Alkaline phosphatase in reparative dentinogenesis

Samuel Seltzer, D.D.S.,” I. B. Bender, D.D.S.,*” and Richard Moodnik, D.D.S.,#*“” Philadelphia, Cnicersity

of Pennsylvania

lYchoo1

of

Irving Pa,.

J. Kaufman,

D.D.S.,“*+

Dentistry

PHoSPHATASE is present in the odontogenic and osteogenic cells and, to a lesser extent, in the calcifying matrix when mineralized tissue There is evidence that this enzyme has a function is being formed or repaired. in both matrix formation and calcification.‘~ 2 Attempts have been made to hasten bone repair by applying alkaline phosphatase to experimentally injured bone. Blum3 applied a mixture of alkaline phosphatase and calcium glycerophosphate to bone fractures and found that healing was more rapid. On the other hand, BourneZ drilled holes into bone and packed them with alkaline phosphatase but saw no evidence of increased bone formation. In a previous article4 we reported somewhat similar experiments on the exposed pulps of dogs’ teeth. Various calcium salts and other substances, including alkaline phosphatase, were tested for their effects on pulp repair. Studies of sections of teeth treated with alkaline phosphatase showed unusually large amounts of predentine around dentine chips which had been accidentally pushed into the exposed pulps by the bur. In none of the teeth t,reated with the other substances was there a like amount. of predentine; nor was there the strikingly picturesque alignment of newly differentiated odontoblasts. The present article reports further studies on the treatment of exposed pulps with alkaline phosphatase; compares results obtained with alkaline phosphatase, acid phosphatase, and a nonspecific protein (albumin) ; and describes some of the phenomena of dentinogenesis observed microscopically in sections of dogs’ teeth.

A

LKALI~E

MATERIALS

AND

METHODS

Cavities were prepared on the labial or buccal surfaces of teeth in five young dogs as follows : Dogs 1 and 2, eight teeth each (canines and carnassials) ; Dog 3, ten teeth (third incisors, canines, and carnassials) ; Dog 4, sixteen tutes

This investigation was supported by a research of Health, United States Public Health Service. *Associate Professor of Oral Pathology and Oral **Associate Professor of Oral Medicine. ***Dental Staff, Albert Einstein Medical Center, ****Lecturer, Department of Operative Dentistry.

859

grant

(D946)

from

the

National

Insti-

Histology. Northern

Division,

Philadelphia,

Pa.

Fig.

1.

Fig.

2.

Fig. 3.-Photornirrogragh of buccolingual section of upper‘ right first molar of w ~lo&. Following exposure, the pulp (I’) LVLLS treatml with a&l phosphatase. There is no rvirlcncv of differentiation of new odontoblasts or matrix formation around dmtine chips (DC ). Inflanmatory cells (PmZ) are present both within blood vrssels (BV) and within tissue. ,, Henra.toxglin and eosin stain. Magnification. Xz’iO ; reduced YL”. ) Fig. 4.-Photomicropraph of bucrolingual section of upper right third incisor of a ~lor

teeth (third incisors, third premolars, canines, and carnassials) ; and Dog 5, fourteen teeth (third incisors, canines, carnassials, and second premolars) . Some of these teeth were unsuitable, either because they were spoiled during sectioning or because no dentine chips were found in the pulp in tissue sections. Thus, the study was based upon observations of the pulps of forty-three teeth. The t,eeth were drilled under a constant stream of water, with either a portable dent.al engine (Dogs 1, ‘2, and 3) or an air turbine” (Dogs 4 and 5). Aft’er retention forms suitable for amalgam restorations had been obtained, the teeth were isolated with cotton rolls, dried, wiped with a 3 per cent aqueous solution of iodine, and dried again. Pulp exposures were then made by drilling through the floors of the cavities with a sterile No. l/L round bur or a Xo. 4 round bur. (The So. $$ round bur was used on half of the teeth drilled in each of three dogs. The No. 4 round bur was used on all other teeth drilled in each of the five dogs.) Hemorrhage was controlled bp wiping with sterile cotton pellets. The substances to be tested were placed on the exposed pulps for one minute. The cavities were then dried, the exposures were covered with sterile asbestos fibers, and the cariCes were sealed wit,h amalgam. The animals were killed by perfusion with fixatives three, five or seven days after the pulp treatments. They were decapitated, and each tooth was removed from the jawbone by means of a band saw. After decalcification, thirteen of the teeth were embedded in celloidin and sectioned, and the rest were sectioned either by the freezing microtome or after being embedded in paraffin. All sections were stained with hematoxylin and eosin for microscopic examination. The following drugst were tested on two exposed pulps in each dog (on a pulp exposed by a No. l/z round bur and one exposed by a No. A round burl : 1. An aqueous mg. per milliliter. 2. An aqueous per milliliter. 3. An aqueous 4. Physiologic In addition, Dog 3.

suspension

of amorphous

alkaline

suspension

of amorphous

acid phosphatase,

suspension of egg albumin, saline solution.

dry alkaline

phosphatase

phosphatase,

100

100 mg.

100 mg. per milliliter.

was tested on two

rxpoced

pulps

of

RESULTS BND DISCUSSION Observations on each of the treated teeth are recorded in Table I. Odontoblast-like cells, matrix, and calcified tissue in varying amounts were found in the pulps of some of the teeth. They could be seen around chips of dentine which had been forced into the pulp by the bur (Fig. 1) but were not evident in any other part of the pulp connective tissue. The “odontoblast-like cells” consisted of fibroblasts and undifferentiated mesenchymal cells which had oriented themselves around the dentine chips. *Kindly tPurchased

supplied from

by Star General

Dental Mfg. Biochemicals,

Co., Inc.,

Philadelphia, Chagrin

Pa. Falls,

Ohio.

TREATMENT

Saline

solution

Albumin

Acid

phosphatase

Alkaline phosphatase (1) Suspension

?Lower

Powder

molar molar molar molar molar premolar

4

Lower Lower Lower Lower Upper Lower Upper Lower

right first le?t right left first right first left first left third left canine right third right first

Upper Upper Upper Lower Upper Upper Upper Upper Lower Upper TIpper Lower

right left right left left right right left left right left left

canine canine canine canine third incisor third incisor canincz canine third premolar first molar third incisor first molar

4”

Severe Hwcre Severe Hcrere Slight Skere Hftrorc Severe Severe Modrrate Moderate Moderate

Upper Tipper Upper Upper Upper Upper Trpper T,ower Upper T,ower I,ower

right loft, right right left left right left right left right

first molar first molar first molar first molar first molar first molar first molar third incisor canine second premolar canine

4 vi 4 J/i 4 4 4 4 4 4 4

Modrrate Slight Sliiht Severe Severe Moderate Moderate Severe Modcrate Revere Swcrc

T,ower T,ower Lower T,ower Lower Lower T,omer [Jpprr Lowc~r

right left left right left left right lrft lfkft

caninr canine canine canine third incisor first molar first molar third incisor cxninr

Upper Tipper *Dogs 1 and 2 killed three and killed seven days after treatment. no chips in the pulp or because the (2)

IXFLAMMATIOX

TOOTH

premola molar

4 4

Scverc Slight Slight Revere Slight Severe Hrverc Severe Severe

Moderate Moderate Severe Severe Moderate Severe Severe Severe Slight

right t,hird premolar Slight left third premolar Revere five days, respectively, after treatment; Ten teeth are not shown in the table teeth were spoiled during sectioning.

CE1,I.S

+ i + .+ -i+ _i + + -

..-

t $ + + + + + -

dogs because

3, 4, and 5 there were

They resembled true odontoblasts in varying degrees and, in fact, seemed to represent different stages in the development of odontogenic cells (odontoblasts) . Their presence at the locus of matrix formation, their tendency to line up in palisade formation (arranged parallel and in continuous contact with each and their morphology (transitional forms approaching a columnar other), shape) identified t,hem as odontoblast-like cells. The association of these cells

yw&

BLKALINE

PHOSPHATBSE

1N REPARATIYE

UESTIXOGENESIS

863

with the production of new dentine was suggested by their arrangement parallel to each other but not lined up with the tubules of the old dentine in the chips (Fig. 2). From these considerations, it would seem that undifferentiated mesenchymal cells can become matrix-forming cells and that the pulp is a reservoir for the supply of such cells. Whether all or only some of the cells in the pulp connective tissue are capable of developing into odontogenic cells can be answered here only by analogy with the corresponding cells involved in bone formation. Pritchard5 summarized evidence indicating that osteoblasts develop from a specific race of connective tissue cells. The more closely the mesenchymal cells resembled odontoblasts, the more orderly was the matrix. The less differentiated mcsenchymal cells produced mat.rix with unoriented tubules or amorphous masses without tubules. The various stages of dentinogenesis could usually be seen toget,her in the same pulp. In all but two of the sixteen pulps in which odontoblast-like cells were observed, matrix was also present (Table I). In spite of the differences in time which had elapsed after treatment, there was no marked difference in the proportion of pulps containing odontoblast-like cells and matrix in the five dogs (Table II). TABIX

IT.

TISSUE

TJESTISOCENESIS

CHANGES

Odontoblast-like Matrix formation resent

*Numerators total number

IN DOGS

DOG NO. 1 (3 DAYS)

I

cells represent of pulps.

EILLED THREE, FIVE, OF EXPOSED PULPS”

DOG NO. I

(5

3/g 3/9 number

of

OR SEVEN

DAYS

2

DOGS NO.

showing

the

tissue

TREATMEKT

3, 4, AND

5

(7 DAYS\ 10/27 12/27

DAYS)

3/7 3/7 pulps

AFTER

change;

denominators

rep-

Both small and large burs had been used to expose pulps because it was thought t,hat the number of dentine chips forced into the pulp might be a function of bur size. Actually, some of the pulps found free of dentine chips had been exposed by the larger bur. However, this failure to find dentine chips was more probably due to a fault in the sectioning technique. Perhaps a more important relationship was that between bur size and frequency of occurrence of severe inflammation. which was somewhat less for pulps exposed by the smaller bur. Xezterity of Inflammation as a Factor in Dentinoyenesis.-Where dentine chips had been pushed into severely inflamed pulp tissue, 36 per cent, of the pulps showed signs of matrix formation, whereas 40 per cent of the moderately inflamed and 62.5 per cent of the slightly inflamed pulps contained new matrix (Table III). Dentinogenesis also occurred in some of the control teeth (treated with saline solution). With respect to degree of inflammation, the results were anomalous, but. the highest incidence of matrix formation occurred in the teeth that showed little inflammation. Similar analysis of the data on odontoblast-like cells bears out the conclusion suggested by the data on matrix formation, namely, that severe inflammation inhibim dentinogenesis.

Volume 15

Nulmber

7

ALKALINE

PHOSPHATASE

IN

REPARATLVE

DE?;TlNOGENESZS

865

distribution of predentine in the pulp is uneven, whether this tissue is intercepted or bypassed in sectioning map be a matter of chance. Although phosphatase within the body probably has a function in the laying down of mineralized tissues, its activity may depend upon a delicate chemical balance within the tissues.’ The mechanical application of the enzyme to the exposed pulp may he erraticall>* effecti\Te, drptlnding upon conditions in the tissue not under the control of t,he experimenter.

Fig. 5.-Photomicrograph posed pulp was treated with in striking palisade arrangement pulp. Abundant matrix (1M) and eosin stain. Magnification, SUMMARY

ASD

of buccolingual alkaline phosphatase. are oriented has been secreted, X270 ; reduced

section around tending “lo.)

of a dog’s lower left canine. The exNewly differentiated odontoblasts (Od) large dentine chips (DC) pushed into ( Hematoxylin to weld chips together.

COSCLTJSIONS

Exposed pulps of forty-three teeth in five dogs were treated with physiologic Sections saline solution, albumin, acid phosphatase, or alkaline phosphatase. of the teeth were examined microscopically three, five, or seven days later for evidence of odontoblast-like cells and matrix formation. 1. Matrix was found only in apposition to dentinc; in the pulp it was found only around dentine chips which had brcn forced in accidentally by the bur. 2. Cndifferentiated mesenchpmal cells and cells in various stages of transition between undifferentiated mesenchymal cells and odontoblasts were found oriented around dentine chips in the pulp. Undifferentiated mesenchymal celLs in the pulp conncctiw tissue can become matrix-forming cells. 3. Reparative dentinogenesis ran occur spontaneously within three days of exposure in pulps which hart been exposed, prorid the inflammation is slight. Severe inflammation inhibited dentinogencsis.