosmetic improvement.

B.

swelling injection

of of

forty-eight hyaluronidase.

hours

duration. Th?

patient

B,

was

Postoperxti~e yleasc~l

n-itl,

Hpaluronidase, 150 TRU in 4 C.C. saline, was injectetl intraorally into the cunt(:r of the swelling. 9 pressure pack was applied for thirty minutes. The patient had a qlight, burning sensation for one minute after the injection. After thirty minutes, measurements were again taken. The lateral dimension had decreased 3 mm. while the intermaxillary space had increased 7 mm., to 28 mm. ‘l’hc shill! appearance of the right cheek had disappeared and the patient said that the swelling felt “smaller.” She was quite pleased with the cosmetic improvement and notitsed that +l,(, could open her mouth wider (Fig. 6, B). After twenty-four hours, the intermaxillary was uneventful after the first day.

space had decreased

to 25 mm.

l<~ov~~ry

476

GEORGE H. HEIVKEL

Case 3.-A 22syear-old man was seen twenty-four hours after removal of an impacted horizontal R-16. He had a moderate amount of swelling in the area of the right angle and body of the mandible. The swelling was soft and only slightly uncomfortable, but the patient was being discharged from the Service, was going home, and was concerned about his condition. His intermaxillary space was 25 mm. (Fig. 7, a). Hyaluronidase, 150 TRU in 1.5 cc. saline, was injected intraorally into the area of swelling. The patient cooperated by allowing the injection series of pictures (Figs. 2, 3, and 4) to be taken. The pressure pack %Gasapplied, as seen in Fig. 4, for thirty minutes. After thirty minutes, the lateral dimension had decreased 3 mm. and the intermaxillary space had increased 3.5 mm., to 28.5 mm. (Fig. 7, B). Recovery was uneventful.

A.

Fig. 7.-A,

Postoperative

B.

swelling of twenty-four hours’ duration. B, Postoperative thirty minutes after injection of hyaluronidase.

swelling

Ch%3 P.--A 21-year-old woman was seen twenty-four hours after removal of an impacted mesioangular L-16. She had a soft, generalized swelling of the entire left side of her face. She felt that her mouth opening was constricted; intermaxillary space registered 25 mm. Hyaluronidase, 150 TRU in 3 cc. saline, was injected intraorally. No pressure pack was applied. This was done in an effort to determine the necessity of pressure supplemental to the increased interstitial pressure caused by the volume of solution injected. After thirty minutes, there was no objective improvement as noted by lateral or vertical measurements. After three hours there was still no objective improvement, although the patient st,ated that the swelling felt “softer” and was less uncomfortable. After twenty-four hours, there was still no objective change; however, the patient Since the hyaluronic acid barrier is not fully was as comfortable as the day before. restored until about forty-eight hours have elapsed, a pressure pack was applied. After thirty minutes of pressure pack, there was a 4 mm. decrease in lateral dimension and an and increase in vertical dimension from 25 to 30 mm. The patient was still comfortable was cosmetically improved.

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Case 5.--A 29-year-old woman was seen forty-eight hours after a Caldwell-Luc vedt we and closetre of an antral-oral fistula with antrostomy beneath the inferior mea She had a gauze: pack through her nose and was on spdemic h;vdrocortisone therapy. The patienl I had considerable generalized swelling of the right cheek and infraort lital regi on and was very uncomfortable. Her intermaxillary space was 26 mm. (Fig. X, ,I f.

B. Fig.

8.-A, .ously. B, -01 sweIIing

Swelling following a Caldwell-Luc operation Ii ‘arty-eight-hour appeammce of patient after f elkming a Caldwell-Luc operation.

performed fmty-eight ire ,,lk! injection of hyaluronidat;e to

478

GEORGE

H.

HENKEL

Hyaluronidase, 300 TRU in 3 C.C. saline, was injected into the center of the swelling and a pressure pack was applied for thirty minutes. When the pressure pack was removed, the swelling had decreased 2 mm. and the intermaxillary space had increased 3 mm., to 29 mm. The results were not striking. The pharmacology of hyaluronidase was reviewed and the hydrocortisone-hyaluronidase incompatibility (q.v.) was noted. The hpdrocortisone therapy was discontinued and the patient observed. After two and one-half hours, the intermaxillary space was unchanged, but the lateral swelling had decreased another 2 mm. After twenty-four hours, the intermaxillary space had increased to 30 mm., although there had been no further decrease in the swelling. After forty-eight hours (Fig. 8, B), the intermaxillary space had increased from the original 26 mm. to the present 35 mm. and the lateral swelling had decreased another 3 mm. (total of 7 mm. decrease). The patient was considerably more comfortable and was cosmetically improved. Case 6.-A Zl-year-old woman was seen twenty-four hours following removal of an impacted R-16. There was marked swelling in the angle region and the patient had difhculty in opening her mouth. to “swell” after any She stated that she had a tendency operation. Hyaluronidase, 150 TRTJ in 3 C.C. saline, was injected extraorally and a pressure pack was applied for thirty minutes. Following treatment, the patient appeared nearly normal. Intermaxillary space was increased 7 mm. No lateral measurement was taken. The patient stated that she felt much better and had experienced no discomfort from the treatment. Case 7.-A 2%year-old man was seen twenty-four hours after removal of an impacted swelling R-8. The patient was heavy, with large buccal fat pads. There was an indurated under the right eye, as well as slight trismus and deviation to the right on opening the mouth.

Hyaluronidase, 150 TRU in 3 cc. saline, was injected extraorally and a pressure pack was applied for thirty minutes. The intermaxillary space increased from 28 The patient became more comfortable. to 35 mm. and the lateral dimension decreased 10 mm. The lateral deviation was improved. Casu S.-A 22-year-old woman was seen twenty-four hours following removal of an impacted L-16. Slight swelling in the angle region and trismus were present. Hyaluronidase, 150 TRU in 3.5 C.C. saline, was injected intraorally. A thirty-minute pressure pack was applied. Following treatment, the patient became more comfortable and stated that her jaw “felt better.” The intermaxillary space increased from 22 to 33 mm. and the lateral dimension decreased 5 mm, Case 9.-A 28-year-old man was seen t,wenty-four hours following removal of an impacted R-16. Considerable soft swelling of the entire right cheek mechanically prevented opening of the mouth. Hyaluronidase, 300 TRU in 4 cc. saline, was injected intraorally and a thirty-minute pressure pack was applied. There was an increase in interThe patient stated that he felt better generally. maxillary space from 32 to 34 mm. The lateral dimension decreased 7 mm. Case lo.-The patient was a 30.year-old woman who, twenty-four hours previously, had had an impacted L-16 removed. There was a soft swelling along the lower border of the mandible and the area was tender and uncomfortable. The patient was given 150 TRU hyaluronidase in 2 C.C. saline, injected intraorally. A pressure pack was applied for thirty minutes.

HYALIJRONIDASE

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DFXTISTRY

‘t’i!)

There was an immediate 2 mm. decrease in dimension and a 5 mm. inCrease in interAfter three hours, there was a further decrease Of 0 mm. in lateral maxillary space. Twetlty-fottr hours after treatment there was no remission. dimension. Case Il.--The patient was a ISyear-old boy wl~ose iurpacted L-S and L-16 had been swelling of the left removed forty-eight hours previously. There was a firm, narneralizrtl cheek, as well as marked restriction to opening the mouth. The patient was given 300 TRU hyaluronidase in ::..i (3.c. saline, and a thirty-minutc~ pressure pack was applied. The patient was considerably more comfortable anti the swelling was soft at the end Intermaxillary space had increased from I:1 to 31 mm. and the lateral of the treatment. dimension had decreased 7 mm. Three hours later there Ras no change, hut twenty-four hours after treatment there was a further decrease of 2 mm. in thr lateral dimension. 7’11~ pat,ient was comfortable but there was no further increase in intrrmaxillary space. Case 12.-A 34-year-old man was seen twenty-four hours after removal of an impacted L-16 with follicular cyst. There was a slight soft swelling along the anterior portion of the mandible. The patient was uncomfortable and had some trismus. He had a parenthesis resulting from trauma to the mandibular nerve during cyst removal. The patient was given 150 TRU hyaluronidase in 3 ~.e. saline. A pressure park was applied for thirty minutes. Treatment was followed by a 5 mm. decrease in lateral dimension. Intermaxillary space increased from 25 to 30 mm. The patient stated that the mandible felt no different (still felt anesthetized) but he was pleased with the improvel( appearance. ‘l’his patient was a reaitlent physician and was enthusiastic about the treatment. Case 13.-The patient, a 20-year-old boy, suffered henlorrhage, followed by ecchpmosis. days postoperatively. Hyaluronidase, 300 TRU in 4 c,c. saline, was injected and a thirt,y-minute pressure pack was applied. Intermaxillary space inrreased fron, There was a 4 mm. decrease in lateral dimension. 24 to RO mm. The patient was more comfortable and conmeticall~- improved. four

C&Se 14.--A 31syear-old woman was seen forty-eight hours after surgical removal of I,-6 and T.-7 and an impactetl L-8. She hat1 hemorrhage for twenty-four hours postoperatively. There was marked trismus, anti the patient was unable to put a teaspoon into her mouth in the morning. It was felt that the firnl, generalized swelling was a conhination of extravanated blood and edema. The patient received 150 TRU hyaluronitlasr in 3 C.V. saline. A pressure pack watt applied for thirty minutes. The patient stated that her mouth still “felt full” but that the constricted feeling and all pain on opening were now absent. Intermaxillary space increased from 19 to 5; mm. and the lateral dimension decreased 5 mm. Cosmetically, the result was Vera goo-1. Case E-An 1%year-old girl was seen twenty-four hours following removal of an impacted TJ-16. A soft mass was present along the body of the mandible and the patient experienced pain on opening her mouth. There also was general disfigurement. Hyaluronidase, 150 TRU in 4 C.C. saline, was injeeted and a pressure pack was appli6.d for thirty minutes. Following treatment, tenderness was absent and the patient felt more comfortable, Intermaxillary space had increased from 16 to 20 mm. and the lateral dimension had (1~. creased 7 mm. The cosmetic result was very good.

Hematomas The rationale for use in injection or other type hematomas is dmos;t the same as for traumatic swellings. In the case of a hema.tomn, however, the

480

GEORGE

H.

HENKEL

l,lood-in2Lctivating factors necessitate use of more of the enzyme. Also; with iTje(>tion llematomas, t’hc onzymc should be injected dircct1.v into the hcmatoma as soon as possible ; thcrc is no period for normal healing. With prompt action, the undesirable disfigurement, discomfort, ant1 psychological trauma can be largely prevented. It would appear that in the case of an injection hematoma, iI1 3 to 5 CC. of the immediate injection of 150 to 300 TRIJ hyaluroni&se physiologic saline is the treatment of choice.” In the year of study at Fitzsimons Hospital, no injection hematomas were observed; the opinion stated here is the one proposed by Benzer.

Local Anesthesia The increa,sed tissue permeability afforded by hyaluronidase lends itself to application in infiltra,tion and block anesthesia. Hyaluronidase is stable when reconstituted in procaine solutions for a minimum of two weeks at room temperature but for over four weeks at 5’ C.” Thus, the enzyme is not suitable today for manufacture in Carpule form; it is quite practical for office use by those who are not opposed to mixing their own solutions. Various investigators have reported a 30 to 50 per cent increase in area of anesthesia with local infiltration techniques.2”, 27,x The onset of operating When hyaluronidase was added to analgesia was also much more rapid. procaine and Pontocaine, the duration of anesthesia was reduced almost 50 per cent. However, when a vasoconstrictor (epinephrine) was added, there was a prolongation of anesthesia time similar to that obt,ained when hyaluronidase was not used in the anesthetic-vasoconstrictor solution.“, 27,” Also, it was found that the vasoconstrictor did not decrease the spreading effect; if there was any change, the spreading was increased even further.25 Although addition of hyaluronidase to the anesthetic solution might increase the area of anesthesia, the rapid onset, and perhaps the depth of anesthesia, the over-all percentage of successful anesthesia probably would not will not penetrat,e a fibrin increase. This is due to the fact that hyaluronidase barrier unless injected directly into it; otherwise, it will follow the path of least resistance and diffuse around the barrier. Likewise, the enzyme will not In short, hyaluronidase may be of benefit render a fascial plane permeable.“’ in certain local anesthesia cases; but it is not a substitute for anatomic knowledge and correct injection procedures.“5

Topical Anesthesia Hyaluronidase enhances the penetration and diffusion of certain substances through the intact mucous membrane.4S It does not seem to be effective in penetrating the intact skin layer,+j although some recent investigations might prove penetration even through intact epithelium. Penicillin in conjunction with hyaluronidase, when inserted into the results in higher blood levels of the antibiotic than when maxillary sirnq5 penicillin is used alone.43 The addition of hyaluronidase will enhance penetration of certain substa.nces, even through an inflamed mucous membrane,“” a fact of special interest to dentistry.

EIYALURONIDASE

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481

Trotter’” mentions several dental uses of Xglocaine/‘hpaluronidase ointInent, and reports uniformly good results. He reports use of a preparation with the formula : Xylocaine 5 per cent Hyaluronidase 0.015 per cent ?Vater miscible base for extraction of deciduous hecth, incision of abscesses, operations on cruptiorl cysts, and in operative dentistry for anesthesia of sensitive dentine. Tn the Fitzsimons Clinic, the commercially available 5 per cent Xylocaine ointment was compounded with 10 TRIi hyaluronidase per gram of ointment. It was found that anesthesia on the mucous membrane was more rap3 and more profound than with the 5 per cent Xylocaine ointment used alone. The post,erior palatal regions of three patients were dried as in preparation for a post,erior palatine injection; 5 per cent Xylocaine ointment was massaged into the mucous membrane on one side for thirty seconds, and $5per cent Xylocaine with IO TRIJ hyaluronidase per gram was nmssaged intro the mucous membrane on the other side. At thirty-second intervals, the areas were tested with a sharp needle. It wa.s found that after one and one-half minutes the area anesthesized with Xylocaine/hyaluronidase ointment was insensit,ivc to the needle prick, while it took at least two and one-half minutes for anest,hcsia to appear on t,he side ;111!‘sthesized wit,h Xylocaine alone. The anesthesia also seemed more profound with the Xylocainr/hyaluronidase. Two deciduous teeth were extracted, using the Xylocaine/hyaluranidase ointment, with no discomfort to the patient. A method of more rapid and deep anesthesia was devised. The screw on :I HB type polishing cup was grasped by a hemostat. The cup was filled with the Xylocaine/hyaluronidase ointment and the cup was pressed rtgainst, drietl mucous membrane. The resulting hydraulic pressure appeared to cause deeper and more profound anesthesia than the method of massaging the ointment illto the membrane. It is believed that hyaluronidase might facilitate more efficient diffusion of other substances through the mucous membrane. The stability of thri various substances combined with hyaluronidase had not, been determined.

Temporomandibular

Joint Disturbances

Problems concerning the temporomandibular joint have disturbed the entire medical field for many years. Dentists, whose consultation is usua,I!y first sought, have had little to offer as palliative or definitive t,reatment. i\s more knowledge was accumulated on the ot,her joints and joint complexes ,,f the human body, more beca,me known about the unique joint of the m;rn~lih!e!.

482

GEORGE

H.

HENKEL

It is not the purpose of this article to discuss the anatomy or physiology of the temporomandibular joint. However, a basic discussion of certain features of the joint is necessary for clarity. Most disturbances of the ternporomanilibular joint are either congenitd 01 developmental, inflammatory, traumatic, or neoplastic in origin.30 Symptoms of temporomandibular joint disturbances vary considerably, namely, clicking and cracking, popping, pain on opening or closing, restricted motion, hypermobility, pain radiating from the joint, subluxation, dislocation, and ankylosis (false or true). Although the clinical symptoms are often readily apparent, the etiology is rarely positively identifiable. A careful medical history, persona.1history, and a comprehensive clinical examination, using all possible diagnostic aids, will assist in diagnosis, but still will often be insufficient. For a given diagnosis, any one or combination of treatments may be prescribed. Injections of sclerosing solut,ions, local anesthetics in oil, cortisone, osteoarthrotomy, condylectomy, meniscectomy, distraction of joint surfaces, diathermy, lysis, osteotomy, infrared therapy, immobilization and rest, occlusal equilibration, and probably many other treatment plans have been and are being used. The dentist is inclined to favor the conservative treatment, while the orthopedic surgeon is prone to utilize the more radical methods, including surgery. Nathau3” in 1954, proposed the use of hyaluronidase in treating certain temporomandibular joint disorders. Hyaluronidase has found some use in orthopedics in treating disturbances in other articulations.5 The rationale for utilization of hyaluronidase is different bhan other treatment of joint problems. With the temporomandibular joint, the rationale has to be modified still further. The condyle of the mandible is composed of typical cancellous bone covered by a layer of cortical bone. The articular fossa consists of a thin, compact layer of bone, while the articular tubercle is composed of spongy bone covered The art,iculating surfaces of these bones are by a thin layer of compacta. covered by a fibrous to fibrocartilaginous tissue, rather than the hyaline cartilage found in most other articulations. A fibrous plate, the articular disc, is interposed between the articulating bones and separates the joint into two compartments: upper and lower. A small amount of viscous synovial fluid is found in the articular compartments. The synovial fluid is felt to be a function of connective tissue. It may be a product of filtration from the blood vessels or a secretion of the synovial membrane (modified connective tissue), possibly both.“’ It is known that hyaluronic acid is a common constituent of synovial fluid as well as of the ground substance of connective tissue.34P39 The permeability of the connective tissue has a normal, constitutional state or tonus which can be altered by various faetors.15 When inflammation is present, tissue tone is disturbed and tissue permeability is altered.“O It is felt that hyaluronic acid is the substance responsible for increased viscosit.y of synovial fluid,3” a condition often seen in pathologically involved joints.

HYALTJRO;L\‘IDASE

IN

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DE?;‘l’IS’rl
1s:<

This disruption of the fluid balance of the synovial fluid may result from I feel that in disturbances of the or contribute to the pathology in the joint. temporomandibular joint the fluid imbalance is at. least contributory to joirlt symptomatology. The blood supply to the articulation is quite adequate ; the condyle receives its blood supply from the internal maxillary artery via the deep auriculatThe posterior artery, which gives off branches to the articular capsule. auricular a,rtery also supplies the condylar head. These vessels form a capillary network close to the inner synovial surface of the capsule, lying itI t,he wa.11 of the capsule, There is no direct blood supply to the avasculat* fibrous or fibrocartilage coverings of the articulating bones or t.o the articular disc ; consequently, the synovial fluid must act, as a lubrirant and as a nutrient supply to these avascular structures.“7 If the fluid balance of the synovial fluid were such that the avascular areas of’ the joint received insufficient lubrication and nutrients for healthy existence, then administration of hyaluronidase might, (1) lyse excessive hyaluronic acid, (2) increase permeability of the capillaries and the synovial layer of the ca,psule to allow re-establishment of the proper fluid balance, and (3) allow Cssue fluids properly to invade the area and thus restore norm:11 tone to the t,issues.‘R, ““3 :jfi The ra.tionale for use of hyaluronidase in trea.ting temporonlandibulrt, for all disorders. Hgaluronidaxt~ joint disturbances does not imply a “cure-all” acts as a palliative agent and as an aid to natural body defensive and rrparative mechanisms. The action might be considered as analogous to t,hat of an antibiotic. Xl1 antibiotic does not usually destroy bact,eria but, rather, is bacteriostatic; that is, it inhibits the growth and multiplication of bacteria to the extent that. i-f effective and if the source of infection were removed, the natural body &fensive mechanisms might destroy t~he bacteria and re-establish body integrity. The action of hyaluronidase is parallel to this. The enzyme cikntlot usually correct t.he basic cause of the disturbance. If the basic pathology wet’(’ eliminated or significantly decrea.sed, however, hyaluronidase would assist the body in its attempt to restore the natural fluid bala,nce, lubrication, and nubition to the joint fluid and to the capsule. The use of hyaluronidase for temporomandibular joint disturbances is 111bi a substitute for knowledge of anatomy, for correct diagnosis, for oc~lus;~I equilibration, for establishment of correct vertical dimension, or for removal of neoplasms or other pathologic entities ; it is not a substitute for surgery in the event of true ankylosis or ot,her conditions in which regenet*ilt,ion of tiss\Le is tleemed impossible. ‘I’hc enxymc is a valuable coml)lcment and supplement to pr*ctsc*ril)t~tl 1Iyaluronidase will often facilitate or perhaps institute repair it1 therapy. cases wherein the return of the damaged joint, tissues to relative normals): is retarded.

484

GEORGE H. HENKEL

The use of hyaluronidase at Fitzsimons Army Hospital has shown definite In the cases promise for relief of many temporomandihular joint disturbances. of hypermobility, there has been a tightening ; with hypomobility, there has been a loosening. Absence of pain during the injection procedure and decrease in painful symptoms after treatment have been noted in most cases. Administration of the enzyme is a relatively simple procedure. In the Fitzsimons Clinic, the patient was first examined carefully and a complete history was obtained. Definitive treatment as indicated by the diagnosisequilibration, mobilization, immobilization, re-establishment of vertical dimension, surgery, etc.-was undertaken as far as possible. If it was then felt, according to the above rationale, that hyaluronidase might be of benefit, an injection was made. Injection was made in several cases when the possible benefit was very doubtful. This was done for the purpose of investigation and because there was always possible benefit, while there was no apparent contraindication. It is believed that at least some of the relapses were due to selection of unsuitable cases. In other cases, relapses were due to uneliminated basic pathology; several of these cases improved after this pathologic condition was corrected. The armamentarium for joint injection was the same as the basic setup shown in Fig. 1, with the addition of a sterile drape, surgical gloves, mouth prop, and surgica,l soap. The patient was draped and the area over the condyles was washed and sterilized. The skin over the condyles was anesthetized with a few drops of local anesthetic solution and the position of the condyle was palpated with a gloved left hand while the patient made various opening and closing movements. When the position of the condyle was determined, a mouth prop wa.s inserted to prevent sudden or reflex closure. No attempt was made to inject specifically into either the upper or lower compartment. A 22to 24-gauge needle in a Lok type syringe was inserted through the anesthetized skin and passed upward, forward, and inward until the head of the condyle was contacted. The needle was then withdrawn slightly and the solution of the enzyme, reconstituted in sterile physiologic saline, was slowly injected int,o the joint capsule. A modified Barton type bandage was t,hen placed, with gauze pad pressure As stated over the condyle areas, for a period of thirt,y to sixty minutes. previously, there was little or no discomfort during or after the procedure. A small volume of saline was used because of limited space available within the capsule. It was felt that a 1 to 1.5 CC. volume of solution, plus the pressure pack, was sufficient to increase the interstitial pressure to the necessary level. Case hist.ories follow. Casx 1.-A 21.year-old white man was seeu as a referral patient from another inHe had a complaint of painful popping and clicking of both eondyles of five stallation. months’ duration. Various treatment plans had been attempted at the other installation, The pain had become more severe during including a “complete grinding of the teeth.” the past month, a month of cold weather.

HYALURONIDAW

IN

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DESTISTRF

tlislocal ion h:ltl t)Yt’I’ I)(‘Thuro was Il~pern~ol~ility of I~oth con~l,vlca Ijut no $l~l~~i~I’(Lllt \‘tlriouh ,‘,I rrotl. ‘t’hv popping :LII~ cracking of tlrc joints ‘ivew :tu~lilblo six feet, away. t.reatment plans were tliscnssotl, rocrltgeno!$anls WUI’C i:tken, :tt)ti the paticbtlt W:IS wl~~rrti~~l to his permanent installation while the case was evaluatcll at Fitzsinlons. After a furt,her an injection into the himself in a ulodifird tllat any opening of two days.

it was dllc:idrtl to attempt review of t,he properties of hyalunonidasc, condyle. When the patient was seen one week later, he presrntetl The pain had brcome so severe during the past w(+I( Barton bandage. The nlodilic(l Barton had hern wor11 for the mouth was intolerable.

Hyaluronidase, 150 TRU in 1.5 C.C. saline, was injected into the right joint capsule and 150 TRU hyaluronidase in 1.5 C.C. saline was injected into the left. A pressure pa~lc was applied to both condylar areas for forty-five minutes. The patient reported no discomfort from the treatment. ‘fht! There was no improvement after the treatment. mo~lific~tl Barton bandage was replaced and the patient, was returned to his homc~ station. The patient was seen after one week and was complet,ely comfortable. He reported that, on the second and third postinjection days he ate hamburger meat without discon~fort. Hy the fourth postinjection day he was comfortable and ate mashed potatoes and l,lWld. Eating shelled corn on that day had caused pain on the right side only. On Ihca On the seventh day the left side was Gxth day the bandage immobilization was removed. c*ompletely comfortable, while the right side “seemed a little tight.” Popping coultl IW hc:nd with a stethoscope, but not with the unaided ear. The patient was seen again after four weeks and was “completely comfortable.” He experienced pain on eating hard food one time two weeks prrviously but the pain halI tlisappearetl after he stopped eating. No popping ~~1~1 I)(: hrxrd with thfa stc~thost~ollt~. had

wivo

The patient was enthusiastic about the treatment and inquired where a further injection, if necessary, after he was discharged from the Service.

he might,

re

The patient was told to report to Fitzsimons if he had any relapse before being itisPharged, or to notify the clinic of any change after discharge. At the time of this report, the pat,ient has given no intli(~afion five and one-half months after the original injection, of relapse. This was the first case in which Fitzsimons (:linic. Due to the excellent llse the enzyme when diagnosis warranted

hyaluronidase injection was attempted at t.he results obtained, it was decided to continur to or suggested utilization.

Case 2.-A 3O-year-old woman was seen with a complaint of painful popping antI The left ride was more painful tha!) (aracking of both condyles of eleven months’ duration. t,he right. Six months previously, there had been a pain radiating from the left ternporomandibular joint into the temporal region. The symptoms were more severe in the morning and, until a few months previous when an occlusal equilibration had been accomplinhr(t, the .\t patient was unable to open her mouth wide enough to brush her teeth in the morning. present, the opening was poor in the morning and improved with exercise during 1 he tlay. The present intermaxillary space was 25 mm. Hyaluronidase, 150 TRU in 1 C.C. saline, was injected into each joint capsule and :, pressure pack was applied. The patient experienced no discomfort from the treatment. Eight hours postinjection, the patient reported that the “scraping of my bones” wa. gone. After twenty-four hours, there was subjective relief, normal eating was possit,l~~. and discomfort was experienced only upon yawning or extreme movement. There \vas son~,~ soreness of the skin and muscle surrounding the joint, probably due to the needle lmn~.tur(.. Vertical dimension had increased 3 mm. to 28 mm. (Xicking could be detected only with the stethoscope. vertical patient

There

was no apparent

popping.

After forty-eight hours, the patient ate Swiss steak without discomfort ant1 ililt dimension had increased to 32 mm. Early morning opening was ronsidered 1)) 1tjtb as normal for the first time in eleven months.

After one week, there was some popping of the right joint but no pain. The patient, could open her mouth and chew any food without tliscomfort. The patient was “completely satisfietl” with the treatment. After one mont,li, the patient statetl she still felt comfortable but that the grating sound had recurred on the left side. The left temporomandibular joint was reinjected with 150 TRU hyaluronidase in 1.5 cc. saline. After one week the grating was barely audible. Three months after the reinject,ion there had been no relapse. Case 3.-A 19-year-old boy was seen with a history of five years’ temporomandibular joint difficulty, especially on the right side. There was an audible pop on 40 mm. opening and general instability and hypermobility of both joints. The right side was painful much of the t.ime, with special difficult7 in eating and smoking. It was not possible for the patient to smoke a whole cigarette; pain in the right temporomandibular joint would become severe before completion. The patient had a marked Class II malocclusion with deep overbite and overjet. The lower anterior teeth closed into the palate, causing soreness of the t,issue. There did not appear to be prematurities in centric, but lateral excursions were prevented by the deeply locked bite. There was neglect of oral hygiene and rampant caries. Occlusal equilibration appeared inadvisable. It was felt that full upper extraction and a denture would be necessary to afford any permanent correction. Complete definitive treatment of this extensive a nature was impossible because the patient was stationed at another installation and was under temporary treatment for a knee injury at Fitzsimons. Definitive treatment was to be deferred until the patient was returned to his home installat,ion or released t,o Fitzsimons for temporomandibular joint treatment. It was felt that temporary relief might be afforded by hyaluronidase. The temporary palliative nature of any proposed treatment was explained to the patient. Hyaluronidase, 150 TRU in 3 C.C. saline, was injected into each joint capsule. The patient experienced no discomfort from the treatment. A pressure pack was applied. Eight hours postinjection, smoking and eating of soft food could be accomplished without difficulty. Thirty-six hours postinjection, there had been a sharp pain radiating from the right temporomandibular joint into the temporal region of thirty seconds’ duration. This was the only difficulty experienced at any time. Forty-eight hours postinjection, there was a slight click in the right temporomandihular joint, but there was no pain. After one week, the patient reported the return of symptoms. The pain was not SO of 300 TRU hyaluronidase in 1.5 severe but was returning on the right side. A reinjection C.C. saline was made into the right joint capsule and pressure was applied. The patient admitted that he had not followed instructions about refraining from unnecessary jaw movement; therefore, after the reinjection the patient was placed in a chin strap and a dental soft diet was ordered. the patient had subjective relief. Twenty-four Four hours after the reinjection, hours after reinjection, the patient was again completely comfortable while eating or smoking. One week after reinjection, the patient noted pain on eating. He was comfortable while not eating and while in the chin strap and was reluctant to take the chin strap off because he felt that the discomfort would return. In this case, the basic disturbance of the joint was left untreated and the hyaluronidase was not expected to effect any cure. If the fluid balance of the joint were restored and the increased nutrition and lubrication of the joint afforded temporary relief of symptoms, the pathologic situation would once again become manifest due to return of the fluid in balance as It was decided to hold the patient to determine the effects of the hyaluronidase disappeared. whether or not treatment of the malocclusion might afford relief.

HYALURONIDASE

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DENTISTRY

A ~OWW overlay splint was constructed to relieve the overclosure and the poor occlusion. The patient was comfortable for one month and then complained of severe the right temporomandibular joint. The splint was adjusted, which gave relief. ‘Ihis situation persisted for one week, when the patient was admitted after (he right condyle while kissing his girl friend. The dislocation was reduced and \\WS sedated and immobilized with Ivy loops and intermaxillary wiring. The wires were removed after one week and the splint was worn once There has been no relapse in two months.

487

to partialI>,

correct

pain about dislocating the patieut more.

Case 4.-A G-year-old woman was seen with a pain in the right ear with radiation downward along the sternocleidomastoid muscle. The pain had been present for six mont,hs There was noticeable clicking and grating. am1 was particularly noticeable on eating. The patient had been seen by the G. I. Clinic (which prescribed iron and soft diet) auti RENT, and had had au x-ray picture of the temporomandibular joint. All findings were negative. Hyaluronidase, 150 TRU in 1.5 C.C. saline, was injected bilaterally and a pressure park was applied. After two days, the patient reported that the left side was sore from the injection. After one week, the patient was a little more comfortable but still had some discomfort on the right side on chewing. Hyaluronidase, 150 TRU in 1.5 c.e. saline, was reinjeetetl into the right joint area. Three days after the reinjection, the patient stated that the pain in her right ear hatI improved considerably. One week after the reinjection (two weeks after the original injection), the patient She stated she could “sleep at night without. felt that the pain was completely gone. She was comfortable on eating. The clicking and grating had disappeared. The tensions.” only concern of the patient now was noticeable movement of the condyle head in normal excursions of the mandible. There has been no relapse in two months. Case 5.-A BY-year-old woman was seen five days after she underwent a CaldwellLuc operation. The patient had pain radiating upward, backward, and forward from the There was some hypomobility but no clicking or popping. right temporomandibular joint. The temporomandibular joint felt tight and the pain was severe. Hyaluronidase, 150 TRU in 1.5 C.G. saline, was injected iuto the right capsule. h pressure pack was applied for thirty minutes. There was some soreness at the site of injection. The pain left after eight hours. The jaws felt looser and opening was accomplished with less discomfort. The patient felt. much better. Case 6.-The patient was a 36-year-old man who had experienced pain in the right The right side was coucoutlyle for three years and in the left condyle for two years. There was a history of pain radiating into the entire face. The aitlerably more painful. patient, hall been treated in Japan by thermal packs which hat1 temporarily localized the pain in t,he temporomandibular joint. The patient ha<1 been seeu bp RENT, with no ear abnorrnalit,ies noted. Rcluilibration had been done six months previously, autl the patient wore a bite plate. Since he had been wearing the plate, the pain was not severe, but there were perioclic atacks. The last attack had occurred the previous night. The patient hail been toll? by his physician that he had rheumatoid arthritis. lI~:llllrt)llillasc~, 300 TRIJ in 2 C.C. saline, was injectetl iutn the right con~lyle; 2 C.C. saline (no c~nzymc~) was inject,ecl into the left condyle as a control and also as an attempt to rule out a psy(~lrosomatic disturbance. The injectiou into the right joint, was acco~nSome pain was note
488

GEORGE

H.

HENKEL

Examination twenty-four hours later revealed no pain on the right side. The patient on the left stated that he felt “completely cured” on the right but still had discomfort side. The pain on the right side had left after nine hours. Examination after forty-eight hours revealetl the same condition. The patient then received 300 TRU hyaluronidase in 1.5 C.C. saline, injected into the left condyle. Only a slight burning sensation was noted on injection. A thirty-minute pressure pack was applied. Forty-eight hours after treatment the patient’s condition remained the same. Teu hours after injection of hyaluronidase, however, there was complete relief on the left side. After one month, the patient reported an episode of pain in the right temporoHyaluronidase, 150 TRU in 1.5 C.C. saline, mandibular joint on the previous evening. was injected into the right side and comfort was obtained within eight hours. One month after the second injection, the patient complained of a dull ache in the Again 150 TRU hyaluronidase in 1.5 C.C. saline was inright temporomandibular joint. Relief was obtained and no relapse occurred within the jected into the right side. ensuing six weeks. Case 7.-A 30-year-old woman reported pain of three years’ duration in the left temporomandibular joint. Her third molars had been removed and equilibration had been done, with some relief. One year before, a periodontist had corrected occlusion and inThe patient described the injection as very jected a “new substance” into the joint. painful, but it had no effect on the temporomandibular joint pain. Nine months previously some infected teeth had been extracted and six months previously the patient had She also had seen a psychiatrist. When seen undergone ethyl chloride spray treatment. at Fitzsimons, the patient was under the care of a “treateur” but had found no relief. She had had severe episodes of pain on the previous evening. Hyaluronidase, 150 TRU in 1 C.C. saline, was injected into each capsule and a pressure The patient found the procedure uncomfortable pack was applied for forty-five minutes. but said that it “really wasn’t painful.” Forty-eight hours after treatment there was no difference in the patient’s condition. There had been no attacks, but the patient said that her teeth felt “on edge.” One week later, the patient still had the “on edge” sensation and had an episode of pain in the left temporomandibular joint. The teeth on the left side were desensitized with sodium fluoride paste and 300 TRU hyaluronidase in 1 c.e. saline was reinjected into the left joint. Five day-s after treatment the patient reported that the “on edge” feeling was gone and the joint pain was less severe. Two weeks after reinjection, the patient felt completely comfortable. Two months after reinjection, the patient stated that her symptoms had returned The left temporomandibular joint was reinjected but were not so severe as previously. with 150 TRU hyaluronidase in 1.5 C.C. saline. Subjective relief was obtained and one month after the last injection there had been no relapse.

Case K-The patient, a 25-year-old man, complained of popping, clicking, and pain in The patient was edentulous and wore a reeeut both joints of about one year’s duration. set of dentures which appeared to be well balanced and to have correct vertical dimension, etc. He stated that this was the first set of dentures that had “felt good.” He had been given sodium psylliate injections into both joints nine months previously, with no relief. Hyaluronidase, 300 TRU in 1.5 C.C. saline, was injected bilaterally and a pressure pack was applied. One week Iater the patient reported that the left side was comfortabIe but that there was no change in the right side. The right temporomandibular joint was reinjected with 300 TRTJ hyalurouidase in 1.5 cc. saline. One week later the patient was free of pain, but t,he clicking still persisted. After three months, there was no change, although the patient was still free of pain.

HYALUROKIDASE

IS

CLINICAL

I)ESTISTKY

Ai;!)

Case 9.-A 22-year-old man once noted sharp, severe pain over both telllporolllalltlil,IIlar joints while eating one and one-half years previously. The pain resolved to a 11u11 ache. The patient had been treated by immobilization one year previously, which relievt,ll The patient had marked overthe pain on the left side. The right side was still painful. bite and orerjet and was locked in acquired centric relation. An overlay splint was inserted in an attempt to correct the overbite and overj& There was no change with the splint in ten days. The patient was then given 150 TRU hyaluroniclase in 1.5 C.V. saline bilat~erall~, aull One week later no change was evident. a pressure pack was applied. The patient was reinjected with 150 TRV and again there was no change within one week.

hy-alurouidase

in 1.5 C.C. saliue

I)ilaterall>-

TWO months later, no relief had been obtained. This is the only case in which there was no improvement. Case lO.-The patieut, a 42.year-old woman, complained that her right jaw “,jumpt~~l She had been aware of this condition fat out of place” when she opened her mouth wide. several years, but it had become progressively more annoying and painful after she ha11 an R-14 filled four months previously. It was believed that the symptoms wrre due 10 the mechanical trauma of the operative work. Hyaluronidase, 150 TRU in 1 cc. saline, was injected into the right capsule aorl :L pressure pack was applied. After twenty-four hours the patient stated that there was less paiu on ext,reme Subjective relief was obtained. There opening and that the popping was not so severe. had been no relapse when the patient was seen tFo and one-half months later. Case Il.-A 36-year-old man complained of soreness and stiffness around the left temporomandibular joint. The condition, which was diagnosed as neuralgia, was no1 The symptoms reappeared four treated and the symptoms disappeared after three weeks. months later and the patient experienced soreness, stiffness, and restrictiou of opening, which were much more severe in the morning. Hyaluronidase, 150 TRU in 1.5 C.C. saline, w-as injected into the left capsule a1111u pressure pack was applied. Three days later the patient stated that the stiffness which had been present in thth morning had disappeared to a great degree, but that there was still some pain ou maximum opening. There was a gain of 15 mm. in intermaxillarp space. The patient was comfort After two months then, able, but still experienced some discomfort on opening his mouth. had been no regression to the original condition, but the patieut, had not obtained ~OIIIplete relief. Case 12.-A 26.gear-old woman complained of throbbing pressure pain in the right The pain occurrod several times (Iail>- ant{ tllc,rf, ear of one and one-half years’ duration. The patient reported occasional popping of was no particular relation to time or activity. the left temporomandibular joint while eat,ing. Hyaluronidase, 150 TRU in 1.5 C.C. saline, was injected into the left capsule anti :I A soft diet was prescribed, Ibressure pack %-as applied. The patient obtained relief within twenty-four hours. One month after treatmeu t she stated that she had been completely free of symptoms since injection. Two mot1t Il.\ after treatment there had been no relapse.

Miscellaneous Uses The uses of hyaluronidase mentioned in previous sections ot’ this articlt> are those which will find the most universal application and those which hav(~ been investigated most extensively. Other clinical applications of the eneym
490

GEORGE

H.

HENKEL

It has been found that certain bacteria are capable of producing a form oral strain of bacteria producing of hyaluronidase.lls 28,2g The predominant the enzyme is an alpha-hemolytic streptococcus, Streptococcus mitkz8 Hyaluronidase in saliva is due to bacterial metabolism.2Y The hyaluronic acid found in the oral mucous membrane and in tooth structure can be attacked by Alteration of the cementing suhthe oral bacteria that produce hyaluronidase. stance of the mucosa or tooth structure may increase tissue permeability, thus mediating the rate and extent of infectionZ8 or decay. Upper respiratory infection, periodontal disease, and dental caries all cause a marked increase in salivary hyaluronidase titer.2g Thus, further investigation may produce an index of incidence of dental disease which will be much more accurate and inclusive than the lactobacillus count or other diagnostic indices available at present,. Plastic surgery has found hyaluronidase useful in the treatment of keloid$ and in protection of flaps and grafts. The enzyme may well be of distinct value for major orofacial surgery. The value of a dispersing agent to prevent ballooning of tissues by local anesthesia for procedures about the face has been well established.5 As noted previously, with the addition of a vasopressor, the depth and duration of anesthesia are not adversely affected. Nutrition of flaps, skin grafts, and bone grafts should also be improved by local infiltration of the enzyme. Edema and hematomas often cause tensions or pressures which endanger the success of the operation. Prophylactic use of hyaluronidase where such a condition might be expected should be considered. As stated previously, however, I do not consider the prophylactic use of hyaluronidase to be a sound procedure when there is wound contamination. It has been hypothesized that certain malignant neoplasms are invasive due to production of hyaluronidase by the cancer cellsZ6 There may be a role of considerable importance for hyaluronidase in the mechanism of invasion of the body by bacteria and other toxic agents.ll’ I5 Sinusitis, although usually not treated by a dentist, may be encountered after such procedures as a Caldwell-Luc opera,tion. Enhancement of penetration of penicillin into the mucous membrane of the paranasal sinuses with resultant higher blood levels of the antibiotic was obtained by Som and his associate.+ with the enzyme. Treatment of postoperative sinusitis with penicillin/hyaluronidase might well be considered. General surgery has found many other uses for the enzyme; similarly, in dentistry, many new horizons are foreseen.

1. Hyaluronidase, an enzymatic spreading factor, is reviewed from physiologic and pharmacologic aspects. 2. Hyaluronidase has applicability in the control of traumatic swellings. 3. Hyaluronidase is a valuable adjunct in the treatment of certain temporomandibular joint disturbances. 4. Hyaluronidase aids in rapid resolution of hemat0ma.s.

HYALURONIDASE

IN

CLINICAI,

DESTISTRY

191

5. Hyaluronidase does not,, in itself, effect healing but, rat,her, allrvi:ltchs certain factors which may delay healing. 6. lfya.luronid;~se demonstrates maximum cft’trctivcuess when coupled with increased interstitial pressure, accomplished by injection of a volume ot’ solution and by pressure packs. 7. Hyaluronidase increases depth, area, ant1 speed of onset of local anest hesia. 8. Hyaluronidase increases absorption of topical anesthetic agents a tltl other topical medicaments. 9. Hyaluronidase may prove to be a potent diagnostic aid, may facilitate cstensive oral surgical procedures, and may contribute valuable knowledge to the host-parasite pr,oblem. 10. Hyaluronidase is a. valuable complement and supplement to many procedures of practical clinical dentistry. References 1. Benditt,

2. 3. 4. 5. 6. 7. 8. 9. .

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Earl P., Schiller, Sara, Wong, Helen, and Dorfman, Albert: Influence of ACTH and Cortisone Upon Capillary Permeability Induced by Hyaluronidase in Rats, Proc. Sot. Exper. Biol. & Med. 75: 782-784, 1950. Rensley, S. H.: On the Presence, Properties and Distribution of the Intercellular Ground Substance of Loose Connective Tissue, Anat. Rec. 60: 93-108, 1934. Renzer. Paul: A Preliminarv Reoort on the Use of Hvaluronidase in the Treatment of Tiaumatic Swellings, O&AL &RG., ORAL MED., AN; ORAL PATH. 4: 1515-1522, 1951. Renzer. Paul. and Sehaffer. Arnold: The Use of Hvaluronidase in the Treatment of Tiaumatic Swellings, URAL SURG., ORAL MED., AND ORAL PATH. 5: 1315-1340, 1952. Rritton, R. C., and Habif, David V.: Current Vses of Hyaluronidase: A Current Heview, Surgery 33: 917-942, 1953. Chain, E., and Duthie, E. 8.: A Mucolytic Enzyme in Testes Extracts, Nature 144: 977-978, 1939. Chain, E., and Duthie, E. 8.: Identity of Hyaluronidase and t,he Spreading Fa(‘toT. Hrit. J. Exper. Path. 21: 324-338, 1940. Capillary Endothelial Cement in Relation tr, Chambers, R., and Zweifaeh, B. W.: Permeability, J. Cell. & Comp. Phpsiol. 15: 2-55-272, 1940. in Treatment of Keloids, Arcah. Aurg. 64: Conway, H., and Stark, R. R.: Corticotropin 47-50, 1952. Use of Hvaluronidase to Prevent Postonerative Comnlicatious. T). Devine. J. C.: Digest 60: 23-24, 1954. L Tjobler, Ruth Elaine: Antigenic Properties of the Hyaluronidases Producer1 b> Hemolytic Streptococci, Thesis, Minneapolis, 1951, University of Minnesota Press. Drinker, C. K., Field, M. E., Ward, H. K., and Lyons, il.: Increased Susceptibility to Local Infection Following C. Blockage of Lrmph_ Drainage, Am. .r. Phvsiol. 112: 74-81. 1935. of Extracts of Certain Organs From Normal an(i Duran-Reinals, F. : The Effect Immunized Animals on the Infecting Power of Vaccine Virus, J. Exprlr. Med. 50: 327-340, 1929. Duran-Reynals, F.: Extent of Local Dispersion of Infection Agents as Factors ill Rc sistance to Tnfection, J. Exper. Med. 61: 617-642, 193.5. Tissue Permeability and the Spreading Factors in Infection,. Rapt. Duran-Reynals, F.: Rev. 6: 197-252, 1942. Effect of Hvaluronitlasr Elster, Samuel K., Freeman, Monroe E., and Dorfman, Albert: on the Passage of Fluid and of T-IX24 Throueh‘1 the Caoillarv %-all. .4111. ,I. I Phpsiol. 156: g29-432, 1949. Fiala, S., and Ruth, K.: Interaction of Hyaluronidase With Thromboplastic (“omponerrt Q of Blood Coagulation, Federation Proc. 11: 344, 1952. Effect of Hvaluronidase on Relief of Trismus in the TemnoroFrank. Onden M.: niangbular Joint, Final Report of Project at T,ettrrman Army Hospital,^ Sarr Francisco, California, 1954. in Pediatric Therapy, T,anctht 257: Gaisford, Wilfrid, and Evans, D. Gi.: Hpaluronidase 505.507, 1949.

492

GEORGE H. HENKEI,

in the Treatment of Soft YO. Gartland, .J. ‘r:, ant1 MacAusland, W. IL., .rr.: Hy;tluroni(lasc Tissue Injury, Arch. Surg. 68: 305-31-1, 1951. in Rheumatic F~vcI', 21. Guerm, F.: Hyaluronidase Inhibition 1)y Sodium Salicylate Science 103: 686-687, 1946. Hvaluronidase Inhibition bv Serum in Human Cancer. Thesis. I 22. Hakanson. Erick Yngve: Minneapolis, 1948, University of Minnesota Press: 23. Hechter, 0.: R,econstlt,ution of the Dermal Barrier in Fluid Diffusion, Proc. Sot. Exper. Biol. L Med. 67: 343-344. 1948. The Clinical Uses of Hyaluronidase in 24. Hechter, O., Dopkeen, S., and Yudell, M.: Hypodermoclysis, J. Pediat. 30: 645656, 1947. The Use of Hyaluronidase With 25. Kirby, C. K., Eckenhoff, J. E., and Looby, J. P.: Local Anesthetic Agents in Nerve Block and Infiltration Anesthesia, Surgery 25: 101-104, 1949. 26. Logan, Archibald Hodge: Hyaluronidase in Human Malignant Neoplasms, Thesis, Minneapolis, 1949, University of Minnesota Press. Use of Hyaluronidase With Local Anesthetic 27. Looby, John P., and Kirby, Charles K.: Agents in Dentistry, J. Am. Dent. A. 38: 1-4, 1949. 28. Lorina, P. L., Lisanti. V. F.. and Chauncev. H. H.: The Production of Beta-Glucdronidase and Hyaluronidase by Streptococcus Mitis, ORAL SURG.,ORAL MED., AND

ORAL PATH. 7: 99%1010,1954. Hvaluronidase-Producina Microoraanisms 29. Mahler. Inga R., and Lisanti. Vincent F.: From Human Saliva, ORAL SURG., ORAL ME;., AND ORAL PATH. 57 1235124;, 1952. Observations on Testicle Extract and Its Effect on Tissue Per30. McClean, D.: Further meability, J. Path. & Bact. 34: 459-470, 1931. 31. Menkin, V.: Newer Concepts of Inflammation, Springfield, Illinois, 1950, Charles C Thomas. 32. Meyer. K.: Mucoids and Glvcooroteins. Adv. Prot. Chem. 2: 249-273. 1945. 33. Meier; K., and Palmer, J. “W.; The Polysaccharide of the Vitreous Humor, J. Riol. Chem. 107: 629-634, 1934. The Isolation of Mucopolysaccharide 34. Meyer, K., Smyth, E. M., and Dawson, M. H.: From Synovial Fluid, J. Biol. Chem. 128: 318-327, 1939. 35. Moore, D. C.: An Evaluation of Hyaluronidase in Local and Nerve Block Anesthesia, Anesthesiology 11: 470-454, 1950. 36. Nathan, A. S.: The Use of Hyaluronidase in Temporomandibular Disturbances, ORAL

SURG.,ORALMED.,AND ORAL PATH.~: 368-377,1954. Oral Histology and Embryology, 37. Orban, Balint: ed. 2, St. Louis, 1949, The C. V. Mosby Company. 38. Possoff, Abraham: External Thermal Applications in Postextraction Therapy, J. Am. Dent. A. 50: 147-156,1955. Hydrolysis of Hyaluronic Acid of Human 39. Ragan, Charles, and DeLamater, Arlene: Joint Fluid in Vivo. Proc. Sot. Exner. Biol. & Med. 50: 349-351. 1942. The Regional Injection of Penicillin in Local Infection, 40. Rose, D., and Hurwitz, ‘M.: New England J. Med. 234: 291-296, 1946. Extract on Distribution and Absorption of Sub41. Sannella, L. S.: Effect of Testicular cutaneous Saline Solution, Yale J. Biol. & Med. 12: 433-439, 1940. and Toxicology of Testicular Hyaluronidase, 42. Seifter, J.: Studies on the Pharmacology Ann. New York Acad. SC. 52: 1141-1155, 1950. 43. Som, M. L., Schneierson, S., and Sussman, M. I.: Enhancement of Penetration of Penicillin Into Inflamed and Normal Mucous Membrane by Hyaluronidase, Proc. Sot. Exper. Biol. 6 Med. 70: 96-99, Jan., 1949. Studies on the Hyaluronidase Inhibitor in Human Blood, Thesis, 44. Tobin, John Robert: Minneaoolis. 1949. University of Minnesota Press. Xplocaine/Hyaluronidase Ointment as a Surface Anesthetic in 45. Trotter, P.&A.:’ ’ Dentistry, Brit. D. J. 97: 94-95, 1954. A Workshop Concerning Histology and Histonathologv of 46. Wellborn. Joseph F.: Supporting and Investing Structures of the Bony Jaws, Unpublish>d Dat’l: Hyaluronidase for Prevention of Edema After 47. Young, H. E., and Kingsbury, B. C., Jr.: Removal of Impacted Mandibular Third Molars, J. Am. Dent. A. 50: 418421, 1955. The Structural Basis of Permeability and Other Functions of Blood 48. Zweifach, B. W.: Cold Spring Harbor Symposium on Quantitative Biology 8: 216-223, Capillaries, 1940. 49. Hydase, Pharmaceutical Brochure, Wyeth Inc., Philadelphia, Pennsylvania. 50. Manual of Alidase .Administration, G. D. Rearle & Company, Chicago, Illinois.