Fibrillin and Expression in Regenerating Frotn Cultured Keratinocyte Autografts: Morphogenesis of Begins At the Dertno-epidertnal Junction and Precedes Fortnation

Michael Raghunath, Thomas Bachi, * Martin Meuli, -r Stefan Altermatt, t l:tita Gobet, t Leena Bruckner-Tuderman,:j: and Beat SteinmalID tDivision of Metabolj c and Molecular Disease and Pedi atric Bum Ccnter of the Ulljvcrsity Children's Hospital Ztirich, "' Electron microscopic Central Laboratory of the University of Ziirich, Switzerland; and t Departlllcnt of Dermatology at the University of Mtinstcr, F.R.G.

The tetnporo-spatial expression of and elas­ horizontailly undulating microfibrils of the neoder­ tin in skin regenerating frotn autologous keratino­ mis which had developed independently. Elastin was cyte grafts was studied in three burned children. Skin first identified in the deeper neodermis 1 month after biopsies taken between 5 days and 17 months after grafting as granular aggregates and 4 months after grafting were investigated by conventional immuno­ grafting on fibrillar structures and surrounding cap­ fluorescence, confocal laser scanning, and electron illaries of the upper neodermis. Association of elastin microscopy. Fibrillin, the major component of 10- with microfibrils in the papillary was not 12-nm microfibrils, appeared 5 days after grafting in detectable before month 17. Our findings suggest a band-like fashion similar to VII at the that the cutaneous microfibrillar apparatus develops prospective basctnent membrane, and then formed simultaneously at both the dermo-epidermal junc­ the characteristic tnicrofibrillar candelabra at the tion and the reticular dermis and is a prerequisite for dermo-epidermal junction by fusion of several fine elastic fiber formation. In addition, it might be a microfibrils to comtnunicating micro project­ driving force for the formation of the papilla-rete ing downward into the reticular layer of the neoder­ ridge pattern. Key II1ol'ds: jiln.illill/micl'ojib,.i/slelasti,d tnis. Four to five months after grafting, several com­ wound It.ealillglbll.l'IIslkemti1loCJlte. J 1111/est Del'mato/ 106: municating micro fibrils were connected to a web of 1090-1095, 1996

ultured keratinocyte autografts (CEA) are c Ul:rent~. y of the human dermis, free micro fibrils form candelabra-like struc­ used to cover burn wounds [1] and other large skm tures and project perpendic ularly to the basal lamina of tll defects [2,3]. During take and development of these denno-epidermal junction (DEl), where tbey seem to be anchored grafts a neoepidermis, a neodermis, and a new [10]. T he fun ction of the extended cutaneous microfibrillar appa­ denno-epidermal junction are established [4]. The ratus is unclear. Mutations of its major component, fibrillin (Fib), a dermo-epidermalC cohesion is largely guaranteed by anchoring 320-kDa glycoprotein [10], cause a heritable fibrils and anchorin g filaments because mutations and functional disorde r, tbe [reviewed in ref. 11 ]. Interestingly. deficits of their major component, coHagen VB and laminin 5, striae distel/sac are frequently seen as a hallmark for dermal incom­ respectively [5], cause a severe blistering skin disease coll ectively petence. In addition, newborns affected with the most severe known as epidermolysis bullosa [reviewed in Ref. G]. Another manifestation of Marfan syndrome, the neonatal Marfim syndrome potential dermal anchoring system is represented by 10-12 lUll, [12,13], consistently show lax and redundant skin; the papillary microfibrils [7,8] that are also widely distt·ibuted iJl the connective dermis is very fragile, and e lastic fibers are fragmented [12]. tissue of the human body. They are ft·equently associated with B Li stering, however, has not been reported, suggesting that ftbrillin­ elastin and thus fo[m ela stic fibers. Alternatively, they occur as free microfibrils do not playa major role for tlte adhesion of the basement microfibrils, rypicall y arranged in bundles [9]. In the papillary layer membrane region to the dermis. To obtain more i.nsigbt into the role of the cutaneous microfibrillar apparatus, we examined the morpho­ Manuscript received Junc 8, 1995; revised December 2 1, 1995; accepted logical dynamics of transplanted keratinocyte autografts, because they for publjcatiol1 January 26, 1996. provide a unique opportwliry to study tile development of microfi­ Reprint requests to: Dr. Michael Raghunath, Institute of Physiological btiUar structu.res during the fOn1latiol1 of new skin. Chemistry and Pathobiochemistry, Universiry of Mllllster, Waldcycrstr. 15. PATIENTS, MATER.lALS. AND METHODS D-48"149 Munster. FRG. Abbreviations: CEA, cultured cpithelial autografts; R T, rOOI11 tcmpera­ Patients A 5-ycar-old girl (R.n.. . ) and a 6-ycar-old boy (S .K.) received ture; DEj, denno-epidermal junction. CEA as primary treatment of burns (full thickness burns of 65'X. and 62% of

0022-202X/96/S10.50 • Copyright 1996 by The Society fo r In vestigative Dermatology, Inc.

1090 VOL. 106, NO. MAY 1996 M IC ROFIBRIL FORMATION IN REGENERATING SKIN 1091

the total body surface area). A 14- yea r-old boy (D.G.) received CEA for Collagen VII Flbrllll" pos t burn revision. In R..R. and S.K., CEA were grafted onto subcutaneous fat after necrosectomy. 1.11 D.G .. CEA were grafted onto a thin layer of dermal remnants after dermabrasion of hyper pigmented areas in the face and removal of hypertrophic at the neck. Scalp excisates (4 X 1.5 cm) w ere taken under local anesthesia for the preparation of CEA. CEA sheets were prepared by a commercial laboratory (B iosurface Technology NS Inc., Boston, MA). For histological follow up after grafting, 4-mm punch biopsies were taken under general or local anesthesia after informed consent of the patients/parents. T he bioptic sed es from each patient was obtained within an area not larger than l O X 10 cm. T he biopsies were taken from the le ft submandibular angle (D.G.), the ventral left thigh (S.K), and the ventral right th.igh (11... .11....). Additionally, time-matched biopsies from mesh.ed, split-skin-thickness skin grafts were available in 11....11.... (dorsal right thigh). Biopsy time points covered days 5-9, 13-14, 21 - 28, 62- 72, and 120-140. In addition, a 17-month (510 d) biopsy was available in D.G. A rime point zero biopsy was taken in all three children from the prepared 7d wound ground immediately prior to transplantation. T he immunohistolog­ ical findings were compared to those obtained from normal skin of 11... . 11.... and S.K. (normal skin of D.G. was not available), as wel1 as to those obtained with non wounded skin from seven adults.

Indirect Immunofluorescence Microscopy Cryostat sections (5 /-un) were stored at - 70°C prior to immunohistochemical analysis. Every 20th section of each series \vas routinely stained w-ith henlatoxyHn and eosin and van G ieson's elastica stain . For immunofluorescence analysis, sections were fixed in acetone at - 20°C for 10 min and air-dried. Unspecific binding sites 7d were blocked by a 30-l1Iin incubation at 11... T in phosphate-buffered saline, pH 7.35, containing l 'y" bovine serum albumin and 5% of normal swine serum/ 5% normal goat serum (both Dako, Glostrup, Denmark). T hi s solution was drained oU:' and replaced by an afri llity-purified polyc1onal ra bbit anti-col1 agen VII antibody recognizing the triple helical region [14] and monoclonal mouse anti-fibrilli l1 antibody 20'1 diluted 1: I O. Aft.er incub ation ovemight at r c in a humid chamber, sLid es were washed 3 X 5 Figure 1. Fibrillill is early expressed at the d ermo- epidermal junc­ min in phosphate-buffered saline, and a mixture of swine anti-rabbit­ tion of regenerating skin. Double immunofluorescence demonstration fl uorescein isothi ocya nate (Dako) 1:30 and goat anti-mouse-Texas R.ed of collagen VII and fibril1in in c''Y0stat sections of normal skin (NS, a and b) crackson [mmunoresearch, West Grove, PA) I :50 was appLied fo r 1 h at and regenerating skin 7 d after grafting of autologous keratinocytes. C ;lI1d d RT. Elastin was visuali zed with a monoclonal mouse antibody to insoluble represent the biops)' fro m patient D.G., e and I fi'olll patient R.R. N ormal elastin (Sigma Chemical C o., St. Louis, MO) for 1 h at RT diluted 1:50. skin shows a brilliant linear Auorescence for co ll agen VLI at the denllo­ Becau se some of the CEA biopsies were fi'agi le, we applied this antibody epidermal junction. Fibrillin shows overlapping distribution w ith coLlagen throug hout the study w itho ut. tryptic pretreatment of the sections. T he same V II in this region bur is also present in a microfibrillar system appearing as antibody was applied in double immunofluorescence studies in com bination candelabra-like structures radiating into the region. with the rabbit polyc1onal anti-fibril1in antibody 5507 (diluted 1: I 00). In T he handles of these candelnbra (ar,."wh eads) connect fine microfibrils \vith this case, bound elastin antibodies were visuali zed with goat anti-mouse­ the horizontall y undulating coarse s. In both 7-d-old autografts, fl uore scein isothi ocya nate, 1 :20, whereas the polyc1o nal fibri ll in antibody co ll agen V II appears already along the dermo-epiden113 l junction mostly as was detected with donkey anti-rabbit-Texas Red, l :200. Preparations were a granular band. Note that basal kcratinocytes show cytoplasmic staining fo r mounted i.n Mowiol (Hoechst, Frankfurt-Hoechst, Germany) prepared in co l1 agen VII (c and e) which is absent in NS. Fibril1in apparently colocali zes Tris-HCl, pH 8.6, and photographs were taken on ASA HP5 black and with co l1 agen V II at the dermo-epidermal junction and appears as inter­ white plus fi lm (ll fo rd) using a Polyvar (Reichert-Jung, Heidelberg, Ger­ rupted zone inlInediately below the basal keratinoc)'tes (d and j), which do man y) epifluo[esccnce equi pment. Double immunofluorescence prepara­ not show in tracellular staining for fibri l1in . In addition. the neodennis, tions were viewed w ith an Axiophot (Zeiss, O berkochen, German y) and mostly consisting of a infiltrate. contains multiple foci of fi briILin photographed with T-Max P3200 black and whi te fillll (Eastman Kodak, syntllesis, appearing as fusiform fibrils (I) or microfib rill ar fragments (d) in Roch ester, NY). random orientation. Scale bar. 50 /-Lm.

Electron Microscopy Biopsies obtained 1]-0111 11....11.... were sp li t, and one-half was immersed in 4% glu taraldehyde in 0.1 M sodium cacodylate fo r 2 4-48 h at 7°C . Specimens were en block-osmicated, dehydrated, and was visu alized o n the h orizontally undulating e lastic fi brils of th e embe dded in EPON 812. Ultrathin sections were contrasted with uranyl reticular layer of the dermis (F ig 1). From this layer, single coarse acetate and lead citra te and viewed with a PhiLips EM 401 electron micr osope at an accelerating voltage of 60 kV. fibrillin-positive fibrils appeared to ascend into th e papillary region of th e dermis an d branched into several m ic rofibrils o f d ecreasing Confocal Laser Scanning Microscopy Sections of one control skin thickness projectin g toward the basement membrane zone . thus and the 17-month biopsy of G.D. were doubly immunolabeled for the forming struceures described pre viously as " cande labra-like" [9,10] simultaneous vi sualization of co l1 agen VII and fib ,"i l1in as described above. (Fig lb). A long the b asem en t m e mbrane, fibrillin immunoreactiv­ Confocal laser scanning microscopy was performed using a Leica (LLT. ity showed ove rl apping dist";bution with collagen VII (Fig 1a). In Heidelberg, FRG), T CS 4D scanner in conjunction with a DM IRBE inverted microscope (Leitz W etzlar, FRG). Images were obtained by the regen eratin g skin, a consistent tempora- spatial distribu tion of seque ntial optical sectioning using the ch:llmels fo r fluorescei n isothi ocya­ fibriUin WaS fo und (Ta ble I). Immunoreactiv e fibriUin alre ady nate and Texas Red, respectively. Processing and merging of the digitized appeared at day 5 (n ot sh own) and day 7 (Fig 1d and f) afte r pictures was achieved using the Imaris (Bitplane, Z Orich, Switzerl and) graftin g simultan eously with co llagen VII (Fig 1 c and e). At this software on a Silicon Graphi cs (Mol.l ll tain view, CA) Indigo 2 work station. time, it appeared conde n sed along the DE] as a thin, partiaJJy The micrographs were printed with a Fuji x Pictrography 3000 digital image g ranular. and interrupted b and, and apparently colo cali zed w it h prin ter (Fuji, Tokyo, Japan). coll age n VI! (Fig 1c and e). FibrilHn was not detected in the cytoplasm of basal ocytes, in contrast to co ll age n VIr (Fig 1c RESULTS and d). Starting at day 9, multiple foci of fi brilJin synth esis in the The Restoration of the Cutancous Microfibrillar Apparatus fibroblast infiltrate of the superficial n e odermis w ere evident as ALter Kcratinocyte Autografting Is Initiated Both At the granular aggregates and fusiform fibrils (Fig 2a). One month after P apillary and the Reticular Dermis In normal skin, fibrillin grafting, th e fibriJlin-positive b and appeared continuo u s, and indi- 1092 RAGH UNATH lOT Ai- THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. Expression of Fibrillin and Elastin in New Skin Developing From Keratinocyte Autografts

Region 1 w (5-7 d) 2 wk (1 3-14 d) 3-4 wk (21- 28 d) 2 m (62-72 d) 4-5 m (120-140 d) 17 m (510 d)"

Basement membrane z one Fibrillin Grall/band Gran/band nand/rootlets Band/ rootlcts Rootlets Rootlets and 2nd order mf Elastin Neg Neg N eg Neg/f.1.int band Thill band Rootlets and 2nd order mf

Superficial neodermis Fibrillin Fusiform mf Fusiform mf Fcw vertical m f Few 2nd order mf Randomly oriented Pcrpcndicul arly Communicating oricnted with ho ri zontal 3rd order m f Elastin Neg N eg Cell-associated Ccll-associated Pcricapill ary 2nd order mf Aggregates Aggregatcs Coat Deeper neodermis Fibrillin Fusifo r111 Inf, Long horizontall y randomly oriented undulating mf Elastin Neg Neg Granular aggregates Granular aggregates Horizontal mf Coarse horizontally fragments undulating mf

" O nly obtained in o ne child; " pcrpcndicuhllo" is defined as vertical projection frolll the plane of the basement membralle towilrd the dermis; " horizol1t:11" is defined as plane parallel to the basement rncmbr'lI1 c zone; neg = negative; gran = granular, mf = microfibril.

vidual fu sifo rm microfibrils became evident with a beguming grafts £i'om the same patient (Fig 31, illserl), w hich contained a horizontal orientation in the reticular neodermis (Fig 2c). Four to 11 0rmal DE] and displayed microfibrillar bundles radiating into the five m onths after grafting, all biopsies showed single fi brils proj ect­ lamina densa (Fig 3j; insert). Elastic fIbrils could easily be detected ing from the DE] perpendicularly toward the ncodermis. A vm-yi ng (Fig 3.D. number of these communicating microfibrils were cOlmected with the horizontall y undulating microfibrils of the dermal (neo)reticular Confocal Laser Scanning Microscopy Highlights the Archi­ layer (Fig 2e). Seven teen months after grafting, microfIbrils at the tecture of the Papillary Microfibrillar Apparatus T he anal­ DE] and all deeper zones appeared coarsened (Fig 2g) , but ys is of a total depth of 3.2 J.Lm confirmed that in nonnal skin the communicatulg ITli crofibrils still appeared reduced in comparison to fmest microfIbrils started exactl y in the same plane as collagen vn nomlal skin. T hey were always found, however, at prospective rete (Fig 4a). In contrast to collagen VII, fIbrillin was not continuously ridges frammg a buddulg papilla (Fig 2g and II) . distributed alo ng the basement m embrane and the region, bm appeared as footlets representing the insertion points of the micro­ Elastin Forms Fibrillar Structures by Deposition on Pre­ fi brils into the basement membrane. T he superior resolution of formed Fibrillin-containing Microfibrils Amorpho us elastin confocal laser scanning microscopy all owed the appreciation of aggregates were not noted in the upper neodennis before 1 m onth architectural details of the microfibrillar apparatus of the papillary after grafting (Fig 2d), whereas the deeper neodermis showed dermis. Several fine microfibrils originating from the basement fibrillar aggregates (not shown). Elastul was consistently present membrane united to form sillgle long microfibrils descending along the basement membrane zone and as pericapillary coating toward the reticular dermis. In contras t, 17 months after graftlllg 4-5 months after graftlllg (Fig 2.D. Anti-elastin antibodies stained the regenerated papillary microfibrillar apparatus appeared mark­ elastin faul t1 y on fibrillin-positive microfibrils in the upper reticular edly compressed. MicrofIbrils o ri ginating from the basement mem­ layer and more pronouncedly in the lower reticular layer of the brane were shorter in comparison with normal skin (Fig 4c and d, neodermis (not shown). Associatio n of elastm with fi brilllll-con­ compare also w ith Fig 2). Long descending microfibrils were tainin g microfibrils in the papillary neodermis was no t seen before sparse, but were always found to frame budding papillae (Fig 4r m onth 17 after grafting (Fig 211). and d, compare also with Fig 2g). Electron Microscopy Confirms Presence of Microfibrils in DISCUSSION Regenerating Skin At an Early Stage O ne week after graft­ ing, the lamin a dens a appeared continuous. T he region below was W e report the morphogenetic stages in the development of the very loosely structured and filled with thul. coll agen fibrils and 1 0 -12-nmmicrofibril system in regenerating human skin. Based ou fuzzy or globular material. Hemidesmosomes were often found our findings, we suggest a model, according to which micro fib rils where basal keratinocytes faced the lamina densa; are generated at the DE], develop downward w hile traversulg thr were rare and thin (Fig 3a). Occasionally, micro fi bril s could be prospective papillary dermis, and finally fuse with an independently identified (Fig 3"). T he papillary neodermis consisted of three formed horizontal microfibrillar web of the prospective reticular layers 72 d after grafting (Fig 3c): th e uppermost was loosely demlis (Fig 5). Furthermore, we show that the formation of packed and contained thin coll agen s and fuzzy m aterial. Here, fIbrillin m icrofibrils precedes the subsequent morphogenesis of the longitudulal fragments of microfibrils were conspicuous. T he mid­ elastic fiber. In tlus regard, it might be interesting to note that dle layer showed tracts of collagen fi brils with intemlingled micro­ elastin appears late in evolution. It is not found in invertebrates fibril bundles (Fig 3c and d) . T he lowest la yer was represented by (reviewed in [15]). In contrast, a homolog of mammalian fibrillim a dense infiltrate of , w hich frequently showed dilated has been recently identifI ed in j ell yfish [16]. It is therefore conceiv­ endoplas mic cisternae (not shown). T he papilla.ry dermis, 120 d able that o ur m odel of microfibrillogenesis as established from after grafting, was dominated by densely packed coHagen fIb rils; the regenerating skin (Fig 5) recapi tulates the phylogenetic order of DE] showed abundant anchoring fibrils and anchoring plaques (Fig appearance of fibrillin and elastin. According to Oll r model, elastin 3e). Microfibrillar bundles were now more difficult to detect in the first covers fibriUin-containing microfibrils of the reticular dermi. compact connective tissue. Elastin was not detectable. For compar­ and later those of the papillary dermis. Altho ugh it remains to b ison, we examined normal skin derived from meshed, split-skin established whether this model holds true also for the normal VOL. 106. NO.5 MAY 1996 MIC ROFIBIUL FORMATION IN I ~GENE RATIN G SKIN 1093

Flbrlllln Elastin

9 d

1 m

4m

17m Figure 3. Electron Inicroscopy reveals early presence of nticrol1- brils but not of clastic fibrils in regenerating skin. All micrographs show the papillary dermis and till! dermo-epidermal junction as character­ ized by hemidcsmosollles (HD) of the basal kcratinocytes and the lamina dens" (LO) of thc basement membrane. n. 7 d aftcr grafting. the lamina densa is continuous, and sparse thin anchoring fibril s (AF, closed nlTo'I'/lCnds) arc visible. The collagen fibers of the papillary dermis arC loosely pncked. b, Figure 2. The organization of the microfibrillar apparatus in occasionally, microfibrils can be identified at tlus early sta ge (opell am/II/­ regenerating skin precedes its investment with elastin. Double hends). c, 72 d after grafting, anchoring fibrils are more numerous bur still immunofluorescence demonstration of fibrilJjn (n , c, e, s) and elastin (II. d,): thin (clllSed nlTOlI'hends). Microfibri ls (MF) arc evident as bundles between It) in skin rcgenerating from keratinocytc autografts. Only papill ary and the collag"n fibers and as fragments below the lamina densa. d, characteristic upper rcticular neodermis arC shown. Nine days after grafting, fibrillin cross section profilcs of microfibrillar bundles arc prescnt in the reticular immunoreactivity is concentrated along the dermo-epidermal junction and ncodcrmis. e, 4 months after grafting, abundant AF arc present, which show bas a streaky appearance in thc papillary dermis (n), and clastin immunore­ cross-striation and connect to anchoring plaques (Ar). Bundles of microfi­ activy is absent (b). After 1 month, individual fibrillin microfibrils arc brils arc visible (MF).): normal skin obtained from a meshed. split-skin graft discemiblc which arc randomly oriented in the papillary region (c) but are from the same paticnt (R.R .) shows normal anchoring fibri ls and microfi­ horizontally oricnted in the dcep reticular neodermis (not visible). Elastin is brils. T hc ;lIsl:,1 shows two microfibrillar bundles runn.ing into the hunin" not detectable (d). After 4 months, fibrillin occupies a narrow zone along dens" (LO, nlTOI//S ). In the reti cular dermis microfibrils appear cmbedded in the dermo-epidermal junction which scnds the first fcw microfibrils towa.rd elastin, the typical aspect of an elastic fiber. Sr o/c "Ol"S , 1 00 n)11. the neodermis (e). The reticular layer ofthc ncodermis shows a well defined layer of horizontally undulating elongated microfibril s. Elastin is barely visi ble either as amorphous aggregates surrounding papillary capillaries or on microfibrils predominantly of the reticular dermis (1). After 17 months the microfibt;l1ar apparatus ha s ga in ed thickncss (g) and an extensive isodesmosin) rathe r than e lastin itself [n addition. proteolyti c association of elastin with the microfibrillar structures of the papillary and ac tivity might influe n cc the amount of elastin incorporated into upper rcticular neodcrmis is evident (h). Note the vcrtical microfibrillar elastic fibrils during skin regeneration. strings at eithcr sidc of a ncwly forming papilla (g. nrrowhends) . Senle hnr, 50 The architecture of the c utaneous microfibrillar apparatus in p.m. maturc skin and its morphogene tic stages in regen e rating skin raise questions rcgarding its function. On the basis of our o bservations. we would Like to propose the term " first ordcl" microfibrils" for developme nt of human skin, it is consistent with data showing that those fine fibrillin-positive structures that evolve from the base­ fibrillin-microfibrils are extractable earli e r than elastin during the ment m embrane zone. "Second order microfibrils" are then development of fetal bovine skin [17] . In a long-term follow-up formed by fusion of several first order mic rofibrils. T h ese second study regarding the development of kerati.nocyte autografts, the order microfibl;ls communicate w ith "third o rder microfibrils," first appearance of short and slendcr elastic fibers was reported to wh.ich undulate horizontally in the re ti cular layer of the d e rmis. occur 2-3 years after grafting and its association with capillaries not The first and second ordcr mic rofibrils represent a vertical traction before 4-5 years ]4] . In contrast, w e observed elastin already 4 system, whic h is an chored at the basem ent m embran e at one side months after grafting surrounding papillary capillaries, and 17 and at h orizontal third order mic roflbl'ils on th e oth er side. T his months after grafting clearly associated vvith microfibrils. This vertical system traverses the papillary layer of the de rmis thereby substantial difference might be explained by the higher sensitivity of spanning approximately 150 p,m (if not more) in 110rmal skin. It is the immunohistological detection of e lastin used he re as opposed to p lausible that tlus system a Uows the papillary de 1"11lis to slide on the the histochemical detection of e lastin (elastica stain), which might reticular dermis to a limited exten t. The investment with elastin be specific for the later occurring elastin cross-links (desmosin, would the n lend recoil properties to the microfibrillar apparatus. In 1094 R AGHUNATH ET AL THE JO UllNAL O F INVESTIGATIVE DE I~ATO L OGY

Figurc 5. Model of thc bifocal development of the microfibrillar nctwork in skin rcgcnerating fr0l11 keratinocytc autografts. This seri es of sketches show a p'lrt o f basal kerarinocytes and the papillary Figure 4. The cutaneous Inicrofibrillar apparatus inserts into the neodermis bordering to the reticular layer of the neodermis. A, microfibrils basement Inembrane and shows a specific configuration in areas of arc formed immediately adjacent to basal keratinocytes al ong the prospec­ papilla formation Normal skin (a,b) and skin after 17 months of tive dermo-epidermal juncti on and simultaneously at the deeper neodermis. keratinocyte autografting (c,d) was double stained for collagen VII B, the fu siform microfibrils at the dermo-epidermaljuncti on fom1 fi rst order (greell j'""yesce"ce) and fibrillin (red jl,,,,resccllce) and analyzed by micro fibrils which fus e and fo rm second order micro fibrils (C) proj ecting confoca l laser scanning. Each micrograph represents an extended fo cus downward. where a la yer of horizontally o ri ented third order mi crofibrils is vi ew calcul ated from I 0 section pl anes (increment 0.320 /Lm). a, tltis simultaneously emerging. The second order microfibrils get into contact tangential section reveals points of origin of fine microtibrils appearing as with third order microfibrils (D) and thus connect the lamina densa with the footl ets (small an''' '''s) in the plane of coll agen VII. The absence of color reti cular la yer o f the neodermis. Sketch E hypothesises that focal expansion mixing suggests that colla gen VII and fibrillin do not share the same of the upper dermis leads to a "denting out" of the epidermis. This initiates structures. h. in this longitudinal section the architecture of the microfibrillar papilla formation and sets the microfibrillar system under te nsion which in apparatus suggests a discrimination of first and second order microfibrils: the turn holds the prospective rete ridges down. Finally. the late st'lge of this al7'()IIJlr rads designate points where several first order microfibrils running microfibrillar system 'lfter investment with elastin is given in F. from the basement membrane unite and thus form a second order micro­ fibril. c. regenerating skin 17 months after gra fting of autologous keratino­ cytes shows compressed and plump tirst and second order microfibrils closely below the basement membrane region represented by collagen VII. drome locu s [1 8]. B ecau se the alfecte d mice d o not show the Two second or third order microfibrils pulling do wnward frame a "bud­ Marfan syndrome phenotype , an unusu al fibrillin mutation can be ding" papilla (/nr;gc ar,.ow) . d. the sa me section contain ed a region with a expec ted. more adva nced papilla fo rmation. Here, second and third order micro fibri ls W e found, to our surprise, that the vertical (transpapiUary) part arc coarser and elongated . T he rete ridge depicted here is surrounded by a of the c utaneous microfibrillar apparatus is primarily gene rated at basket-like network of coarse second order micro fibriJs . It is conceivable the D E] despite a massive fibrob last infiltrate that accounts fo r the th at increasing tensional forces in this paft of the skin cause an invagination of the interpapillary space. The epidermis (E) is not visible. Scale bar, 25 /Lm . major mass o f the papillary n e ode rmis during the first 1-2 m o n ths. The re markably close spatial relationship b e twe en basal keratino­ c ytes and fibrillin immunoreactivity would, in analogy to what is known about collagen VII [6], sugge st that, besides m esen chym e­ addition to ex ogenous sh ea.ring forces, focal e xpansion of the uppe r d erive d cells, basal ke ratinocytes arc al so involve d in fibril1in n eode rmis would set the ve rtical trac tio n system (first and second synthe sis, although the y showed no intracellular staining for fibril­ orde r mjcrofibrils) unde r e ndoge nous (ve rtical) te nsion and could lin as opposed to colJagen VII . There is, howe ve r, evide n ce on both give rise to p apilla formation. [n thjs case, the fibrillin microfibril cDNA and protein leve l that c ultured ke ratinocytes are cap able of syste m w o uld hold the prosp ective re te ridges down like strings o f fibrillin synthesis (Raghunath e/ ai, unpublish e d). Finally , tlle a parachute and thus establish the papilla/re te ridge patte rn. At sep arate formation of microfibrils at the D E] raise s questio ns about present, w e arc not aware of data concerning th e d e velopme nt of the role of preforme d extracellular m atrix guiding microfibril the p apilla/re te patte rn in M a rfan patients b esides clinical and formation and possible linking prote ins mediating the insertion of histological signs of d e rmal fragility. [t is worth m entioning that the mjc rofibrils into the baseme nt m e mbrane. The furthe r e lucidation tight skin mouse phe notype could rece ntly b e linked to the murine of this phenomenon will certainly augme nt the unde rstan ding of fibrillin-l g en e locu s, the human equivalent of the Marfan syn- the fun ctional anatomy of the skjn as well as provide m o re insig hr VOL. 106, NO.5 MAY 1996 MI CR.OFIBR.IL FOR.MATION IN R.EGENER.ATING SKIN 1095

intO the development and formation of l11i crofibrils III other body 7. Low fN . Microfibrils: fine fi larnctl l:Ous components oft'hc ti ss ue spa ce. Aria! Ref tissues. 142:131 -1 37. 1%2 8. M :l ddox OK , S~k:1 i LY. Keene DR. G I:Ulvillc It \ XI: Connective ti SS li C microfi­ bril,. } Billl C helll 264:2138 1-21385. 1989 9. Flcischmajcr I~ ! J:1 cobs L. Schw3rtz E. Sakai LY : Extracclluh'lr ll1i crofibrils (10 Tire jibrillill allli/JOdies lIIere SCllerollsly g illell by Drs. Maurice G"'!Fc), (Mnl) 210; nlll in dhl1nc.tcr) ~rc increased in localized ;H1,d systemic skin. LA b Olllahn, NE) olld C (/)' Kiell)' (PAl, 5507; MnJ/chesle,; UK). I,Ve are ;lIdeflled 10 IIII'es' 64:79 1-798. 1991 N icole fI'fiihllleilll , L. Blllgcr, nllli S II ,.(//lIIe S'(/Ilb/i for (//11110rk, nllli '" !I!{s. !'IJ!iillcr '10. Sakai LY. Keene DR., Engvoll E: Fibrillin, a new 350 kD glycoprotein. is a component of extro ccllular microfibrils.} C ell BioI 103:2499-2509. 198 6 alld Ms. Scherrer-Herll;ch (DC(I(//7I11 e,II 4 Derlllnlolo.~)', Ulli llers il), of Ziirich) JII' 11 . Pycritz It.E: T he Marf:1 11 syndro me . III : Royce PM. Stcinnwnn B (cds.). Co"" Ntil1c Ille prepnrnlioll of C1'),oSlnl scclioJ/s. T he i11llrtillable hel(l of Ms. L ,'ilili (ElecrnJII Ti.(wt' fUul1u H(',.j /tJlJ/(~ Di... ,Jrdt'l"S, iVlolcm/a,., Gl'tII.:/ir alJd JHt'diral A .• pt'CIS, Wilcy­ microscopic Celllmi Lnbomlol")" Ziirirh) is dce(ll)1 np(I ,winrcd. This /IIork /IIns Liss. New York, 1993. pp 437-468 SIIpp0l1ed b)' gmllis frOIll ,h" Silliss Nnl;oJ/ al SeicJ/ee FCllllldnlioJ/ 10 B.S. (32- t 2, R.3ghlln~th M. SlIpcrri-Furg<1 A. Godfre y rv1. Stcil1l11:1l1n 13: Decrcased deposition 27884.8911 + 2) oJ/d ille GenJ/nJ/ Sci""ce FO/ l/l dnrioJ/ (DFG gm J/I Ra 44713-1). of fihri llin and dccorin in ncon:Hal Marf.,n syndrome fi brobla sts, H"III Crllet 90:5 11 -5 15. 1993 13. Godfi'e)' M. Raghunath M. C isler G. Bevin s C L. DcPaepc A. DiRocco M. Gregoritch j . 111l 41iz ll n li K. Kaplall P. Kuroki Y . Silberbach M, Supcrti-Furga A. vall T hicnc n M-N, Vctter U. Steinmann .8: Abnormal morpholob'Y of R EFERENCES fibrillin 1I1icro fibrils in fibroblast cul turcs fr o m paticllts wirh neonatal Marfan syndrome. A lii} 1'.,11"' 14 6:14 14-1421.1995 I. Gallico GG. O'Collllor NE. Compton CC. Kehinde O. Green 1-1: A pcr1l1an em '14, Bruckncr- Tudcrman L. Schnvdc.r UW. \V inrerh41lrcr KH. Bruckner P: Tissue covcr;\gc orb r g~ burn wounds with in1tologo\\s cultured hml\"n epithelium. N form of type VII collagen human skin and dermal fibroblasts in culrures. E II.~I}Mc£/3 11 :448-45 1. 1984 r;0111 2. Odcsscy R: Multi ccll rcr experiell ce with cultured epithe li al :1 utog-raft for trea t­ [Jllr} BiorlwlII 165:607-6 11 . 1987 ment of burns. J Hum Cn n' Rd",lJil ·13: 174 - 180, \1)92 15, Rosenbloom J. Abram s WH.., Mecham R : 4: The el:,stic 3. Siwy .13K: Cultured epid ermis: Indiana University Medic;,l Center's experi ence.) fibe r. PASEB} 7: 1208-12 18, 1 9~3 BII'" Care Rcllllbil 13: 124 -'126. 1992 t 6. Rcbcr-Mlillcr S, Spissing:cr T. Sdluchcrt P, Spring j, Scllmid V: An cxtracellul ar 4. Compton CC. Gi ll )M. Bradfo rd DA. R.ega uer S. Gallico GG. O'Connor NE: l11:ttrix pro tein ofjell ylish homologous to 1l 1~l11ll1alia ll fibrillin form s different Skin regenerated from cultured epithelial