University of ConnecticutOpenCommons@UConn

SoDM Masters Theses School of Dental Medicine

June 1984

Identification and Characterization of Wound-Associated Soluble SubstancesElizeu Alvaro Pascon

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Recommended CitationPascon, Elizeu Alvaro, "Identification and Characterization of Wound-Associated Soluble Substances" (1984). SoDM Masters Theses.100.https://opencommons.uconn.edu/sodm_masters/100

IDENTIFICATION AND CHARACTERIZATION OF WOUND-ASSOCIATED

SOLUBLE SUBSTANCES

ELIZEU ALVARO PASCON, D.D.S.

A THESIS

SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF DENTAL SCIENCE

AT

THE UNIVERSITY OF CONNECTICUT

1984

APPROVAL PAGE

MASTER OF DENTAL SCIENCE

IDENTIFICATION AND CHARACTERIZATION OF WOUND-ASSOCIATED

SOLUBLE SUBSTANCES

Presented by:

ELIZEU ALVARO

Major Advisor:

Associ.ate Advisor:

Associate Advisor:

Associate Advisor:

D.D.S.

William E.<DowdenAssistant ProfessorDept 0 Endodonti s

Sevgi lB. RodanAssistant ProfessorDept. of Oral Diagnosis

THE UNIVERSITY OF CONNECTICUT

1984

ii

TO LEILAH, LUMENA, ELIZEU, JR., AND FABIANA

iii

ACKNOWLEDGEMENTS

I wish to convey a special debt of gratitude to

Charles Nicholas Bertolami, Assistant Professor, who not

only provided the guidance for the realization and

materialization of this project, but offered me an

atmosphere of enthusiasm and learning possible only through

a true and real friendship.

I thank the members of my research committee, Dr. Sevgi

B. Rodan and Dr. William E. Dowden for their advice in the

completion of the research project, and Professor Kaare

Langeland for his continuing support.

My sincere appreciation and thankfulness to Professor

Jose Gustavo de Paiva - University of Sao Paulo - for his

consideration and confidence.

I wish to thank all the members of the Department of

Endodontics who gave me encouragement. Special thanks to

Mrs. Maryann McCarthy for her untiring efforts in typing

this thesis.

iv

TABLE OF CONTENTS

INTRODUCTION 1-2

LITERATURE REVIEW 3-9

Background 3-4

Reasons for studying buffer-soluble glycoproteinsfrom open-wound granulation tissues 5-6

Soluble extracellular granulation tissueconstituents as regulatory molecules 7-9

MATERIALS AND METHODS 10-17

Wounding 10

Sampl ing 10-11

Grafting 11-12

Tissue preparation, storage, and extraction 12-13

Analytical Techniques 14

Gel filtration 14

Electrophoresis 14-15

Isoelectric focusing (IEF) 15

Amino acid analysis 16

Collagen detenninations 16-17

Effects of extraction period 17

Inhibition of non-specific protease activity 17

Measurement of NANA, and protein 18

Statistical methods 18

v

RESULTS 1·9-22

Gel filtration 19

SOS-PAGE 19-20

Protein and N-acethylneuraminic acid (NANA)determinations 20

Collagen determinations 21

Total collagen content 21

Collagenase treatment of extract supernatants 21-22

Effects of extraction period and non-specificproteases 22

Isoelectric focusing and amino acid composition 22

DISCUSSION •.•••...•.•...••...••••......•••....•••...••.•••• 23-28

CONCLUSIONS ••••••..••........•••....••..•..•.••....•...•••• 29

LITERATURE CITED ...••....•...•......•.......•..•.....•..••. 30-36

FIGURE TEXT AND FIGURES 37-44

TABLE I 45

TABLE II 46

vi

The purpose of

INTRODUCTION

this study was to identify,

characterize, and partially purify certain highly soluble

glycoproteins from experimental wounds in rabbits. An

attempt was made to determine whether the changing

concentrations of such substances could be used to

objectively monitor the repair process.

Soluble glycoproteins derived from open wound

granulation tissues have not been studied previously; but,

comparable substances have been identified in extracts from

polyvinyl sponge-induced inflammatory granulomas. 1- 5 In

granulomas, these substances experience predictable changes

in concentration as the tissue ages.

In the present work, soluble glycoproteins were

extracted from open wound granulation tissues at increasing

postwound intervals and were monitored and characterized by

methods used previously for the study of granuloma-derived

granulation tissues. The major purification technique

involved gel filtration using Sephacryl SF 200. Crude and

purified preparations were characterized by sodium dodecyl

sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE),

isoelectric focusing (IEF), amino acid analysis,

glycoprotein susceptibility to a monospecific clostridial

collagenase, and measurement of soluble collagen, N-

acetylneuraminic acid (NANA), and protein. The use of these

techniques allowed the investigator to: 1) establish if

wound-derived soluble substances could be used to monitor

repair over a given interval, and 2) determine if open-wound

granulation tissue profiles resemble those from other tissue

sources (i.e., inflammatory granulomas). If soluble

glycoprotein profiles could be confirmed as having

characteristic configurations for wounds of specified age,

they would have value as objective reflections of healing

status.

2

LITERATURE REVIEW

Background

Normal healing of open full-thickness wounds of

mammalian integument occurs by the process of repair rather

than by true regeneration. 6,7 As a result, the tissues of

healed open wounds are neither structurally nor functionally

identical to those of the pre-injury state. The inevitable

scarring associated with normal healing can produce severe

impairments. 6,7 Efforts at directing repair so as to limit

scarring and contraction or to facilitate re-

epithelialization have been hampered by lack of objective

methods for assessing the effects of new agents or

techniques on healing processes. This deficiency is made

more apparent when healing occurs abnormally; aberrations in

wound repair are relatively common. 6,8 Keloids and

hypertrophic scars, for example, are debilitating

derangements to which open wounds

susceptible. 6,8 In addition, failure

are particularly

of wounds to re-

establish surface integrity and functional actiVity due to

infection, nutritional deficit, systemic disease, or drug

therapy has been well-documented. 6 By the time such

abnormalities become clinically eVident, effective

therapeutic intervention may be precluded. If wounds that

are at risk of healing abnormally could be identified during

incipient aberrancy, and if early treatment could be

3

initiated, a return to normal patterns of repair might be

accomplished. Objective criteria for monitoring wound

repair would also allow the effects of various wound

treatments to be more appropriately evaluated.

4

Reasons fQL Studying Buffer-Solubl~ Glycoprote1n~ fLQmOpen-W2Ynd Granulation Tissues

Numerous methods have been suggested for enhancing the

rate or quality of wound repair 9- 12 , but few methods have

found application in clinical practice. This disparity may

result from using sUbjective clinical criteria for

monitoring the healing process and from inevitable

investigator bias. Attempts have been made at more

objectively quantitating healing in experimental animals by

measuring rates of re-ePithelization 13 , wound

contraction,14-16 connective tissue reconstitution,16-17

collagen synthesis, 18-20 glycosaminoglycan (GAG)

production,21-23 and determination of fibroblast growth

kinetics;24 but, because species variation is significant,25

the relevance of such research to human open-wound repair

remains unclear. Certainly, methods of analysis which

require surgical removal of wound tissues are rarely

suitable for studying healing in humans. This investigation

is an experimental animal study which evaluates the

contention that highly soluble substances are present in

open wound tissues and can be used to accurately monitor the

healing process.

The existence of wound-derived soluble glycoproteins

whose concentrations change as a function of postwound

interval has never been established; but, similar substances

5

have been reported for granulation tissues of non-wound

origin (i.e., inflammatory granulomas).1-5 The close

resemblance between granulation tissue derived from

inflammatory granulomas and that from open, full thickness

skin wounds,26 justifies the speculation that a similar

assortment of glycoproteins would be identified.

Buffer-soluble granuloma glycoproteins were originally

studied to assess changes in cellular function mediated by

exposure of cells to extracellular matrix constituents. 1

When rat granuloma tissue extracts were added to cultures of

matrix-free embryonic chick tendon cells, a dramatic

suppression of collagen and other protein synthesis

occurred. 1,2 In addition, the secretion of collagen into the

medium was diminished and the normal tendency of tendon

cells to cluster during incubation was inhibited. 1,2

Another biological effect was a 10-fold drop in phagocytic

index when peritoneal macrophages were exposed in vitro to

extracts prepared from 14- and 42-day polyvinyl sponge

granulomas. 3 Subsequently, efforts at isolating and

identifying the active components of such extracts revealed

that granuloma age was

specific gel filtration

accurately

2patterns.

reflected by highly

The characteristic

changes in elution profiles observed as granuloma tissues

matured led to speculation that soluble glycoproteins play

an active role in directing tissue development. 2

6

Soluble Extracellular Granulation Tissu~ ~nstituents

~ Regulatory Molecules

Feedback regulation of reparative tissue development

has been identified as an important function for the soluble

glycoproteins of inflammatory granulomas. 2 ,27 After crude

extracts of granuloma granulation tissues were shown to

inhibit collagen synthesis and secretion,1,2 subfractions

were isolated which exhibited inhibition at extremely low

concentrations Among the many soluble

extracellular substances that might function in this

capacity, two categories of molecules, NANA-containing

glycoproteins and glycosaminoglycans (GAG), have received

particular attention.

NANA was originally implicated as a regulator of cell

function when normal and transformed cells were shown to

have different total sialoprotein contents. 28 Changes in

the synthesis/secretion of collagen and other proteins as

well as alterations in cell surface attachment mechanisms,

cellular aggregation phenomena, lymphocytic circulation,

expression of cellular antigenicity, and sensitivity of

smooth muscle cells to serotonin-induced contraction have

all been envisaged as being NANA-dependent. 29 - 33 A

neuraminidase-sensitive glycoprotein involved in collagen

synthesis and in phagocytosis has also been described. 34

Such research has been used to support the conclusion that

NANA-containing glycoproteins exert potentially important

7

influences on the changing developmental state of

granulation tissues. 2

GAG have also been envisaged as being important for

modulating cellular behavior in deve1opmenta1 35- 42 and in

21-23wound healing systems. In developmental models such as

newt limb regeneration 35, chick embryo cornea formation,36

limb bud and axial chrondogenesis,37 and chick heart and

brain deve1opment,38,40-42 the unsu1fated GAG, hyaluronic

acid, has been found to facilitate cellular division and

SUbsequent hyaluronate removal by

migration while

differentiation. 35 ,36,39

directly suppressing

tissue hyaluronidase causes a cessation of migration and

permits aggregation and differentiation to proceed in the

proper sequence for tissue organization. 35 ,42 In

healing open wounds, identical changes in GAG composition

occur. 21 - 23 In addition, a wound associated hyaluronidase

differentiation

whose activity correlates with the onset of

has been identified. 21 ,22 These

cellular

studies

support an analogy between reparative and developmental

systems and lend credence to the hypothesis that the

synthesis and degradation of hyaluronic acid help to control

and

explain

differentiation,proliferation,cellular migration,

synthesis. 35 - 42 Possible mechanisms to

hyaluronate's effects have been advanced by Ivaska. 5

Predicted changes in the concentrations of readily

extractable glycoproteins of developing granulation tissues

8

were used in this study to monitor the progression of normal

repair in experimental wounds. Since soluble glycoproteins

from open-wound tissues have not been studied previously, an

analogy between the open wound -and the granuloma systems

needed to be verified. This study sought to confirm

that soluble glycoproteins could be isolated from open wound

granulation tissues and that time-dependent changes in

soluble glycoprotein composition would occur.

9

MATERIALS AND METHODS

Woynding

Forty 2.0-3.0 kg New Zealand white rabbits (Oryctolagus

cuniculus) were caged separately and maintained on a Purina

laboratory Rabbit Chow HF 5326 diet (Ralston Purina) with

food and water taken ~ liQ. For wounding and harvesting

procedures, rabbits were anesthetized by intramuscular

injection of ketamine HCI (Bristol), 35 mg/kg and xylazine

(Haver-Lockhart), 5 mg/kg and flanks were shaved and

depilated with Nair (Carter-Wallace Inc.) Bilateral flank

wounds, 6 em x 6 em were made in all animals (Fig. 1). Skin

and superficial fascia were excised to the level of the

panniculus carnosus muscle with resected skin being frozen

and retained. Flank dressings consisted of petrolatum

gauze, gauze roll, and a light plaster cast.

Sampling

A differential wound topography permits recognition of

two distinct zones. 20- 22 Most of the open defect contains a

prOVisional tissue referred to as "central granulation

tissue". Along the wound's periphery, between central

granulation tissue and the original unwounded skin, a bi­

partite zone exists which is composed of a deep layer of

granulation tissue and a superficial sheet of migrating

10

epithelium. This bi-layered region constitutes "whole edge

tissue". All samples were harvested from the central

granulation tissue region and were free of peripheral

epithelium. Sampling was performed on postwound days 3, 7,

9, and 14. For each animal, the tissues from the left and

the right wounds were pooled and extractions subsequently

performed. Excision of underlying panniculus carnosus muscle

was avoided at the time of harvesting.

Grafting

Skin grafting 1s a common therapeutic maneuver believed

to produce a significant alteration in patterns of wound

repair. Two animals were, therefore, studied for which any

changes in soluble glycoprotein profiles induced by the

application of full-thickness autogenous skin grafts could

be defined. These animals received a unilateral 6 em x 6 em

flank wound that was dressed as described above. On the

seventh day after wounding, the opposite side was wounded

and the freshly harvested skin applied as a full-thickness

graft to the week-old wound. The deep surface of the graft

was defatted to expose clean dermis, this procedure greatly

enhances the success of grafting. 43 Abdominal skin was

avoided as a source of graft material because resultant

closure of such donor sites creates undesirable tensions on

the flanks and might retard the natural course of

contraction. Petrolatum gauze, gauze roll, and plaster cast

1 1

were again used as dressings. On the fourteenth day after

original wounding (7 days after grafting), grafts were

removed and sUbjacent granulation tissue harvested; results

of previous work show that translocation of granulation

tissue to grafts is minimal. 43 The donor site was allowed

to heal for a total of 14 days and then its granulation

tissue harvested as described for open wounds.

Tissue Preparation, Storage, anQ Extraction

Harvested tissues were frozen in liquid nitrogen,

lyophilized, milled and then stored at 40 C until required.

Extl~acts were prepared by dissolVing 100 mg of milled tissue

powder in 4.0 ml of 0.05 M Tris (hydroxylmethyl)

aminomethane-HCl (Tris-HC1), 0.005 M CaC1 2 , pH 7.6, buffer

and gently stirred at 4° C for 16 hrs. as previously

described. 1,2 Samples were then centrifuged at 14,700 X g

for 5 min., insoluble pellets discarded, and the

supernatants saved for analysis. An identical extraction

was used for normal skin. Normal rabbit serum was analyzed

after dialysis against the extraction buffer. Data for

norma] skin and serum were compared with those obtained for

granulation tissues.

All samples were studied by means of gel filtration,

sodium dodecyl sulfate (SDS)-polyacrylamide

12

gel

electrophoresis (PAGE), isoelectric focusing, amino

analysis (for fractions of sufficient purity),

measurement of soluble collagen, hydroxyproline,

acetylneuraminic acid (NANA), and protein.

13

acid

and

N-

Analytical Technigues

~ Filtration

Gel filtration was performed by introducing a 500 ul

aliquot of each crude extract to a 1.6 cm x 90 cm column of

Sephacryl SF 200 (Sigma) which had been equilibrated with

the extraction buffer. Elution was performed with the same

buffer at a flow rate of 0.66 ml/min. Effluent was

monitored at 280 nm with a Buchler Fracto-Scan UV monitor;

3.2 ml fractions were collected. Peak fractions were

dialyzed against cold distilled water, lyophilized, and

stored for later analysis.

Elution profiles were described on the basis of peak

elution volumes. Changes in the profiles were expected to

reflect the changing developmental state of the granulation

tissue.

Electrophoresis

Crude extract supernatants as well as peak fractions

obtained by gel filtration were electrophoresed on 1~ SDS­

-5.6% polyacrylamide gels as described by Fairbanks, Steck

et. al. 44 Staining for protein was performed with 0.05%

coomassie blue and for carbohydrate with a periodic acid

Shiff (PAS) procedure. 44 All gels were photographed and

then scanned with a Corning model 760 computing

densitometer. Protein standards consisting of lysozyme,

14

a-lactoglobulin, ferritin, ovalbumin, bovine serum albumin,

and a-galactosidase were used. Electrophoretic patterns

allowed molecular weights, approximate carbohydrate

contents, and relative purity to be determined.

Densitometric scans of gels were compared for protein

migration distances and for differences in densitometer­

computed band areas.

~lectrlc fQQ.y.UnK (lEF)

Isoelectric focusing was performed as described by

Righetti and Drysdale45 using a 40% ampholin~ solution (Bio

Lyte 3/10) having a gradient of pH 4-9. Staining was

performed with coomassie blue and all gels were then

scanned. Using pI calibration standards (Pharmacia),

isoelectric points over the range pH 4-9 were determined

for purified extract subfractions. Since previous

worl{ has established the isolectric points and molecular

weights of soluble glycoproteins of inflammatory

granulomas,3 such data provided another ~asis for comparing

extracts from open wound granulation tissues and from

granulomas. Changes in isoelectric patterns occurring as a

function of postwound interval would, concej.vably, be useful

for monitoring the emergence and/or disappeal~ance of various

proteins as wound gr~nulation tissue matures and remodels.

15

Amino ~d Analysis

Those fractions that could be purified sufficiently so

as to yield a single band by SDS-PAGE were sUbjected to

amino acid analysis. Analyses were performed after

hydrolysis at 1100 C in 6 N Hel for 24 hrs. in evacuated

nitrogen-flushed tubes by the University of Connecticut

Health Center Amino Acid Analysis Facility.

Amino acid compositions of wound-derived soluble

glycoproteins were used in conjunction with SDS-PAGE and IEF

in an attempt at protein identification. Comparisons were

made between experimental substances and glycoproteins of

known composition.

Collagen Determinations

SolUble, collagen-derived peptides present in crude

extract supernatants or in isolated subfractions were

identified by evaluating extract protein susceptibility to

collagenase. Hydroxyproline content was also measured.

Samples were treated with a purified monospecific

clostridial collagenase (EC 3.4.24.3) (Millipore) and

resulting alterations in 5DS-PAGE patterns ide~tified.

Samples were divided into fractions of 250 ul; each received

162.6 ~g of N-ethylmaleimide (NEM) to inhibit nonspecific

protease activity. Half of the samples were then treated

• i- ...W1. v.1 23.2 ug of active collagenase and the remainder with

the same amount of heat-inactivated collagenase. All

16

r ti i t incubated at 37° Ceac on m x ures were for 2 hrs.

Collagenase-mediated changes in electrophoretic patterns

were monitored using the SDS-PAGE technique for collagenous

polypeptides of Noelken, Wisdom et. al. 46

Hydroxyproline levels were determined for all samples

by the alkaline hydrolysis of Huszar, Maiocca et.al. 47

~fects Qf Extraction Period

To assess the effects of varying the duration of wound

tissue extraction periods on gel filtration elution profiles

and on gel electrophoretic patterns, extract supernatants

derived from wound granulation tissue aged 3, 7, 9, and 14

days ~ere incubated in the Tris-HCl extraction buffer at 40

C for 4, 8, 12, and 16 hours respectively. Any differences

arising in the profile or electrophoretic pattern for a

given tissue reflected the influence of extraction duration.

Inhibition Qf Non-Specific Protease Activity

For each of the extract supernatants derived from the

wound tissues described above, N-ethylmaleimide (NEM) was

added to a final concentration of 2.5 mM for the purpose of

inhibiting non-specific protease activity. Resultant

elution profiles and electrophoretic patterns were then

studied and compared with NEM-untreated extracts.

17

Measurement Qf NANA, and Protein

NANA levels were estimated by the method of Aminoff. 48

Samples and standards contained in a volume of 0.5 ml were

incubated with 250 ul periodate reagent at 370 C for 30 min.

Sod i.urn arsen i te (200 ul) and th10barb i tur 1c ac id reagen t (2

ml) were added and samples and standards heated at 1000

C

for 7.5 minutes. Acid-butanol (4 ml) was added, mixtures

centrifuged, and NANA-chromophore read in the butanol layer

at 549 nm.

Lowry protein was measured using the modified folin­

phenol reagent method reported by Peterson. 49

Statistical ~~~

Data for assays of tissue samples pooled according to

postwound interval or graft status were expressed as means +

one standard error of the mean (S.E.M.). Statistical

significance was determined by applying t-tests for

independent variables to the difference between means.

S i g n i f i canc e was ass i g ned at alevel 0 f p rob abiIi t y ~ 0 • 0 5 •

For gel filtration, PAGE, arld IEF experiments,

re9resentative elution profiles and gels are presented.

18

RESULTS

~ Filtration

A time-dependent change in the elution profile of

supernatants derived from rabbit skin granulation tissue was

observed (Fig. 2). Two major peaks (A and C) were seen for

each day studied, but the relative proportion of the profile

attributable to a given peak varied. Peak A was the most

prominent constituent of days 3, 9, and 14, and possessed an

approximate molecular weight of 93,000 (Fig. 3). This peak

was markedly diminished on postwound day 7, but increased

progressively throughout the second postwound ~eek. A minor

peak (E) was observed in less mature granulation tissue

(postwound days 7 and 9) and was particularly evident in

grafted granulation tissue (Figs. 2 and 4). Peak C had an

elution volume consistent with a population of substances of

differ'ing molecular weights. This possibility is suggested

by the eventual emergence of 3 discrete peaks 1n the C

position at 14 days.

SDS--PAGE

SDS-PAGE patterns of tissue extracts exhibited a

surprising degree of similarity ,·egardless of postwcund day

(Fig. 5). At least five comparable bands were identified

fo~ each extract, however, densitometric scanning

demonstrated important differences (Fig. 6). Although

19

band 3 was invariably the major component, its relative

predominence was markedly diminished on day 7 when it

comprised only 27~ of the scan (Fig. 7). Subsequently, the

the percentage of the scan attributable to band 3

progressively inoreased, reaching a maximum on day 14 of

59.1% (Fig. 7). When Sephacryl SF 200 Peak A was isolated

and electrophoresed, a single homogenous band was identified

which corresponded to band 3 of the crude extracts (Fig. 5).

The other SDS-PAGE bands (1, 2, 4, and 5) constituted

relatively low percentages of the scan on each postwound

day. Band 5 peaked on postwound day 7 when it comprised

15.4% of the scan. Bands 1, 2, and 4 were essentially

unaffected by increasing postwound intervals (Fig. 7). When

gels were stained for carbohydrate, all the coomassie­

staining bands were PAS positive.

~tein an~ ~etylneuraminic~ (NANA) DeterminatiQn~

The described changes in elution profiles and 5DS-PAGE

patterns were accompanied by increasing concentrations of

extractable protein throughout the healing process (Table

I). NANA levels were uniformly low on all postwound days

except day 7 when the concentration of extractable NANA was

4- to 6.5-fold greater than at any ether postwound interval

(Table I). Isolated Peak A material was found not to

possess significant amounts of NANA.

20

Collagen Determinations

Iotal Collagen Content: An estimate of total collagen

content was made for uncentrifuged crude extracts by

measuring the release of collagen cleavage products after

treatment with purified, monospecific clostridial

collagenase. Release of cleavage products increased in

direct proportion to collagenase concentration until a

plateau was reached at 8.9 ug/mg dry tissue (Figure 8).

Collagen measured in this manner was found to be negligible

at postwound day 3, but increased thereafter, peaking on

day 9 and remaining elevated until day 14 (Fig. 8).

Although grafting appeared to diminish the collagen content

of subjacent granulation tissue, the difference between

grafted and ungrafted tissue samples was found to be

statistically insignificant (p ~ .05).

~lagenase Treatment Qf Extract ~~a~s; No

differences in electrophoretic patterns were observed

between samples treated with active or heat-inactivated

clostridial collagenase. Similarly, SDS-PAGE of samples

incubated with N-ethylmaleimide (NEM) to inhibit non­

specific protease activity were indistinguishable from those

that were NEM-untreated.

Hydroxyproline determinations of extra~t supernatants

were also performed. For the periods studied, variably low

levels of hydroxyproline were e~tracted (Table I) that did

21

not reflect total wound collagen content (Fig. 8).

Effects Qf Extraction Period ~ Non-Specific Proteases

Under the conditions of the experiments (4 0 C), neither

varying the extraction time over a range of 4-16 hours nor

inclusion of NEM in extraction mixtures produced any

detectable change in gel filtration profiles or

electrophoretic patterns.

IsoelectrtQ Focusing an~ AminQ ~id ComgQsition: Only

Peak A (Band 3) could be isolated with sufficient purity to

justify IEF and amino acid composition determination. The

isoelectric point was found to be 6.9; the amino acid

composition is shown in Table II.

22

DISCUSSION

The elution profiles of open wound granulation tissues

changed as a function of postwound interval. The pattern of

change was dramatic for Sephacryl SF 200 Peak A. Peak A was

present in high concentrations on postwound day 3, was

markedly suppressed on postwound day 7, and then

progressively increased during the second postwound week

(days 9 and 14).

When isolated Peak A glycoprotein was studied by gel

electrophoresis, it was identified as a single, homogeneous

protein corresponding to the third band in elect~ophoretic

patterns of crude extract (Fig. 5, compare gels 1-13 with

gel 14). By performing densitometry on each gel, the

proportion of the pattern attributable to Band 3 could be

determined at increasing postwound intervals (Figs. 6 and

7). By this method, Band 3 was shown to vary over time in a

manner identical to that seen for Peak A by gel filtration,

I.e., initially high levels of Peak A on day 3 were

depressed on day 7 and progressively increased on days 9 and

14 (compare changes in Peak A, Fig. 2 with changes in band

3, Fig. 7).

The cause of Peak A/Band 3 (PA/B3) depression at the

end of the first postwound week is unknown, but the

observation that NANA was increased on day 7 may be

significant. NANA-containing glycoproteins are believed to

23

29­exert numerous important effects upon cellular function.

33 They undergo marked changes in concentration as tissues

2 50age,' and they may function in the feedback regulation of

granulation tissue development. 2 Purified NANA-containing

glycoproteins have been shown to suppress protein and

collagen synthesis/secretion at very low concentrations

(10-6 M).2 Since Bands 1,2,4, and 5 were not diminished on

day 7, the nature of PA/B3 depression may be rather

specific, whatever its ultimate cause. Importantly, these

studies assessed protein content, not rate of synthesis, so

subtle changes in protein synthesis and/or secretion might

exist on other postwound days that would not be recognized

by tile methods employed.

Impressive changes in Peak C were also observed; Peak C

gradually increased in quantity and eventually separated

into three well-defined peaks (Fig. 2). This phenomenon may

indicate a time-dependent structural modification such as

a·gg,re"ga·tionj the concorni tant formation of small er, Peak C-

de~ived remnants may also occur. The inability to

distinguish differences in Peak C patterns by 5DS-PAGE over

time might result from reduction of disulfide bonds by

dithiothreitol and disruption of Peak C into its essential,

non-agg~egated components. In any event, the emergence of

sev~ral discrete peaks in the C position of 14-day elution

profiles implies possible value for Peak C as a monitor

substance.

24

The identity of PA/B3, Peak C and t~e other soluble

glycoproteins remains unknown. Since electrophoresis

patterns were not altered when extracts were treated with

collagenase, none of the major bands appear to be collagen­

derived peptides. The absence of hydroxyproline from PA/B3

and the entirely different pattern of time-dependent change

observed for PA/B3 when compared to total wound collagen

argue against collagen as a major soluble constituent.

Soluble glycoproteins from open-wound granulation

tissues have not been studied previously, but similar

substances of non-wound granulation tissue origin have been

reported. 1-3 These earlier works may provide insight

into the nature of the materials described by this study.

Bole, Jourdain et. al. 3 isolated a saline soluble

glycoprotein from 14- and 42-day polyvinyl sponge granulomas

in rats by chromatographic fractionation of extracts on

DEAE-cellulose and Bio Gel P-150. A protein peak identified

as DEAE-celluJose C/Bio Gel P-150 II exhibited

chromatographic and electrophoretic characteristics similar

to PA/B3, but was much smaller (MW=48,500). The material

isolated by Bole, Jourdain et. al. 3 significantly depressed

phago~yto3is by peritoneal macrophages in culture.

Similarly, Aalto and Kulonen 1 described a 0.3 M Tris-HCl,

0.0015 M CaCl, pH 7.8, extract from viscose sponge-induced

inflammatory granuloma tissue that suppressed the synthesis

25

of protein and collagen by embryonal chick tendon cells.

Subsequently, a 0.5 M Tris-Hel, 0.02 M EDTA, 0.075 M boric

acid extract of rat granuloma tissue was identified that had

the same effect and that possessed a gel filtration pattern

which accurately reflected granuloma age. 2 When Aa1to,

Poti1a et. a1. 2 subjected these 7- and 21-day granuloma

extracts to Sephadex G-200 gel chromatography, they

identified three peaks (designated Sephadex G 200/1, II, and

III) that appear identical to Sephacryl SF 200 Peaks A, B,

and C obtained by the present analysis (Fig. 2). Sephadex G

200/1 and PA/B3 occupy similar chromatographic elution

positions and both increased with advancing age. 2

Isolation of

cellulose anion

Sephadex

exchange

G 200/1 followed by

chromatography2 produced

DEAE-

t~o

protein peaks which eluted at 0.15 M NaCl, a major ~rotein

subfraction (DEAE 1) and a lesser protein subfraction (DEAE

2)~ Although high levels of NANA were associated with

Sephadex G 200/1, NANA was confined exclusively to the

lesser subfraction (DEAE 2). The major subfraction (DEAE 1)

was totally free of NANA and appears to be comparable to

Sin~e the specific identification of the soluble

glycoproteins de~ived from open wound granulation tissue or

from rat granulation tissue has not been accomplished, the

possibility of a serum origin for such substances must be

26

considered. The probability of simple serum contamination

of tissue samples seems small since such contamination would

not explain the changing proportions of soluble constituents

observed as the wounds matured. In addition, the

predominant serum protein, albumin, is free of carbohydrate

and possesses a sUbstantially lower molecular weight

(63,900) than the predominant PAS-positive glycoprotein

identified in wound extracts (PA/B3 MW=93,000). Naturally,

PA/B3 could represent a higher molecular weight serum

component such as a or a macroglobulins or

immunoglobulins, or even albumin functioning as a car-ri.er

and bound to some other glycoprotein. Certainly, the amino

acid composition and isoelectric point of PA/B3 is rather

a1 bumino id • In any even t, a serum 0 r ig i 11 for this

substance would in no way invalidate its use in monitoring

the progression of normal repair given a consistent

relationship between the healing process and PA/B3

concentration. The selective uptake of serum components by

maturing wound tissues could provide a basis for the

observed effect.

The major change provoked in gel filtration patterns by

the application of skin grafts appears to be an accentuation

of Peak B. This phenomenon might be t~e result of a

retention of less mature tissue characteristics since Peak B

was more prominent in tissues harvested from younger wounds

(Days 7 and 9). In any event, the limited graft study of

27

this project supports the contention that a method known to

dramatically alter the normal healing process (i.e.,

grafting) also produces distinct changes in glycoprotein

profiles.

28

CONCLUSIONS

1) The profiles of soluble substances identified in

this study varied in a characteristic manner as a function

of postwound interval. Such profiles have apparent value as

objective reflections of healing status.

2) One soluble substance, Peak A/Band 3 (PA/B3) was

identified a~ a single, non-collagenous PAS-positive

protein, having a molecular weight of approximately 93,000

and an isoelectric point of 6.9.

3) Cullagen was not a significant component of

soluble extracts.

4) Increased levels of N-acetylneuraminic acid on day

7 may be related to an observed suppression of PA/B3.

5) Peak C substances also varied over time and may

have value as monitor substances.

6)

procedure

provoked

profiles~

Full thickness, autogenous skin grafting (a

known to dramatically alter wound healing)

distinct changes in soluble substance elution

29

LITERATURE CITED

E.: Inhibition of protein

cells by extracts from

tisue. F.E.B.S. Lett.,

1 • Aalto, M., and Kulonen,

synthesis in tendon

experimental ganulation

49:70, 1974.

2. AaIta, M., Potila, M., and Kulonen, E.: Glycoproteins

from experimental granulation tissue and their

effects on collagen synthesis in embryonic chick

tendon cells. Biochem. Biophys. Acta, 587:606,

1979.

3. Bole, G.G., Jourdain, G.W., and Wright, J.E.:

Isolation and chemical characterization of a

granuloma glycoprotein that inhibits macrophage

phagocytosis. J. Lab. Clin. Med., 86:1018, 1975.

4. Rajamaki, A., and Kulonen, E.: Purification of a

structural glycoprotein from collagenase-digested

experimental granuloma. Biochem. Biophys. Acta,

243:398, 1971.

5. Ivaska, K.: Effect of extracellular glycosaminoglycans

on the synthesis of collagen and proteoglycans by

granulation tissue cells. Acta Physiol. Scand.

Suppl., 494, 1981.

6. Peacock~ E.E., and Van Winkle, w.: HQyn~ ~~~ 2nd

Ed., W.B. Saunders Co., Philadelphia, 1976.

30

7. Bertolami, C.N.: Oral Wound Repair. In: Clinical

Dentistry, J.W. Clark (ed.), Harper and Rowe

PUblishers, Philadelphia, 1982.

8. Ketchum, L. D. , Cohen, I.K., and Master, F.W.~

Hypertrophic scars and kelo1ds.

Surg., 53: 140, 1974.

Plast. Reconstr.

9. FaUlk, W.P., Stevens, P.J., Burgess, H., Matthews, R.,

Bennett, J.P., and Hsi, B.L.: Human amnion as an

adjunct in wound healing.

1980.

Lancet, 8179:1156,

10. Calver, R.F., and Stanley, J.K.: Enhancement of

secondary wound healing by local tissue nutrition.

Clin. Trials J. (Lond), 17: 144, 1980.

11. Shafer, W.G., Beatty, R.E., and Davis, w.B.: Effect of

dilantin sodium on tensile strength of healing

wounds. Proe. Soc. Exp. BioI. Med., 98:348, 1958.

12. Ringsdorf, W.M., and Cheraskin, E •.. Vitamin C and

wound healing. Oral Surg •., 53:231, 1982.

13. Krawczyk, W.S.: A pattern of epidermal cell migration

during wound healing.

1971.

J. Cell BioI., 49:247,

14. Abercrombie, M.. , Flint, M.H., and James, D.W.:

Collagen formation and wound contraction during

repair of small excised wounds in the skin of

rats. J. Embryol. Exp. Morph., 2:264, 1954.

31

15. Abercrombie, M., James, D.W., and Newcombe, j.F.:

Wound contraction in rabbit skin studied by

splinting the wound margins. J. Anat. (Lond.),

94: 170, 1970.

16. Grillo, H.e., Watts, G.T., and Gross, J.: Studies in

wound healing. I. Contraction and the wound

contents. Ann. Surg., 148: 145, 1958.

17. Grillo, H.C.: Aspects of the origin, synthesis, and

evolution of fibrous tissue in repair. In:

Adyances in Biology Qf ~ Skin. Pergamon Press,

London, 5: 128, 1964.

18. Diegelman, R.F., Rothkopf, L.C., and Cohen, I.K.:

Measurement of collagen biosynthesis during wound

healing. J. Surge Res., 19:239, 1975.

19. Stein, H.D., and Keiser, H.R~: Collagen metabolism in

granulating wounds. J. Surg. Res., 1 '1 : 22'7, 1971.

20. Bertolami, C.N., and Donoff, R.B.: The effect of skin

grafting upon prolyl hydroxylase and hyaluronidase

activities in mammalian wound repair. J. Surge

Res., 27:359, 1979.

21. Bertolami, C.N., and Donoff, R.B.: Iiyaluronidase

activity during open wound healing in rabbits: A

preliminary report. J. Surge Res., 25:256, 1978.

22. Bertolami, C.N., and Donoff, R.B.: Identification,

characterization, and partial purification of

mammalian skin wound hyaluronidase. J. Invest.

32

Derm., 79:417, 1982.

23. Alexander, S.A., and Donoff, R.B.: The

Proc.

glycosaminoglycans of open wounds. J. Surge Res.,

29:422, 1980.

24. Diegelmann, R.F., Cohen, I.K., and McCoy, B.J.: Growth

kinetics and collagen synthesis in normal skin,

normal scar, and keloid fibroblasts in yitrQ. J~

Cell Physiol., 98:341, 1979.

25. Grillo, H.C., and Gross, J.: Studies in wound healing.

III~ Contraction in vitamin C deficiency.

Soc. Exp. BioI. Med., 101:268, 1959.

26. Gabbiani, G., Hirschel, B.J., Ryan: G.B., Statkov,

P.R., and Majno, G.: Granulation tissue as a

contractile organ: A study of structure and

function. J. Exp. Med., 135:719, 1972.

27. Aho, S., and Kulonen, E.: Effect of silica-liberated

macrophage factors on protein synthesis in cell­

free systems. Exp. Cell Res., 104:31,197'7.

28. Ohta, N., Pardee, A. B. , McAuslan, B.R., and Burger,

M. M. : Si81ic acid contents and controls of normal

and malignant cells. Biochem. Bioph,y.s. Acta,

158:98, 1968.

29. Ginsburg, V• , and Neufeld, E: • F • : Complex

heterosaccharides of animals. Ann. Rev. Biochem.,

38:371, 1969.

33

30. Winzler, R.J.: Carbohydrates in cell surfaces. Int.

Rev. Cytol., 29:77, 1970.

31. Spiro, R. G. : Glycoproteins. Ann~ Rev. Biochem.,

39:599, 1970.

32. Marshall, R.D.:

41 :673, 1972.

Glycoproteins. Ann. Rev. Biochem.,

33. Rosenberg, A., and Shengrund, e.L. (eds.): Biological

Roles of Sialic Acid, p. 375, Plenum Press, New

York, 1976.

34. MII. , Ronnemaa, rr

.L • , and Kulonen, t:' •1:1 •• Effect of

neuraminidase of the synthesis of collagen and

other proteins.

1974.

Biochem. Biophys. Acta, 342:247,

35 • Toole, B • P • , and Gr 0 s s, J.: The ext rae e11u1arma t r~ i x

of the regenerating newt limb: Synthesis and

removal of hyaluronate prior to differentiation.

Dev. BioI., 25: 57, 1971.

36. Toole, B.P", and Trelstad, Hyaluronate

production and removal during cornea development

in the chick. Dev. BioI., 26:28, 1971~

37. Toole, B.P.: Hyaluronate turnover during

chondrogenesis in the developing chick limb and

axial skeleton. Dev. BioI., 29:321, 1972.

38. Orkin, R.W~, and Toole, B.P.: Hyaluronidase activity

and hyaluronate content of the developing chick

embryo heart. Dev. BioI., 66:308, 1978.

34

39. Toole, B.P., Jackson, G. t and Gross, J.: Hyaluronate

morphogenesis. Inhibition of chondrogenesis in

Vitro. Prac. Nat. Acad. Sci., 69:1384, 1972.

40. Orkin, R.W., Jackson, G., and Toole, B.P.:

Hyaluronidase activity in cultured chick embryo

skin fibroblasts. Biochem. Blophys. Res. Comm.,

77:132, 1977.

41. Polansky, J.R., Toole, B.P., and Gross, J.: Brain

hyaluronidase: Changes in activity during chick

development. Science, 183, 862, 1974.

42. Polansky, J.R., and Toole, B.P.: Hyaluronidase

activity during thyroxine-induced tadpole

metamorphosis. Dev. Bl01., 53:30, 1976.

43. Donoff, R.B., and Grillo, H.C.: The effects of skin

grafting on healing open wounds in rabbits. J.

Surge Res., 19:163-167, 1915.

44. Fairbanks, G., Steck, T.L., and Wallach, D.F.H.:

Electrophoretic analysis of the major polypeptides

of the human erythrocyte membrane. Biochem.,.

10:2606, 1971.

45. Righetti, P.G., and Drysdale, J.W.: Isoelectric

focusing - Laboratory techniques in biochemistry

and molecular biology. Work, r.s., and Work, E.

(eds.), 5:337-590, North Holland, 1976.

46. Noelken, M.E., Wisdom, B.J., and Hudson, B.G.:

Estimation of the size of collagenous polypeptides

35

by sodium dodecyl sulfate - polyacrylamide gel

electrophoresis. Anal. Biochem., 11:131, 1981.

47. Huszar, G., Maiocco, J., and Naftolin, F.: Monitoring

of collagen and collagen fragments in

chromatography of protein mixtures. Anal

Biochem., 105:424, 1980.

48. Aminoff, D.: Methods for the quantitative estimation

of N-acetylneuraminic acid and their application

to hydrolysates of sialomucoids. Biochem. J.,

81: 384, 1961.

49. Peterson, G.L.: A simplification of the protein assay

method of Lowry et. ale which is more generally

applicable. Anal. Biochem., 83:346, 1977.

50. Lehtonen, A.: The mucopolysaccharides of aging

experimental granulation tissue. Acta Physiol.

Scand. Suppl., 310, 1968.

36

FIG. 1

A • F 1 a (l k W0 U tl d b 0 undar i e s •

B. Excision of skin and superficial fascia.

c. Open wound at the level cf the panniculuscarnOSLlS.

D. Incised granulation tissue.

37

FIG. 2

Elution profiles of soluble wound extract

constituents on Sephacryl SF 200 as a function

of time. Normal skin; 1.8mg, 3-day granulation

tissue; 2.0 mg, 7-day granulation tissue;

2.5 mg, 9-day granulation tissue; 2.9 mg,

14-day granulation tissue.

38

Ec

3.0

\\\\\

\. -"*"''''''' ,\\\\

\'""-

"'"'--

~~---4"'O---80-r-i---1.."..20---....160--'-2~60....-----2M)-r-iElution Volume (ml)

Normal Skin3d GT 1.8mg7d GT 2.0mg9d GT 2.5mg

14d GT 2.9mg

FIG. 3

Molecular weight determination of

Peak A/Band 3 (PA/B3) material.

39

0.8

0.6>eu~

0.4

0.3

0.2

0.1

"u..LYSOZymc

~ f3 -Lactoglobulin

~

Pepsin

Ovalbumin

Bovine Serum Albumin

PA/B3

f3 -Galactosidase

10 20 30 40 50 60 70 80 100

Molecular Weight >< 103

FIG. 4

Elution profiles of 2.9 mg, 14-day open-wound

granulation tissue and 3.8mg, 14-day grafted

granulation tissue.

40

Ec~ 2.N

tDoceu€o.,.0c(G)

~..,asCDa:

Vt

+

O~----y--------,..----y-----,.-----y------

Elution Volume (ml)

----14d GT 2.9mg-1 ~d grafted GT 3.8mg

FIG. 5

SDS-PAGE of wound tissue extracts.

A. Gels 1-4, 3-day granulation tissue;

Gels 5-6, 7-day granulation tissue;

Gels 7-8, 9-day granulation tissue;

Gels 9-10, 14-day granulation tissue (open);

Each gel contains 25-30 ul of crude tissue

extract.

B. Gels 11-13, 14-day gr·anulation tissue

(grafted) ;

Each gel contains 25-30 ul of crude tissue

extract.

C. Gel 14, isolated Sephacryl SF 200 Peak Aj

Gels 15-16, isolated Sephacryl SF 200 Peak Cj

Gels 17-19, overloaded crude extracts derived

from 7-, 9-, and 14-day open-wound tissues.

41

.,

granulation tissue extract

on SDS-polyacrylamide.

Representative

g·.day central

electrophoresed

FIG. 6

densitometric

42

scan of

(1)(.)cccs.0L-

oen.0«Q)

>

90 80 70 60 50 40 30 20 10 0

Migration Distance (mm)

FIG. 7

Time-dependent changes in electrophoretic

banding pattern of crude wound tissue

extracts. Abscissa: Postwound interval;

Ordinate: Percentage of scan attributable

to a given band.

43

70

c: 5«S()

en"t-

o(1) ........ Band 1(»

as ----- Band 2+oJ 30c Band 3(1)0 ~.-.- Band 4'-Q) Band 5a. 20

10- --: ::::-~-;:- -;:...,....,....... ..... ­.,,, ••..••• .....-..-......_.~:r"""e&,;;...- •...... .. -.

O-t----.,----r---.,.-----r'----3 14

Postwound Interval

FIG. 8

Total wound collagen content in grafted

wounds as a function of time. Due

to the large standard errors for open

and grafted wounds at 14 days, the

apparent difference between these wounds

on postwound day 14 was not statistically

significant.

44

J6

1.5

14

13

!2

"'0 11G)

• JO•G)

G) 9ex:C •G) 7...,0'- 6a.0 j

E4

J

2

0

0

f

WOUND COLLAGEN CONTENT

............ __ T

-............ I...............~

I•.L

2 J 4 .s 6 1 I 9 10 11 12 13 ,~

Post Wound Day

open

grafted

TABLE I

SOLUBLE PROTEIN, NANA, AND

HYDROXYPROLINE OF WOUND TISSUE EXTRACTS.

POSTWOUND LOWRY PROTEIN NANA HYDROXYPROLINEDAY (mg/ml extract) (ug ± SEM) (ug/mg dry tissue

± SEM)

~ 3.52 ± .02 0.20 ±.O4 0.34 ±.O8..)

7 3.89 ± .29 0.78 ±.O2 0.35 ±.O8

9 5.00 ± .38 0.12 ±.O4 0.55 ±.12

i4 5.76 ± .04 0.22 ±.O3 o~ 4!~ ±.10

14 (Graft) 7.55 ±.22 0.62 ±.12

45

TABLE II

Amino AQ14 Analysis a

~acryl Sf 2QQ ~ A/SDS-Page~ 3

Glu 115 Val 61 Tyr 45

Lys 111 Gly 57 His 38

Leu 107 Al·g 56 Iso 32

Asp 90 Thr 51 Meth 8

Pro 66 Ser 51 Hyl

Ala 62 Phen 50 Hyp

aResidues/1~OOO Residues

46