A New Murine Model for Mammalian Wound Repair and Regeneration

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C L I N I C A L I M M U N O L O G Y AN D I M M U N O P A TH O L O G Y

Vol. 88, No. 1, J uly, pp. 35– 45, 1998Article No. II984519

A New Murine Model for Mammalian Wound Repair and Regeneration

Lise Desquenne Clark, Robert K. Clark, and Ellen Heber-Katz

T h e W i s t a r I n st i t u t e, 3 6 0 1 S p r u c e St r e et , P h i l a d el p h i a , P en n s y l v a n i a 1 9 1 04

ear hole closure (5). This sugg ests t ha t the capa city forRegeneration is generally considered to be a phe- regeneration has not been completely lost.

nomenon restricted to amphibians in which ampu- There are, of course, cells and t issues in mammalstated limbsreform andregrow.Wehaverecently noted

that are continually being replaced. This occurs eithera strain of mouse, the MRL, which displays a remark-

(a) by cell duplication producing, for example, liver (6able capacity for cartilagenous wound closure and

7) a nd blood vessels t hrough th e process of an giogeneprovides an example of a phenomenon previously con-

sis (8) or (b) by the generation and differentiat ion ofsidered to be a form of regeneration. Specifically,

cells from stem cells, a process th a t produces new epithrough-and-through ear punches rapidly attain fullthelia l cells (9, 10) a nd lymphoid cells (11). Howeverclosure with normal tissue architecture reminiscentin t h es e c a s es , t h e a rc h it ec t u ra l s c a f fo ld in g is o f t enof regeneration seen in amphibians asopposed to scar-

ma inta ined and hence there is no gross t issue replacering, as usually seen in mammals. H istologically, wemen t , i . e . , regen era t io n . O n t h e o t h er h a n d , c ert a inhave demonstrated normal cell growth and microanat-cells and t issues exist throughout life without beingomy, including angiogenesis and chondrogenesis, asreplaced and do not proliferate. These include nerveopposed to control C57BL /6 mice which have ear holes

that contract minimally but do not close. Finally, this cells, hea rt t issue, th e eye lens, a nd the ca rt ilage of thephenomenon is a genetically definable quantitative nose a nd ear (12).trait. 1998 Academic Press We describe herein a mouse model of wound repair

K e y Wo r d s : mice; wound healing; regeneration. an d regeneration which ha s a llowed us to begin to explore the molecular, cellular, and genetic bases of thisphenomenon. F ull-thickness t hrough-a nd-thr ough ea r

INTRODUCTION punches of a bout 2 mm in diam eter ha ve genera lly beenused a s a mea ns of long-term id ent ificat ion of mice kep

in c o lo n ies . T h is is b a s ed o n t h e fa c t t h a t t h es e ea rThe biological r esponse t o tra uma tic injury in higherpunch holes generally do not close over th e lifetime oorganisms falls into two categories: regeneration andt h e a n ima l a n d p ro v id e a c o n v en ien t a n d p erma n enw ound repair. Regenera tion involves the gross replace-ma rker. We noted in t he ca se of the MRL m ouse stra inment a nd restora t ion of adult t issue mass with norma lh ow ev er , t h a t t h es e ea rp un ch es w ere t ra n s ien t , w it ha rchitectur e and fun ction a nd, in the extreme ca se, fullfull closure occurring with in 4 w eeks w ith norma l t isorgans, whereas wound repair involves the migrat ions u e a r ch i t ect u r e a n d w i t h ou t s ca r r i n g. Th a t t h i s iof fibrobla sts t o the w ound site, forma tion of gra nula -probably not only wound repair but regeneration is inion t issue, a nd t he laying down of collagen in a disor-d ic a t ed b y t h e p res en c e o f n o rma l d erma l regro w t hg a n i ze d f a s h ion w i t h t h e f or m a t i on of s ca r t i ss u e.with organized extracellular matrix (ECM) depositionRa rely d o h a ir fo l l ic les a n d s w ea t gla n d s ret u rn a n dnormal vascula ture, and cart ilage regrowth th at is simnormal ar chitecture a nd function is not fully restored.i l a r t o t h a t i n t h e n o r m a l e a r . I n c o n t r a s t , e a r h o l eI t is gen era l ly o b s erv ed t h a t t h e c a p a c i t y fo r t is s u ewounds in cont rol C57B L/6 mice (a nd for t ha t ma tt erregeneration in mammals is l imited, especially com-in every other s tra in of mouse we a re a wa re of) neverpared to tha t in a mphibian s, where entire limbs ca n beclose completely. Histological differences can be seenregenera ted a fter a mputa tion (1). This a pparent lack ofb et w een t h e M RL a n d t h e C57B L/6 mice a s ea r ly a sma mma lian regenera tive capa city ha s largely directedday 2, and these differences include rate of reepithelihe focus of s tudy towa rd w ound repair, th e ar chetypala l iz a t ion , a n n u la r s w ell in g, ra p id con n ect iv e t is su eresponse to injury in ma mma ls.proliferat ion, a ngiogenesis, a nd chondrogenesis.While th e ab ove describes th e generic mode of w ound

The MRL mouse has been intensively s tudied as ahealing in mammals, there do exist several instancesmodel for systemic lupus erythematosis (SLE), and, inof l imited regeneration reminiscent of that occurringits mut a nt form, t he lpr/lpr s tra in displa ys gross an dn amphibians (2). They include the regrowth of the

ips of fingers (3), ant lers (4), and a n example of rabbit ra pid lymphoprolifera tive disease, serum a utoa nt ibod

35 0090-1229/98 $25.00Copyright 1998 by Academic P res

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36 CLARK, CLARK, AND HE BE R-KATZ

FIG. 1. The kinetics of ear punch hole closure. Two-millimeter holes wer e punched in ears on da y 0 and , for ea ch stra in of mouse, holew ere measu red a t days indicat ed on t he horizont al a xis. Average hole diamet ers a re show n (n Å 4).

es, and autoimmunity as it ages (13– 15). This lpr de- H i stology. Ea rs were removed with scissors by cuttin g a t t he ba se of th e pinna . They w ere fixed overnightfect is d u e t o a mu t a t ion in t h e fa s gen e w h ich lea d s

o an ina bility to media te apoptosis of lymphocytes (16, in 10%buffered forma lin. To facilita te sectioning, theywere held fla t during fixa tion by invert ing t he lid of a17). While this defect might suggest t ha t a n un checked

growth of cells due to lack of apoptosis is the underlying processing ca ssette on the base an d sandw iching thefla tt ened ea r by applying gentle pressure with a rubbercause of th e r egenerat ive capacity, both MRL/lpr a nd

M RL// mice, w it h a n d w it h ou t a d efect iv e fa s mole- b a n d .Once fixed, ea rs were bissected across the widescu le , i n f a ct h a v e t h e s a m e r eg en er a t i v e c a p a ci t y .

B reeding st udies involving the generat ion of F1 a nd point of the hole using a dissecting microscope an d aNo. 10 sca lpel blade. The t wo ha lves w ere then gluedb a c kcros s s t ra in s b et w een M RL a n d C57BL /6 mice

ha ve shown t ha t there is a genetic basis for this wound together w ith collodion, aga in using the dissecting microscope to obta in perfect alignment of the cut edgesrepair/regenera tion tr a it .and hole margins. Because the collodion would most

METHODS likely dissolve in rea gents used to prepare specimensfor pa ra ffin embedding, t he ear s w ere sutured t ogeth er

A n i m a l s . MR L /Mp – /// (MRL//) mic e a n d M RL/ using 5-O silk on a 1 1

2-in. s traight Keith AbdominaMp – lpr/lpr (MRL/lpr) m ice w ere obta ined from The cutt ing needle with a triangular point .J a ckson La bora tories (B a r Ha rbor, ME ). C 57B l/6 (B 6) Tissues w ere embedded in par a ffin a nd sectioned somice were obtained from Taconic Laboratories (Ger- t h a t t h e c u t ed ges c o n t a in in g t h e h o le ma rgin s w erema ntown, NY). The anima ls were then bred and ma in- in t h e p la n e o f t h e s ec t io n . S l id es w ere s t a in ed w it ha in ed u n d er s t a n d a rd con d it ion s a t t h e Wist a r In s t i- h em a t o xy li n a n d e os in or w i t h G o m or i t r i ch r om eute Animal Facility. s t ra in .

Phenotyping. A 2-mm through-an d-through holeRESULTS

w a s m a d e i n t h e c e n t e r o f t h e c a r t i l a g e n o u s p a r t o fboth ears of 6-week-old mice using a metal ear punchThe Wound Healing Phenotype: Kinetics and Gross

(Fisher Scientific, P itt sburgh, Ca ta log No. 01-337B).Aspects

The holes were measured a t the t ime of wounding a ndfollowed for wound closure using a grid-etched reticle C57B L/6 and MRL mice were ea r punched using a

s t a n d a r d m et a l ea r p un ch t o cr ea t e a w e ll -ci r cu m(Bausch and Lomb, 71).

FIG. 2. P hotographs of the hea ling ear wounds. C57BL/6 (left) an d MRL/lpr (right) ears were punched bilatera lly in the center of theears creating a 2-mm through-an d-through w ound a nd followed for 33 da ys. Fr om top to bottom, one can see the progression of hole closurrom day 1, day 9, day 20, to day 33.



37R E GE NE R ATI ON I N MI CE




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scribed circular surgica l wound of about 2 mm in diam- shown limited progression beyond the cut cart ilagemargins and have a dist inct paucity of epidermal haireter a t a site at w hich the thickness of th e ea r is consid-

era b ly les s t h a n a mill imet er . Th e a n ima ls u s ed in i- fol licles a n d s eb a c eou s gla n d s . In con t ra s t , t h e M RLwounds show marked progress toward full closure dueially were within the 8- to 12-week-old young-adult

a ge ra nge so tha t early developmental considera t ions to extensive dermal prolifera t ion and are well suppliedw it h h a ir fo l l ic les a n d s eb a c eo u s gla n d s in t h e n eww ould not be a n issue. As can be seen in F ig. 1, by da y

15 maximal closure wa s a chieved in t he C57BL/6 with growth zone. EC M is laid down so as to preserve normaa rchitecture, the und erlying connective tissue is hypera 30%reduction in the original hole diameter, and this

rema in s st a b le. In c on t ra s t , t h e M RL a c h ieves a n 85% p la s t ic, a n d t h e epid ermis is r ic h a n d t h ick . Th e ea r

car t ilage la yer h as not s ignifican tly extended into t hered u c t io n in h o le d ia met er b y d a y 15 w it h c o mp let eclos u re b y d a y 25. Reexa min a t ion of t h e ea rs o n d a y w o u n d s it e b ey on d t h e in i t ia l cu t ma r gin .

In Fig. 5, C57BL/6 and MRL ea rs a re shown on da y81 s h o w ed n o fu rt h er c h a n ges . In F ig. 2 , t h e c lo s edMRL w ound is evident a nd it is difficult to identify the 81 aft er w ounding. Here numerous ingrowths of car t i

lage th at ar e absent from t he C57B L/6 ear can be seenoriginal s ite of the hole s ince there is no fibrosis orsca rr in g. in t h e MRL ea r . Th e ca rt ila ge in grow t h s a re sur

rounded by numerous a dipocytes which norma lly makeup the minor subcutaneous or hypodermal layer conTh e Wound H eali ng Phenotype: H istological Aspects necting ear cart ilage to dermis. I t is not clear why fatcells ha ve come to be such a prominent cell type by t hisOne possibility considered for the complete hole clo-

s u re in M R L m i ce w a s t h a t t h e r e w a s a d ef ect i n t h e t i m e poi n t .a b i l i t y o f t h eir ep it h el iu m t o migra t e a c ro s s t h e c u t

T h e P a ttern of I n h eri ta n c e S een in Wou n d H ea li n g i sedge of the dermis a nd cart ilage, thereby a llowing un -Q u a n t i t a t i v e nh ibited conn ective tissue prolifera tion. Hist ologic sec-

ions of healing ear punch holes for the first 2 da ysOur init ia l findings on the heredita ry na ture of the

were examined (Fig. 3). This possibility was rejectedwound hea ling tr a it can be seen in Fig. 6 (top). In t hese

w h en i t w a s o b served t h a t n ot on ly d id epit h el iu mstudies, mice were ear punched a t 6 w eeks of a ge an d

p ro mp t ly migra t e a c ro s s t h e M RL w o u n d s , b u t t h a tw ere exa min ed a t 2 a n d 4 w eek s a f t er ea r p u n c h in g

his cha nge occurred 1 day earlier for MRL mice (dayThe 4-week ear hole size of the MRL mice ranged from

2) th a n for C57BL /6 mice (da y 3, not sh own). I ndeed,0 to 0.4 mm, wh ile the ea r hole s ize of the C57B L/6

epithelium completely covered virtua lly a ll wounds ex-mice ra n ged from 1. 2 t o 1. 6 mm. Th ese t w o h ea l in g

a mined a fter da y 1 for MRL m ice. Epithelium coveredphenoty pes were nonoverla pping. F ifteen F1 mice bred

a ll C57B L/6 wounds exa mined a fter a nd including dayfrom MRL 1 C57B L/6 ha d ea r holes int ermedia te be

3, except one w ound from da y 5 (Fig. 4a) wh ich show ed

t w een t h o s e o f t h e t w o p a ren t s ( ra n gin g fro m 0. 4 t ohe continued presence of eschar with migrat ing epi- 1.1 mm ).helium fa iling to bridge the cut edge.

In an init ial experiment, t wo ba ckcross populat ionsAs ears were prepared for histology, two grossly ob-

were created by using (MRL fema le 1 C 57B L/6 m a leserva ble differences w ere noted betw een C57BL /6 a nd

F 1 f e m a l e s a n d m a t i n g t h e m t o t h e p a r e n t a l m a l e sMRL. The first wa s th a t, for a ll time points, t he tissue

(Fig. 6, bottom). The backcross population to MRL dissurrounding t he wounds in the MRL ea rs wa s severely

p la y ed a cu rv e s k ew ed t o M RL-t y p e h ea l in g. In t h eh y p eremic w h en comp a red t o t h a t of t h e C57B L/6

ba ckcross popula tion t o C57BL /6, the progeny s howedw ounds. Also, sta rt ing on day 4, an d contin ung on each

a curve w ith its mea n displaced to C 57B L/6-ty pe (i.e.succeeding da y, a prominent a nnular swelling w as ob-

poor) hea ling. The healing t hus a ppea rs to be a qu a nti-s erv ed a ro u n d t h e M RL w o u n d s t h a t w a s a b s en t fo r

t a t i v e t r a i t .C57B L/6 w ounds.

Consistent with the grossly observable differences

DISCUSSIONbetween the w ounds, histological exa minat ion a t eachimep o in t ( F igs . 4a , 4b , a n d 4c ; d a y s 5, 10, a n d 20)

A C u r io u s R esu l t showed a ma rked difference in the degree of a ngiogene-sis , cell prolifera t ion, connective t issue mat rix forma - These studies a rose from t he serendipitous finding

t h a t t h e n u m be ri ng b y e a r p un ch in g of M R L m i ceion , fib rob la s t migra t ion , a n d E CM d ep os it ion oc-curring in the t wo stra ins. Also, the presence of ha ir proved to be ineffective as several weeks a fter punch

ing, the ear holes disappeared. P erhaps th e most s tr ikfollicles with accompanying sebaceous glands withinh e h ea l i ng w ou n d s w a s n ot e d f or b ot h M R L a n d i ng ch a r a c t er i st i c w a s t h a t t h e ea r h o le w ou ld cl os e

w it h o u t d et ec t a b le s c a rr in g. E a r p u n c h in g, in w h ic hC57B L/6 w ounds but a ppeared more prominent an dn u mer ou s i n M RL t h a n i n C 57B L /6 w ou n ds . a w e ll -ci rcu m scr ib ed t h r ou gh -a n d -t h r ou gh ci rcu la r

w o u n d o f a b o u t 2 mm in d ia met er a t a s i t e in w h ic hAt a ll of these t ime points , C57B L/6 w ounds ha ve




FIG. 4. Da y 5, 10, and 20 wounds. For a ll da ys indica ted for C57BL/6 tissue (A), MRL// tiss ue (B), a nd MRL/lpr t issue (C). All sectiona re sta ined with hemotoxylin a nd eosin (pictures a re representa tive of four edges examined). (a) Da y 5 (A, B , C: 401, top; and 201, bott om)Swelling at the MRL wound site is extensive with neovascularization (401, a rrowhead s) and ma rked derma l fibroblast prolifera tion (201a rge ar rowhea ds). Adnexa e in th e new epithelium can be seen here (201, sma ll arr owheads). AC 57BL /6 wound tha t ha s failed to epithelializes shown here, although this is not typical (201, *). (b) Day 10 (A, B, C: 401, top; and 101, bottom): There is ma rked neovascularizat ion401, ar rows) a nd fibroblas t prolifera tion (101, arrows) seen in the MRL wound extending out beyond the borders of the wound where the

cut cart i lage edges a re seen. Not e t he ext ent t o w hich dermal cells have migrat ed out beyond t he w ou nd margin marked by t he cart i lageedge for the t wo MRL ears compared to th e C57BL/6 ear. (c) Da y 20 (A, C:201; B:401-upper pa nels a nd 51-lower pan els): The prominenprolifera tion of fibroblast s in the dermis a nd t he a ppear a nce of a blastema -like structure ha s led to significant closure of th e MRL woundas originally marked by t he cu t edges of cart i lage at t he r ight and lef t margins of t he p hot ograp h (51-between arrows). By comparisonhere is lit t le extension of C57BL/6 tissue into t he w ound spa ce. The h omogeneity of fibroblast prolifera tion a nd EC M deposition is mos

striking in MRL (B:401-upper panel).




FIG. 4—Continued

FIG. 5. La te-sta ge ear w ound closure. Wound sites (n Å 3) 81 da ys a fter wound ing for C57BL /6 (top) an d MR L/lpr (bottom) mice werea ligned using a d issection microscope and su tur ed together to ensure sectioning through th e former w ound for MRL/lpr tissue. Ca rt ilag enouslands (sma ll ar rowhead) present t hroughout the MRL/lpr section a re surrounded by prominent adipocytes (large a rrowhead ). Theseatures ar e a bsent from t he C57BL/6 tissue. The tissue s ections a re st ained with G omori t richrome. Ma gnification, 101.




FIG. 6. The a na lysis of frequencies of wound closure on da y 30 in parenta l a nd crossbred populat ions. Histograms of day 30 earpunchhole diameters can be seen for t he following genotypes: C57BL/6 a nd MRL/lpr par enta l mice a nd (MRLxB6)F 1 mice (top) a nd the firsbackcross to each parental strain (bottom).

h e t h ick n es s of t h e ea r is con s id era b ly les s t h a n a a n d u n derly in g con n ect iv e t is su e w a s h y p erp la s t ic a n dappeared t o form a blastema -like str ucture. The bla smillimeter, is a general met hod of ident ifying an d num-

b erin g mice o v er a lon g p eriod of t ime. I n a l l ot h er t ema h a s b een d es cribed in t erms of a mp h ibia n l imbregen era t ion . In t h e a d u lt a n ima l , i t is t h e b egin n in gmouse stra ins, these ear punches do not heal shut an d

generally lea d to shar p an d well-defined circula r holes of n ew t issue ma ss derived from a dult mesenchymatissue at the limb stump w here proliferat ion, dedifferw it h c irc u mferen t ia l h ea l in g. T h is is u n lik e w h a t is

seen with rodent trunk excision wounds which show entiat ion, an d redifferentia t ion occur a s part of the process of regeneration (19, 20). The major cell types thacontra cture of the wound bed an d scar ifica tion (18).

Rodent body skin is loose, unlike mouse ear skin, w hich undergo dedifferentia tion include dermis and cart ilage( 21) . W it h t h e n ew d erma l gro w t h s een in t h e M RLs connected to a layer of cartilage. Thus, wound con-

ra ct u re is a l l b u t imp os s ib le in ou r s t u d y w h ere t h ere mou s e, a n giogen es is w a s s een a s ea r ly a s d a y 4. H a irfollicles and sebaceous glands were seen in this news n o w ou n d b ed a n d t h e a d ja c en t t i ss ue i s fir m l y

a t t a ch ed t o t he u nder ly in g ca r t ila g e. g row t h a r ea bu t it is n ot clea r w h et her t hese a d nexa emigrated, were pulled into the new epithelial growthH is t ologica l s t u d ies s h o w ed t h a t n orma l t is su e in -

growth a nd remodeling with epithelial a nd fibroblast or actually represented newly regenera ted structuresIn the first 3 w eeks, th ere wa s m inimal evidence foproliferat ion w ere the cause of the closure. In t he MRL

s t ra in , n ew epit h el iu m clos ed t h e circu mferen t ia l t h e ca r t i la ge ext en d in g in t o t h e n ew grow t h a rea . H o wev er , ex a m in a t ion of t h e w ou n d s it e a t 3 m on t hwound ma rgin wit hin 2 days a nd becam e strat ified and

corn ified . E CM w a s la id d ow n in a n o rd erly fa s h ion , s h ow ed n ew ca rt i la ge fillin g t h e w o u n d s i t e in w h a t




appears to be the process of reorganization. This is in Wound Repai r ver sus Regener ati on —Gr oss and

M icroanatomi c Aspects cont ra st t o w ha t is seen in the C57BL /6 wounds wh ereclosure is minimal.

The type of cell replacement seen in wound healingViewing hole closure from a purely geometric per-

c a n gen era l ly b e d iv id ed in t o t w o c a t ego ries : w o u n dspective, we note that ingrowth of t issue must be ac-

repair and regeneration. Wound repair, a fter injurycompanied by continuous remodeling. Oth erwise, th e

begins w ith a fibrin clot wh ich provides a provisionais su e w o u ld b u n ch u p a n d b ecome irregu la r ly en -

ma t r ix fo r c el l migra t io n a n d relea s e o f c h emo t a c t icfolded. What we observe, however, is a grossly normal

factors leading to recruitment of inflammatory cellsfla t t issue layer. Wound repair/regenera tion studies

Epithelial cells migrat ing from the wound margin ex-done from dista l extension w ounds, such a s fingert ip press integrins which can recognize the matrix comporegr owt h, do not ad dress t his issue (3). There ha ve been nents , thus propelling the cells forward. Collagen isextensive studies a ddressing t he ra te of closure of skin laid down in an irregular fashion and the provisionawounds in mamma ls dat ing ba ck at least to the classic matrix is replaced by a collagenous scar (25, 26). Asw o rk o f Ca rrel on gu in ea p ig a n d h u ma n s k in (22). concisely stated by J . Gross,Int erestingly, th e proport iona l ra te of closure is similar

‘‘Mam ma ls in contras t t o some amphibians (but not a ll) substi-a nd occurs w ithin 1 month, t hough in th e case of skint u t e rep air for regenerat ion t o close fu ll t hickness t rau mat ichere is a wound bed, upon which a provisional matrixwounds. The basic mechanisms involve pulling the wound edges

forms a nd epithelium regrows, an d w ound contra cture over the excised a rea a nd simulta neously filling in t he defectd o min a t es . In t h e w o rk rep o rt ed h ere, t h ere is n ew w it h a t emporary cellu lar , vascu lar , and ext ra cellular mat r ix-

cont aining granu lat ion t issu e w hich, w it h t ime, is largely ab-issue ma ss which extends int o the w ound with no guid-sorbed or converted t o an ava scula r, spa rsely cellular fibrousance or support from an underlying layer of tissue andscar . The rela tive degree to wh ich each of these processes is usedno realis t ic possibility of contra cture. Interestingly, is dependent on species and region of the body. For example,

Ca rrel a n d H a rt ma n n ( 22) n o t ed t h a t t h e ra t e o f c lo - in the huma n excision wound, there is slow, or no, centripetalmovement of the dermal edges in man y a reas of the body; granu-sure seen in mamm alia n skin wounds was th e sam e aslat ion tissue wh ich gra dua lly converts t o scar fills the d efect.h a t s een in s a la ma n d er regen era t ion .The fina l fibrotic pat ch in huma n skin a nd t he minima l residua lscar in most mat u re animal sp ecies lacks t he normal dermalcollagenou s p at t ern, and t he epidermis covering t he scar fai ls

T h e M R L M ou se t o develop ap p endages su ch as hair and t he several t yp es ofglands.’’ (1).

The MRL mouse (H-2k) is derived from an inter-Regenera tion, on t he other ha nd, involves the gross

breeding of the LG mouse (75%; H-2d/f), the AKRreplacement and restorat ion of t issue mass with nor

mous e (12.6%; H -2k), t he C 3H mous e (12.1%h-2k), a ndma l a rchitecture a nd, in th e extreme case, full orga ns

h e C 57Bl/6 mouse (0.3%H -2b) (13) a nd w a s s elected Renegeration can be seen in vertebrates such as urooriginally for its la rge size. MRL/lpr, a m uta nt deriveddeles (salamanders and newts) but is not seen widely

from this colony, showed enlarging spleen a nd lymphin ma mma ls . Th e ma in t h ru s t of t h es e s t u d ies, d o n e

node with a ge, lymphoprolifera t ion w ith a berrant con-p rima ri ly in a mp h ib ia n s pecies , h a s focu s ed on t h e

r ol of apoptosis in germ ina l cent ers, an d a high suscep- blastema —the t issue structure tha t conta ins undifferibility to aut oimmune disease with aut oan tibodies, a n entiated mesenchymal progenitor cells. A covering o

arthritis-like syndrome, and glomerulonephritis. This wound epithelium is formed by the m igra tion of epiderwa s shown t o be the direct result of a retrotran sposon ma l cells ar ound t he w ound (27). I t is from this s t rucnsert ion into the second intron of the fa s gene in t he t u re t h a t t h e n ew l imb regen era t es .pr str a in (16, 17, 23). However , the ra pid an d complete A noted study of mammalian regeneration involves

w ound closure tha t w e describe here is unrelat ed to fa s the closure of through-an d-through (1 cm diametersince the MRL// mou s e h a s t h e s a me h ea l in g c h a ra c- ra bbit ea r holes (5). Ra bbit ea rs, like mouse ears, ha ve

erist ics . Furthermore, wound closure is unlinked to a cen t ra l c a r t i la ge la y er a n d t h e gros s a n d microa n ahe lympadenopath y (R Å 0.4) a ssociat ed wit h lpr m ice t omic s eq u ela e ob s erv ed a re s imila r t o w h a t w e h a v e

a n d t h e a u t o a n t ib o d ies ma d e t o h is t o n e p ro t ein s b y seen in t he MRL mouse, i .e. , the ra bbit exhibits comh ese a nima ls (dat a not sh ow n) (13– 15, 24). This la ck plete closure with norma l skin a rchitectur e a nd chron

of fa s involvement h a s been confirm ed in ma pping stu d- drogenesis . Ea r w ounds in dog a nd sheep showed ines usin g MR L/lpr a nd C57B L/6 ba ckcross m ice show- complete hole closure a s seen in th e C57B L/6 mousen g a clea r gen et ic b a s is for t h is regen era t ion t ra i t , Th e ra b b it ea r h a s b een u s ed t o s t u d y t h e ef fect s o

unlinked to th e fas genet ic locus (McBrea rt y et al., sub- locally a pplied growt h fa ctors (28, 29). Wounds weremitted for publication). One characteristic of the MRL crea t ed d ow n t o t h e ca r t i la ge la y er a n d recomb in a nmouse is i ts large size; h owever, th ere is no evidence huma n fibroblast gr owt h fa ctor a pplied to the w ound

led to relat ively greater am ounts of granula t ion t issueha t th is tra it is linked to adult body weight (R Å 0.12).




4. G oss, R. J . , P roblems of ant lerogenesis. C l i n . O r t h o p. 69, 227–neovascula rizat ion, and an increase in the EC M. Inter-238, 1970.es t in gly , t h e s a me exp erimen t d o n e in mic e s h o w ed

5. G oss, R. J . , and G rimes, L. N., Epiderma l downgrowt hs in regenittle or no effects of th is gr ow th fa ctor (30).erat ing ra bbit ear holes. J . M or p h o l . 146, 533– 542, 1975.

Following up on reports of regrowth of amputated6. Fa usto, N., Hepat ic regenera tion. I n ‘‘Hepa tology : A Textbook in

fingertips in children, wherein a na tomically norma lLiver Disease’ ’ (D. Zakim and T. D. Boyer, Eds.), 2nd ed. , pp

fingertips including na ilbeds ha ve spora dica lly been 49– 65, Sa unders, P hiladelphia, 1990.observed, Borgens examined the effect of precision sur- 7. Micha lopoulos, G . K., a nd DeFrances, M. C. , Liver regenerationgical a mputa tions on the foretoes of B 6C3H mice (3). Science 276, 60– 66, 1997.

B riefly, it wa s observed tha t a mputa tions distal t o the 8. Folkma n, J . , an d Klagsbrun, M., Angiogenic factors. Science 235

442– 447, 1987.ast phala ngeal joint led t o predicta ble regrowt h of toe9. P otten, C. S. , an d Morris, R. J . , Epithelial stem cells in vivo. I nips, but , in most cases the nail did not grow back or

‘‘St em C ells’’ (B . I . Lord a nd T. M. D exter, E ds.), pp. 45– 62, Thew h e n i t d i d w a s a b n o r m a l . A m p u t a t i o n s a s l i t t l e a sCompany of Biologists Limited, Ca mbridge, 1988.

100 mm proximal (into the middle phalange) led t o no10. Mar tin, P . , Wound healing —Aiming for perfect skin r egenera

regrow t h , a n d a n ob v iou s b la s t ema w a s n o t p res en tt ion. Science 276, 75– 81, 1997.

even when regrowth occurred.11. Spa ngrude, G . J . , Heimfeld, S. , a nd Weisman , I . L. P urification

Though it does appear that a regeneration blastema an d cha ra cterization of mouse hematopoietic stem cells. Scienc

s formed in this murine wound closure model, it is not 241, 58– 62, 1988.

c lea r t h a t t h e s a me ev en t s t a k in g p la c e d u rin g l imb 12. Wornom, I . L. , a nd Buchma n, S. R. , Bone and cart ilaginous tissue. I n ‘‘Wound Hea ling: Biochemical an d Clinica l Aspectsregeneration in the amphibian are occurring here. What(I. K. Cohen, R. F. Diegelmann, and W. J . Lindblad, Eds.), pps useful in this case is tha t we h a ve identified a mouse356– 383, S a unders, P hiladelphia, 1992.

strain, unlike other mouse stra ins, in which regenera-

13. Murphy, E. D., a nd Roths, J . B . , Autoimmunity a nd lymphoproliion is readily seen and quant ified and which has al- ferat ion: I ndu ct ion by mu t ant gene lp r and accelerat ion by aow ed a genetic dissection of this tr a it. In a second study male-associat ed fact or in st rain B XSB . I n ‘‘Genetic Control o

(31), we have made use of an extensive microsatellite Autoimmune Disease’ ’ (N. R. Rose, P . E. Bigazzi, and N. L. Warner, Eds .), pp. 207– 220, Els evier, New York, 1979.map in the mouse to determine the underlying genetic

14. Cohen, P. L. , a nd Eisenberg, R. A., Lpr and gld: Single gene modba sis of this t ra it a nd ha ve identified mult iple loci w ithels of systemic a utoimmunity a nd lymphoprolifera rtive diseasean array of potentially interesting candidate genes. AA n n u . R ev . I m m u n o l . 9, 243– 269, 1991.

min imu m of seven loci were id ent ified a t a 20-cM resolu-15. Theofilopoulos, A. N., Im munologic genes in mouse lu pus modion, none of which corresponds to a ny previously ident i-

els. I n ‘‘The Molecular Pathology of Autoimmune Diseases’’ (Cfied locus in volved in MR L a ut oimmun ity (17). One ca n- B ona, K. A. S iminovitch, M. Za nett i, a nd A. N. Theofilopoulosdidate gene of interest is the retinoic acid receptor g. Ed s.), pp. 281– 316, Ha rw ood Aca demic, La nghorn e, 1993.

Retinoic acid (RA) is an initiator and important compo- 16. Wat an abe-Fukuna ga, R. , Br a nna n, C. , Copeland , N. G. , J enkinsN. A., and Nagata, S. , Lymphoproliferation disorder in mice isnent in development and regeneration and acts on epi-

exp lained by defect s in Fa s a nt igen t hat mediat es a p op t osish elial cells, binding t o retinoic a cid g (RARg) receptorsN a t u r e 356, 314– 316, 1992.

n the cytoplasm (32). In amphibians, RARd (the RARg17. Wa tson, M. L., et al ., Genetic analysis of MRL-lpr mice: Relationhomologue) is highly abundant in the limb regeneration

ship of t he Fas ap op t osis gene t o disease manifest at ions andblastema (32, 33) and RA is known to a ctiva te t he sonic renal disease modifying loci. J . E x p . M ed . 176, 1645– 1656, 1992hedgehog, hox, msx, and wnt genes also act ivated in 18. Russell, P . S. , and B illingha m, R. E. , Some aspects of the repaimammalian embryonic development (34). process in ma mma ls. P r o g . S u r g . 2, 1– 72, 1962.

19. Cha lkley, D . T., The cellular basis of limb regeneration. I n ‘‘Regeneration in Vertebrates’ ’ (C. S. Thornton, Eds.), pp. 34– 58ACKNOWLEDGMENTSUn iv. of Chicago P ress, Chicago, 1959.

This w ork w as generously su p port ed by t he G . H arold and L eila 20. Simon, H-G ., Kitta ppa, R. , Khan, P . A., Tsilfidis, C. , Liversa geY. Ma thers Cha rita ble Founda tion. We than k J am es Ha ndelman for R. A., and Oppenheimer, S. , A novel family of T-box genes inhis intense interest a nd vita l support, David E. Sa rfat ti for his ongo- urodele am phibian limb development an d regeneration: Ca nding enthusiam for wound repair, and E lizabeth B lankenhorn for crit i- da te genes in vertebrat e forelimb/hindlimb patt erning. Develop

cally reviewing the ma nuscript. We also wish t o tha nk Doug Tha yer ment 124, 1355– 1366, 1997.and Elsa Aglow for their superb technical assistance. 21. Muneoka, K., Fox, W. F., a nd Br ya nt , S. V., Cellula r contribut ion

from dermis a nd cart i lage t o t he regenerat ing l imb blast ema inaxolotls. Dev. Biol. 116, 256– 260, 1986.REFERENCES

22. Carrel , A., a nd Ha rt ma nn, A., Cicat r izat ion of w ou nds. I . Therelation between the size of the wound a nd th e rat e of its cicat ri1. Gross, J . , Get t ing t o mamma lian w ou nd repair a nd a mp hibianzation. J . E x p . M e d . 24, 429– 450, 1916.limb regenera tion: Amechanist ic link in the early events. W o u n d

Repair Regen. 4, 190– 202, 1996. 23. Adachi, M., Wat a nabe-Fu ku naga, R . , and Naga t a, S. , Aberrantranscription caused by the insertion of an endogenous retrovi2. S tocum, D. L. , Tissue r estoration: Approaches a nd prospects.

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I m m u n o p a t h o l . 40, 276– 282, 1986. 30. Uhl, E . , et al., B asic fibroblast growth factor a ccelera tes w oundhealing in chronically ischaemic t issue. B r . J . S u r g . 80, 977–25. Cla rk, R. A. F. , Wound repair: Overview a nd genera l considera-

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spinal cord a nd limb regeneration. D e v. G r o w t h D i f f er . 37, 133–29. P ierce, G. F. , et al. Platelet-derived growth factor (BB homodi-mer), t ra nsforming grow t h fact or-B 1, and basic fibroblast grow t h 147, 1995.

Received D ecember 11, 1997; accepted wit h revision J an ua ry 9, 1998

A New Murine Model for Mammalian Wound Repair and Regeneration

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Transcript of A New Murine Model for Mammalian Wound Repair and Regeneration