62 DISCUSSION

also needs to be clarified if ALP released from remote reactive cells diffused and pooled at certain loci within the local environment.

In conclusion, although the origin and biologic sig- nificance of ALP are still not well understood, the observations on the extracellular ALP activity made in this study may be used as a simple and reliable histochemical process marker for skin wound healing.

zyme histochemical and cytochemical study. Br .I Dermatol 104:521, 1981

4. Smith YR, Klitzman B, Ellis MN, et al: The effect of nicotin- amide on microvascular density and thermal injury in rats. J Surg Res 47:465, 1989

5. Fawcett DW: A Textbook of Histology. New York, NY, Chap- man and Hall, 1994. P 180

6. Lasarov I: Enzyme act&ties of leukocytes in skin cut wounds with various wound ages. Z-Rechtsmed 100:157, 1988

7. Agren MS, SGderberg TA, Reuterving CO, et al: Effect of topi- cal zinc oxide on bacterial growth and inflammation in full- thickness skin wounds in normal and diabetic rats. Eur J Sum 157:97, 1991

References 8. Tengrup I, Hallmans G, Agren MS: Granulation tissue formation and metabolism of zinc and Conner in alloxan-diabetic rats. Stand J Plast Reconstr Surg 22!il, 1988

1. Low MG, Finean JB: Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. Biochem J 167:281, 1977

2. Sognnaes RF: Calcification in Biological Systems. Washington, DC, Am Assoc Adv Sci, 1960, pp 217-243

3. Higgins JC, Eady RAJ: Human dermal microvasculature: En-

9. Tengrup I, Ahonen J, Rank F, et al: Cytochemical study of granulation tissue in zinc treated rats. Acta Chir Stand 146:243, 1980

10. Thies RS, Bauduy M, Ashton BA, et al: Recombinant human bone morphogenetic protein-2 induces osteoblastic differenti- ation in W-20-17 stromal cells. Endocrinology 130:1318, 1992

J Oral Maxillofac Surg 55:62-63, 1997

Discussion

Extracellular Alkaline Phosphatase Activity as a Possible Marker for Wound Healing:

A Preliminary Report

Alfred0 Aguirre, DOS, MS State University of New York at Buffalo, Buffalo, New York

Depending on the extent of cellular death, wound healing occurs either by primary union (healing by first intention), or by secondary union (healing by second intention). The complex but systematic orchestration of the myriad of pro- cesses that govern wound healing have been extensively documented. Remarkable advances have been accomplished in our understanding and elucidation of the basic mecha- nisms associated with wound healing. These include the con- tribution of mediators of acute inflammation and the role of growth factors in such processes as cell migration, cell proliferation and differentiation, angiogenesis, fibrosis, and wound collagenization.’ This knowledge has led to the accu- mulation of a wealth of information at the histological, ultra- structural, biochemical, molecular, and clinical levels. The article under discussion focuses on an histochemical obser- vation, the heretofore unreported time-dependent and local- ization-specific changes of extracellular alkaline phosphatase (ALP) in wound healing of rats.

ALP is a membrane-bound metalloenzyme that is released in physiological or pathological conditions of a wide range of organs. The organs most commonly associated with ALP release in pathological conditions are bone and hepatobiliary disease, but the enzyme may also be expressed in certain malignancies. The association of release of ALP with more generalized healing has not been as clearly established. ALP represents a superfamily of several distinct enzymes, each primarily associated with different organs. Four structural

genes encoding ALP have been cloned, sequenced”’ and mapped to human chromosomes.5-7 Four sources of ALP have been identified; tissue nonspecific, intestinal, placental, and germ cell (placenta-like). The tissue nonspecific ALP gene, expressed in osteoblasts, hepatocytes, lung, kidney, early placenta, endothelial cells, leukocytes, and other cells is located on the short arm of chromosome 1, bands p36.1- ~34. Adult intestinal ALP is encoded by a locus on chromo- some 2, bands q34-q37 and is expressed in the brush border of the mucus membrane. Loci that encode the ALP of the mature placenta and the closely similar (placental-like) germ cell ALP are also located on chromosome 2. The multiple family members originating form single genetic loci (iso- forms) differ in a single amino acid (deletion) or a carbohy- drate (addition or removal) from the parent isoenzyme.8a9

For more than half a century, ALP has received wide attention as a clinical diagnostic tool. Currently, the measure- ment of ALP in serum is amongst the most frequently per- formed tests in medicine (especially during the initial exami- nation of patients complaining of a variety of symptoms). Isoenzyme analysis may be carried out to determine if liver or bone is the source of elevated levels.” It is evident that determination of ALP concentration in serum has a solid position in clinical chemistry. What is not evident today is the role that extracellular ALP may play in wound healing.

Wound repair can be thought of as a culmination of the following three major overlapping events: inflammation, proliferation, and remodeling.” The sequencing and orches- tration of the wound healing process is very closely regu- lated. An exquisite integration of dynamic reciprocity among cells, cytokines, and matrix elements takes place.”

The serendipitous observation of Alpasian and associates concerning the time-dependent and site-specific localization of extracellular ALP during wound healing by secondary intention underlines the sequential characteristics of this pro-

ALFRED0 AQUIRRE 63

cess. The question raised by these authors is a legitimate one; is the presence of extracellular ALP part of the orderly cascade of events that occur during the proliferation and remodeling processes, or just a by-product of cells involved in this intricate phenomenon?

It seems to be that the most logical sources of extracellular ALP in wound healing would be neutrophils, macrophages, and/or endothelial cells. The rich vascular proliferation sug- gests that the endothelial cells might be the principal source of extracellular ALP. However, from this report a close scru- tiny of the tissue sections failed to show the genesis of the copious quantities of ALP. The authors propose to use in situ hybridization to discern if neutrophils, fibroblasts, and/ or endothelial cells are responsible for the abundance of ALP in the granulation tissue. In situ hybridization is a powerful tool to visualize the spatial localization of messenger RNA,13 and would provide valuable information concerning the type of cells involved in the transcription of ALP. However, it would not account for ALP that originated from distant reser- voirs. The investigators are encouraged to consider a chemi- cal approach to examine the isoenzymic status of the ALP pooled in the granulation tissue. Furthermore, the qualitative demonstration of ALP in wound healing should be comple- mented with a quantitative procedure to determine if differ- ent levels of ALP enzymatic activity exist in the various stages of healing. It would also be interesting in the future to assess the extracellular ALP profile in anomalous wound repair (ie, pyogenic granulomas, hypertrophic scars, and ke- loids). By comparing this with normal wound healing, we might achieve greater insight into our understanding of the process of wound healing.

In summary, the current report describes the previously unreported accumulation of extracellular ALP in the granula- tion tissue associated with wound healing in rats. The data presented is clear and convincing regarding the time depen- dent and specific localization of extracellular ALP in granu- lation tissue. I agree with the investigators’ conclusion about the uncertain origin and biological significance of extracellu- lar ALP in wound healing. However, at this time, the prag-

matic value of extracellular ALP as a marker for wound healing remains unclear.

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