complications associated with bovine corneal endothelial cell … native endothelium had been...

Complications associated with bovinecorneal endothelial cell-lined homografts

in the catCharles F. Bahn, Donald K. MacCallum, John H. Lillie,

Roger F. Meyer, and Csaba L. Martonyi

Cultured bovine corneal endothelial cells were subcultured onto feline corneas from which thenative endothelium had been mechanically removed, and, transplanted into cats via penetratingkeratoplasty. Although the transplants remained thin and. clear in the immediate postoperativeperiod, correlative clinical and morphologic analysis disclosed evidence of a host responsedirected against the heterologous endothelium by the ninth postoperative day. Eyes withrotational autografts or transplanted homografts did, not disclose evidence of a similar hostresponse. (INVEST OPHTHALMOL VIS SCI 22:73-90, 1982.)

Key words: corneal endothelium, cell culture, corneal transplantation,transplant rejection, retrocorneal membrane

The maintenance of corneal transparency isdependent on a population of corneal endo-thelial cells sufficient to affect deturgescenceof the corneal stroma.1 Reduction in thenumber of corneal endothelial cells in thehuman,2 rhesus monkey,3 or the domesticcat4—species with a limited endothelial cellregenerative capacity—results in a compen-satory hypertrophy of the remaining cells andretention of an intact endothelial cell mono-

From the Department of Ophthalmology, University ofMichigan Medical School, Ann Arbor (C.F.B.,C.L.M., and R.F.M.), and Department of Anatomyand Dental Research Institute, University of MichiganMedical School and School of Dentistry, Ann Arbor(D.K.M. and J.H.L.).

This research was supported by grants from the NationalInstitutes of Health (DE-02731), Research to PreventBlindness, the Michigan Eye Bank Research Fund,and the Office of the Vice President for Research Uni-versity of Michigan.

Submitted for publication July 2, 1980.Reprint requests: Charles F. Bahn, M.D., M.S., De-

partment of Ophthalmology, Box 012, University ofMichigan Hospital, Ann Arbor, Mich. 48109.

layer. If the endothelial cell population isgreatly reduced, the effectiveness of the de-turgescent mechanism is lost and stromalthickening and opacification occur as the cor-nea hydrates.5 Epithelial bullous formationwith recurrent epithelial erosions, stromalscarring, and blindness are the final result ifan adequate population of endothelial cellscannot be restored. Clinically, endothelialcell replacement is achieved by homotrans-plantation of a healthy donor cornea.

Recent experiments have demonstratedthat corneal transplants bearing culturedhomologous6' 7 or heterologous8 corneal en-dothelial cells remain clear and appear tofunction normally. These experiments haveimportant clinical implications because theability to use cultured corneal endotheliumto repopulate corneal surfaces prior to penet-rating keratoplasty would increase the num-ber of corneas suitable for transplantation aswell as provide a means of standardizing theendothelial population of donor preparations.The purpose of this article is to report the

0146-0404/82/010073+18$01.80/0 © 1982 Assoc. for Res. in Vis. and Ophthal., Inc. 73

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74 Bahn et al.Invest. Ophthalmol. Vis. Sci.

January 1982

HETEROLOGOUS ENDOTHELIAL TRANSPLANTS

Fresh Feline Corneo Donor Fresh Bovine Endothelium Donor

Endothelium ,0 \ .Denuded and \ j h J/Oiscorded ^ = = * 5 '

8 mm Button

PenetratingKerotoplasty(Living FelineRecipient)

Fig. 1. Schematic flow diagram illustrating thetechniques used to produce bovine corneal endo-thelial cell-lined corneal transplants.

results of a series of feline corneal transplantsrepopulated with bovine corneal endothe-lium that had been grown in monolayer cellculture. Clinical and morphologic evaluationof the transplants demonstrated a host re-sponse directed against the grafted heterolo-gous endothelium—a complication that hasnot been previously reported.

Materials and methods

A schematic illustration of the sequence used toproduce heterologous cell-lined feline homograftsis presented in Fig. 1.

Bovine corneal endothelial cell culture. Primaryendothelial cell cultures were established frombovine eyes that were obtained within 6 hr afterdeath. The eyes were trimned of excess tissue andwashed with running tap water prior to sequentialrinsing of the corneas and adjacent scleras withantibiotics (Neosporin Ophthalmic Solution; Bur-roughs Wellcome Co., Research Triangle Park,N.C., and Aureomycin [50 jug/ml]; Lederle Lab-oratories, Pearl River, N.Y.). Corneas were ex-cised with a 2 mm scleral rim and placed in plastic

cups fabricated to conform to the corneal contour.Endothelial cells were dislodged with a siliconerubber spatula after a 5 to 7 min incubation at 37°C in sterile, pH 7.5, Ca++- and Mg++-free Earle'sBalanced Salt Solution (EBSS) (Gibco, Grand Is-land, N.Y.) containing 5 mM ethylene diamino-tetraacetate (EDTA) and 0.25% trypsin (1:250)(Difco, Detroit, Mich.). Cells from one set of eyeswere collected in Minimum Essential Medium(MEM) (Gibco) with 10% calf serum containing 50yug/ml gentamicin (Garamycin; Schering Corp.,Kenilworth, NJ.) and 160 U/ml nystatin (Myco-statin; E. R. Squibb, Princeton, N.J.). The approx-imately 0.5 x 106 cells, when cultured in 25 cm2

flasks maintained in 5% CO2 and air, reachedconfluence in 6 to 8 days. Cultures were fed every3 days with the same medium except the nystatinwas omitted.

At confluence the endothelial cells form ahighly ordered monolayer (Figs. 2 and 3) and con-tinue to produce a basement membrane (Fig. 4)that contains Type IV collagen9 and one or moreglycoproteins common to basement membranes.10

A comprehensive report of bovine corneal endo-thelial cells in culture and the deposition of an invitro basement membrane has been submitted forpublication."

Graft preparation and heterologous endothe-lial cell repopulation. Fresh donor cat eyes wereobtained from animals sacrificed by an overdose ofintravenous sodium pentobarbital. The eyes wererinsed in sterile saline and the corneas and adja-cent scleras were flushed with antibiotic (Neospo-rin Ophthalmic Solution). Scleral rim preparationsof the cornea were placed in plastic cups moldedto the contour of the cornea. The endothelial sur-face was covered with MEM containing 5% de-xtran (D-MEM; Sigma Chemical Co., St. Louis,Mo.) (M.W. 40,000) and buffered to pH 7.3 with15 mM HEPES, 10 mM TES, and 10 mM BES(Calbiochem, La Jolla, CA). The native endothe-lial cells were removed by gently swabbing theendothelial surface with a moistened cotton-tipped applicator. After a thorough rinse inD-MEM the denuded corneas were kept in theplastic cups during the initial stages of repopula-tion with cultured bovine corneal endothelium.

First or second subcultures of bovine cornealendothelial cells were removed from culture flaskswith 5 mM EDTA and 0.125% trypsin as de-scribed below. The action of the trypsin was stop-ped by adding the cell suspension to an equal vol-ume of D-MEM containing 10% calf serum. Analiquot was taken for cell counting, and the re-


Volume 22Number 1 Heterologous cell-lined corneal homografts 75

main ing cells were centrifuged and subsequentlywashed once in serum-free D-MEM. The cellswere diluted with serum-free D-MEM to yieldapproximately 1 X 105 cells/60 fx\. The 60 /A in-oculum was added to the denuded corneas and thepreparations were incubated in a humidified at-mosphere for 2 hr. After four gentle rinses inD-MEM to wash off nonadherent cells the corneaswere incubated for an additional 12 to 18 hr in 10ml of D-MEM containing gentamicin (100 /Ug/ml)and amphotericin B (4 /ig/ml). The addition ofthe dextran to the MEM effectively minimizedstromal swelling.

The optimal protocol for subculturing bovinecorneal endothelial cells onto feline homograftswas experimentally determined and found to bedependent on the status of the particular mono-layer culture. In most instances endothelial cellswere removed from flasks with 0.125% trypsin and5 mM EDTA 24 to 48 hr after subculturing at a 1:2split ratio, a process that both facilitated detachingcells from the flask bottom (2 min at 20° C) andenhanced early spreading of the cells onto Desce-met's membrane. Cultures that had been conflu-ent for 1 to 2 weeks required incubation at 37° C in0.25% trypsin and EDTA for 5 to 7 min to achieveefficient cell detachment. We believe the 1 to 2week postconfluent cells attached with almostequal efficiency as the recently subcultured cellsdescribed above, but these cells required a muchlonger period of time (10 to 12 hr) to spread ontothe feline Descemet's membrane. The signi-ficantly lower amount of trypsin (0.005%) and 10mM EDTA used successfully by Gospodarowiczet al.12 required a 60 to 120 min incubation at37° C before cells began to detach in our systemand therefore were not used for the subculturingof endothelial cells onto feline corneas.

In vitro specular microscopy, alizarin red stain-ing1 (Fig. 8, a), and scanning electron microscopyof representative feline corneas from which theendothelium had been removed by swabbingdemonstrated an infrequent remaining brokencell; however, the membranes appeared remark-ably free of cells considering the nature of the de-nuding technique. Similar studies of the repopu-lated grafts indicated approximately 50% to 60%(Figs. 5 and 8, b) of the attached cells had begun tospread onto the surface of Descemets membraneat 2 hr and that a complete, although irregularlyarranged (when compared with the native en-dothelium), monolayer was formed by 12 to 18 hrafter adding the cultured bovine cells (Figs. 6, 7,and 8, c).

Tntiated-thymidine labeling of heterologousendothelial cells. Primary bovine corneal endo-thelial cell cultures were subcultivated at a 1:2split ratio; 24 hr later they were labeled with 12.5fjcCi (2.5 yaCi/ml) of :!H-thymidine (methyl-;iH-thymidine, specific activity 71.8 Ci/mmol, NewEngland Nuclear Corp., Boston, Mass.) in MEMwith 10% calf serum for 24 hr. The medium con-taining !H-thymidine was decanted, and the cul-tures were washed five times with EBSS beforebeing cultured in MEM with 10% calf serum for anadditional 24 hr. (This protocol results in nuclearlabeling much greater than that desired when thelong-term survival of grafted endothelial cells is animportant consideration. These particular heavy-labeling conditions were chosen for this study todocument the bovine origin of the endothelial cellslining the graft even in the face of further celldivision.) The labeled endothelial cells were sub-cultivated onto denuded feline corneas by thesame protocol as described above. Two labeledcorneas were fixed in alcohol-formalin- acetic acidas controls at the time of surgery (2 hr seeding, 12hr culture). Four labeled corneas were trans-planted as described below. Nine days postopera-tively the animals were sacrificed and the anteriorchambers of eyes bearing transplants were per-fused with fixative. A 3 to 7 mm wide strip was cutacross the corneal equator, and Descemets mem-brane was dissected free, carrying with it as littlestroma as possible. The wound margins separatedin all cases so that complete equatorial strips couldnot be mounted. The resulting host andheterologous cell-lined strips were dried downonto gelatin-coated slides. The slides were coatedwith NTB-3 (Eastman Kodak Co., Rochester,N.Y.), exposed for 7 days before being developedin D-170, fixed, washed, and stained with hema-toxylin.

Feline keratoplasty. Five sets of animals werestudied. (1) Rotational autografts were used to con-trol the surgical procedure. (2) Homografts (de-rived from either freshly enucleated eyes at thetime of surgery or from corneas cultured underidentical conditions to those used for heterologouscell-lined grafts) were used to simulate the homo-graft procedures employed in human cornealtransplantation and as a control for the effects ofculturing corneas prior to transplantation. (3) Ho-mografts from which the native endothelium hadbeen removed and then maintained in culture for24 hr served as controls for recipient endothelialregeneration and other phenomena, especiallyimmune phenomena, that might have resulted


76 Balm et al.Invest. Ophthalmol. Vis. Sri.

January 1982

Figs. 2 to 7. For legends see facing page.


Volume 22Number 1 Heterologous cell-lined corneal home-grafts 77

from the homograft stroma or Descemet's mem-brane. (4) Homografts lined with heterologous cul-tured bovine corneal endotheliuni representedthe principal experimental series. (5) Homograftslined with bovine corneal endotheliuni (one eye)and rotational autografts (contralateral eye), someof which were treated postoperative]}' with top-ical steroids, were used to further study thehost response to the heterologous cell-lined ho-mografts.

All animals were examined prior to surgery andthose with significant signs of ocular infection wereexcluded from the study. Cats used as cornealdonors or as transplant recipients in short-term (24hr) experiments received no treatment on arrival.Cats used for 2 week experiments received oraltetracycline, intravenous fluids as needed, anddistemper vaccine upon arrival. Care for the gen-eral health, housing, and maintenance of the ani-mals was provided by the Unit for LaboratoryAnimal Medicine.

Except for 300 mg of aspirin given orally 8 hrbefore surgery, the animals were fasted for 12 hrprior to keratoplasty. Heparin (1000 U) was admin-istered subcutaneously 1 hr before surgery. An-esthesia was preceded by the intramuscular in-jection of 1 mg/kg of xylazine (Rompun; CutterLaboratories, Berkeley, Calif.) and subcutaneous

atropine sulfate (0.4 mg/kg). Ten minutes later 8to 10 mg/kg of ketamine (Vetalar; Parke-Davis &:Co., Detroit, Mich.) was administered intramus-cularly. This technique provided surgical anes-thesia for approximately 20 min. Additional intra-muscular ketamine (5 to 8 mg/kg) was adminis-tered during the operative procedure as necessaryto maintain an adequate level of anesthesia.

While the recipient eye was being prepared forsurgery, the donor cornea with a rim of sclera wasplaced endothelial side up on a Teflon block. Withan 8.0 mm trephine the donor corneal button wasexcised from the central cornea in a punch fashion.One button from each donor pair was placed in atransparent plastic chamber filled with D-MEMwhere it was examined and photographed with amodified laboratory specular microscope.1' Theremaining corneal button was prepared in anidentical fashion except that it was not examinedby specular microscopy.

The anesthetized cat was taped to a woodenoperating frame in the supine position. The unop-erated eye was taped closed, and a plastic surgicaldrape was applied to the eye selected for kerato-plasty. Silk sutures were placed through the lidsfor retraction, and a pediatric wire lid speculumwas used to retract the nictitating membrane. Silktraction sutures were placed through the episclera

Fig. 2. Phase-contrast micrograph of second passage bovine corneal endothelial cells 5 daysafter subcultivation. The cells, some of which are binucleate, form a highly ordered monolayer.(X425.)Fig. 3. Scanning electron micrograph of a culture comparable to that depicted in Fig. 2,emphasizing the close apposition of the endothelial cells. (X1600.)Fig. 4. Transmission electron micrograph of an endothelial cell 3 weeks after subcultivation.The cell has deposited a basement membrane (BM) on the culture flask surface (indicated bythe thin electron opaque line at the bottom of the micrograph). (x32,400.)Fig. 5. Scanning electron micrograph of bovine corneal endothelial cells 2 hr after subcultivat-ing them onto a denuded feline cornea. The cells illustrated remained attached to Descemet'smembrane through four gentle rinses in medium, but only a few cells have begun to spreadonto the membrane surface. As described in the text, this type of attachment was characteristicof cells subcultivated after having been confluent for 1 to 2 weeks. (X650.)Fig. 6. Scanning electron micrograph of bovine cornea! endothelial cells 14 hr after subcultiva-tion onto a feline cornea. The cells that had attached during the initial 2 hr of subcultivation(Fig. 5) have subsequently spread onto the surface of Descemet's membrane, forming anintact, irregularly arranged cell monolayer. When recently subcultivated endothelial cellswere used to repopulate grafts, extensive spreading onto the graft surface, as illustrated here,was evident at the end of 2 hr (see also Fig. 8, b). (X1000.)Fig. 7. Scanning electron micrograph of native feline corneal endotheliuni demonstrating thehexagonal (or pentagonal) arrangement characteristic of native corneal endothelium. Comparethe arrangement of these cells with the subcultivated bovine cells in Fig. 6. (X1050.)


78 Bahn et al.Invest. Ophthahnol. Vis. Sd.

January 1982

8a b cFig. 8. Feline corneas stained with alizarin red S immediately after removal of the nativeendothelium (a), 2 hr after adding bovine cells (b), and 12 hr after incubating a specimensimilar to that illustrated in b (c). (Approximately twice actual size.)

superiorly and inferiorly for fixation of the globe.The anterior chamber was entered through thecentral cornea with an 8.0 mm trephine. Heparin(1000 U/ml in balanced salt solution without pre-servative) was immediately introduced into the an-terior chamber to prevent fibrin formation. Thehost corneal button was excised with curvedcorneal scissors. In rotational autografts the cor-neal button was rotated 180 degrees and suturedinto position with interrupted 9-0 monofilamentnylon sutures. In homografts and heterografts theappropriate donor corneal button was transferredto the recipient bed and sutured into position in anidentical fashion. Throughout the surgical proce-dure the anterior chamber was frequently irri-gated with heparin. At the end of the surgical pro-cedure the wound was examined for evidence ofwound leaks, and additional sutures were placedas necessary. Atropine drops (1%) and antibioticophthalmic ointment (Neo-polycin; Dow Chemi-cal Co., Indianapolis, Ind.) were applied to theeye at the end of the procedure. The. contralateraleye was subsequently prepared, and the appropri-ate keratoplasty procedure was accomplished.Surgery was performed with standard microsur-gical instruments and a Zeiss surgical microscope,with the surgeons performing alternate kerato-plastics. The average operating time was 60 min/eye.

At the completion of surgery the operated catswere placed in protective cages to recover fromanesthesia under close observation. Atropine drops(1%) and antibiotic ophthalmic ointment were ad-ministered to both eyes daily for 1 week. Animalstreated with steroids received 0.1% dexametha-

sone (Decadron; Merck, Sharpe & Dohme, WestPoint, Pa.) drops three times daily.

Clinical techniques of analysis. Penlight exam-ination of the anterior segment was performeddaily to evaluate external ocular inflammation,gross clarity of the donor and host corneas, depthof the anterior chamber, and integrity of thewound margin. Slit-lamp examination (and appro-priate serial photography), pachometry, and to-nometry (MacKay-Marg) were performed on anes-thetized animals weekly and at the time of sac-rifice.

Animals were sacrificed either 24 hr after sur-gery or between the ninth and fourteenth postop-erative days, depending on the experiment andthe status of the graft. The 24 hr period was usedto analyze the conditions imposed on the graft andhost cornea by the surgical procedure. The 14 dayperiod was selected because a preliminary animalseries indicated that significant pathologic changesin the heterologous cell-lined grafts began tooccur at this time (see Fig. 23).

Morphologic techniques of analysis. Trans-planted corneas were fixed by perfusing the an-terior chamber with 20 ml of 2.5% glutaraldehydein pH 7.3, 0.1M cacodylate buffer containing 4%sucrose and 2 mM CaCl2. The globe was then im-mersed for an additional hour in the same fixativebefore the cornea was removed and processed forsequential scanning and transmission electron mi-croscopy. This involved postfixation in buffered1% OsO4, dehydration in graded ethanols, andcritical-point drying in CO2. In most instances re-trocorneal membranes were partially removed bydissection when immersed in fixative to permit



Table I. Clinical summary of rotational autograft series

AnimalNo.

1

2

3

4

5

Transplant

Rotational autograft OU





Sacrifice(clays after surgery)

1

1

1

14

14

Corneal thick-ness (mm)

OD 0.62OS 0.65OD 0.60OS 0.65OD 0.60OS 0.65OD 0.62OS 0.62OD 0.50OS 0.65

Slit-lampappearance

Clear OU

Clear OU

Clear OU

Clear OU

Clear OU

Complications

—

—

—

—Replace inferior

suture

OD = right eye; OS = left eye; OU = both eyes.

Table II. Clinical summary of homograft series

AnimalNo.*

6

7

8

10

13

9

11

Transplant

Fresh OU

24-hr cultured OU

24-hr cultured OU

24-hr cultured OU

24-hr cultured OU

Denuded and 24-hrcultured OU

OD Denuded and 24-hr cultured

OS Rotational autograft

Sacrifice(days after surgery)

1

1

1

14

14

14

30


OD 0.70OS 0.70OD 0.75OS 0.70OD 0.82OS 0.80OD 0.58OS 0.65OD 0.70OS 0.70OD 1.1OS 1.4OD 1.4

OS 0.58

Slit-lampappearance Complications

Clear OU —

Clear OU —

Clear OU —

Clear OU —

Clear OU Replace suture

Severe stromal —edema OU

Severe stromal —edema

Clear —

OD = right eye; OS = left eye; OU = both eyes.

*Two animals (one with bilateral fresh homografts and the other with bilateral 24-hr cultured homografts) were exiuded from this seriesbecause of severe ocular infection that developed at day 9 postoperatively. The infection, which was documented by scanning electronmicroscopy, resulted in the profound decoration of both the host and graft endothelia with white blood cells. An additional animal wasexcluded because of wound dehiscence.

examination of the heterologous endothelia] cells.The specimens were sputter-coated with gold-palladium and were scanned. If the cornea was tobe studied subsequently in section, it was rinsedseveral times in acetone and propylene oxide andthen infiltrated with several changes of epoxy re-sin. After polymerization, the cornea was seriallysectioned at 60 (xm in thickness.w Areas of interestwere subsequently cut out for 1 jum thick sectionsand further trimmed and processed for transmis-sion electron microscopy.

Results

The corneal thickness of the nonstandar-dized cat population used in this study

ranged between 0.55 and 0.65 mm (average,0.59 mm; n = 20), and the intraocular pres-sure ranged between 26 and 40 mm Hg (av-erage, 30 mm Hg; n = 20). The observedwide variation in intraocular pressure wasprobably because of the depth of anesthesiaat the time the intraocular pressure was mea-sured. Twenty-four hours after surgery theintraocular pressure ranged between 6 and20 mm Hg (n = 6). At 9 to 14 days after sur-gery the intraocular pressure ranged be-tween 14 and 30 mm Hg (n = 18). Differ-ences in intraocular pressure that could beattributed to the type of transplant (autograft,


80 Bahn et at. Invest. Ophthalmol. Vis. Sci.January 1982

Table HI. Clinical summary of heterologous cell-lined homograft series

AnimalNo.

16

17

15

18

19

20

Transplant

Heterograft* OU

Heterograft OU

Heterograft OU

Heterograft OU

Heterograft OU

Heterograft OU


1

1

9

9

9

9


OD 0.90OS 0.90OD 0.80OS 0.80OD 0.60OS 0.58OD 0.78OS 0.78OD 0.80OS 0.80OD 0.72OS 0.90

Slit-lampappearance

Mild stromaledema OU

Mild stromaledema OU

KP, C & FKP, C & FRCM, KP, C & FKP, C & FKP, C & FRCM, KP, C & FRCM, KP, C & FRCM,KP, C & F

Complications

—

Replace sutureand rejectiont

Rejection OU

Rejection OU

Rejection OU

OD = right eye; OS = left eye; OU = both eyes; KP = keratic precipitates; C & F = cell and flare in auqeous; RCM = rectrocor-neal membrane.

•Cultured bovine corneal endothelial-lined feline homograft (denuded of native endothelium).

f Rejection based on clinical appearance.

homograft, homograft lined with bovine en-dothelium) were not observed.

The clinical findings for the autograft seriesare summarized in Table I. All autografts re-mained clear throughout the 14 day postop-erative period. The corneal thickness of theautografts matched that of unoperated eyes,and there was only minimal evidence ofinflammatory reaction.

Morphologically the process of autograft-ing caused some tearing of Descemet's mem-brane and slight damage to both the host andgraft endothelial cells adjacent to the woundmargin. Otherwise the 24-hr animals wereunremarkable. At 2 weeks the endotheliumof both the host and graft had recovered thearea immediately adjacent to the wound mar-gin. The host-graft junction of all transplantseries was similar and is described in con-junction with the heterograft series below.

The clinical findings for the homograftseries are summarized in Table II. All homo-grafts, except the ones from which the en-dothelium had been removed, remainedclear throughout the 14 day postoperativeperiod. A mild inflammatory response in theform of conjunctival erythema was observed,as was a moderate increase in corneal thick-ness. Homografts lacking endothelium de-veloped severe and persistent stromal edemawith minimal inflammatory response.

Scanning electron microscopy of the ho-mografts demonstrated identical findings to

those observed in the autograft series. Occa-sional cells were observed adhering to boththe graft and host endothelia. These adherentcells were thought to be white blood cellsthat originated from the secondary aqueoushumor. The denuded grafts were free of en-dothelial cells. Two small (10 /am2) foci of celldebris and fibrin were observed on the de-nuded surface of one graft. The fibroblasts atthe wound margin did not migrate onto thedenuded membrane to any greater extentthan that observed in autografts and homo-grafts bearing endothelium.

The clinical findings of bovine corneal en-dothelial cell-lined feline homografts arepresented in Table III. Clinically these ani-mals presented a picture compatible withendothelial rejection. They demonstratedkeratic precipitates restricted to the graft en-dothelium, varying degrees of retrocornealmembrane formation, and aqueous cell andflare of mild-to-moderate intensity. Serialexaminations performed on the seventh andninth postoperative days demonstrated thatthese findings were progressive. The hostendothelium was never involved. Slit-lampphotographs of healthy homografts and graftswith compromised heterologous bovine en-dothelium are presented in Figs. 9 to 14. Ex-cept for mild edema overlying the involvedendothelium, the stroma of heterologouscell-lined grafts remained clear. (Reliableexamination of the transplants could only be



Table IV. Clinical summary of rotational autograft and contralateral heterologouscell-lined homograft series

AnimalNo.

22

23

241

25|

Transplant

OD Heterograft*OS AutograftOD HeterograftOS AutograftOD HeterograftOS AutograftOD HeterograftOS Autograft


9

9

9

9


0.880.560.680.580.750.620.600.60

Slit-lampappearance

KP, RCMClearKPClearKP, RCMClearKP, RCMClear

Complications

Rejection t—

Rejection—

Rejection—

Rejection—

OD = right eye; OS = left eye; KP = keratic precipitates; RCM = retrocorneal membrane.

*Cultured bovine corneal endothelial—lined feline homograft (denuded of native endothelium).

t Rejection based on clinical appearance.

(Steroid-treated animals receiving 0.1% dexamethasone three times per day, both eyes.

performed with the slit lamp; simple penlightexamination of the anterior segment revealedno abnormalities.)

Morphologically the grafts examined at 24hr after surgery resembled both the autograftand homograft series with the following ex-ceptions: (1) The heterologous bovine cellsthat covered the grafts were irregularlyshaped (Fig. 6) and identical in appearance tothe cellular array formed by rabbit en-dothelium subcultured onto denuded rabbitcorneas.6'7 (2) A variable number of roundedendothelial cells either adhered to the bovineendothelial cell surfaces or appeared to be inthe process of being incorporated into thecell monolayer. Examination of endothelialcell -lined corneas cultured for 3 days (n = 3)or transplants 3 days after surgery (n = 2)demonstrated a uniform covering of flat-tened, irregularly shaped cells (unpublishedobservation). Conformations of adherent cellson the endothelial surface, such as those ob-served on bovine endothelial cell-linedgrafts (described below), never occurred dur-ing the immediate postoperative period.

Nine days after surgery the heterologousendothelium was intact and the bovine cellshad achieved a greater degree of ordering or"mosaic packing" when compared with the 24hr grafts. Although the host endotheliun wasalways unremarkable except for an occasionaladherent white blood cell, the heterologousbovine donor endothelium lining the graftswas festooned with either solitary cells or

clumps of adherent cells (Figs. 15 to 16). Thesurfaces of the cells within these cellular ag-gregates were highly plicated with both filo-podia and lamellipodia extending between ad-jacent cells and to (and occasionally through)the endothelial surface (Fig. 17). When thecell aggregates were viewed in section, apopulation of mononuclear cells with exten-sively folded cytoplasmic processes were ob-served (Fig. 18). The lineage of every cellwithin the aggregates could not be deter-mined. However, a high proportion of thecells had cytologic characteristics of mono-cytes or lymphoblasts (Fig. 19). Granulocyteswere occasionally observed, most often in-sinuated between or beneath the lining endo-thelial cells. The subcultivated bovine endo-thelial cells continued to deposit additionalbasement membrane onto the pre-existentfeline Descemet's membrane (Fig. 20). Theprocess of recovering the denuded feline Des-cemet's membrane by subcultivated bovineendothelial cells appeared in many places tobe a more complex process than that of form-ing a simple monolayer. Two or three layers ofcells, occasionally with additional basementmembrane interposed between the cell lay-ers, were sometimes observed.

Examination of 3H-thymidine— labeled bo-vine endothelial cells on feline corneas dem-onstrated numerous, heavily labeled nucleiacross the surface of the graft (Figs. 21 and22). Heavily labeled cells were distributed ina somewhat more patchy fashion in the 9 day


82 Bahn et al.Invest. Ophthalmol, Vis. Sci.

January 1982




grafts when compared with the preoperativecontrols. In the 9 day grafts, cell nuclei inthose cells separating clusters of heavily la-beled cells usually possessed a dusting ofsilver grains (noticeably above background),indicating that they represented several sub-sequent cell generations derived from the bo-vine cells used to initially seed the graft. Hostfeline endothelial cell nuclei were not labeled.Admixing of feline host and bovine graft en-dothelia was prevented by the barrier natureof the wound margin as described below.

Scanning electron microscopy of the hostgraft junction in all transplant series demon-strated an elevated ridge of circumferentiallyarranged cells having the appearance offibroblasts. Variable numbers of white bloodcells adhered to these cells, which were inturn frequently covered by a meshwork offibrin (Figs. 24 and 25). When the host-graftjunction was viewed in cross section by eithertransmission electron microscopy or light mi-croscopy, the surface ridge of cells (fibro-blasts) was observed to occupy a shallowstromal trough of variable width primarily lo-cated between the interrupted Descemet'smembrane (Figs. 26 to 28). Few if anyinflammatory cells were observed in thestroma of donor or recipient corneas. How-

ever, leukocytes were frequently found onthe surfaces of both host and graft (autograft,homograft, and heterograft) endothelial cellsimmediately adjacent to those wound mar-gins that were extensively decorated withadherent leukocytes (Fig. 25).

A "floating" type retrocorneal membranethat had its point of attachment at the host-graft junction was a nearly universal findingin heterologous cell-lined homografts. Themembrane was composed of a layer of cellsthat were identical in structure to thosefound at the host-graft junction in all the dif-ferent transplant series (autografts, homo-grafts, etc.). The cellular portion of the mem-brane was usually covered on its posteriorsurface by a meshwork of fibrin, containingoccasional admixed red and white blood cells(Fig. 29). Retrocorneal membrane formationoccurred only in association with heterolog-ous cell—lined transplants.

The thickness of heterologous endothelialcell-lined grafts and the relationship be-tween retrocorneal membrane formation andendothelial compromise is illustrated in Fig.23. These data, obtained from a preliminaryanimal series, were responsible for the four-teenth postoperative day being selected forstudy in the succeeding experimental series.

Fig. 9. Slit-lamp photograph of a healthy, homologous feline corneal transplant 2 weeks afterkeratoplasty. The graft is thin and clear.Fig. 10. Slit-lamp specular reflection of the endothelium lining a homologous transplant 2weeks after penetrating keratoplasty. (Approximately X80.)Fig. 11. Slit-lamp photograph illustrating thickening of the posterior corneal surface (arrow) ina heterologous (bovine endothelial cell-lined) transplant 9 days after keratoplasty. The overly-ing corneal stroma is also thickened.Fig. 12. Slit-lamp specular reflection of the heterologous bovine endothelium 9 days afterkeratoplasty. The large densities (diagonal arrows) are keratic precipitates. The heterologousendothelial cells are more sparsely distributed than the endothelial cells of the homologousgraft (see Fig. 10), and several folds are evident in the posterior corneal surface (horizontalarrows). (Approximately X80.)Fig. 13. Slit-lamp photograph of a heterologous transplant 9 days after keratoplasty demon-strating many small keratic precipitates (horizontal arrow) as well as laiger (confluent) keraticprecipitates (vertical arrow) on the bovine endothelium. Keratic precipitates as well as mild-to-moderate aqueous cell and flare characterized all of the heterologous transplants.Fig. 14. Slit-lamp photograph of a retrocorneal membrane (arrows) behind a heterologoustransplant 9 days after keratoplasty. These membranes occur only in grafts bearing heterolog-ous (bovine) endothelium.



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Volume 22Number 1 Heterologous cell-lined cornea! homografts 85

As is evident from the plotted corneal thick-ness, both a breakdown in the corneal endo-thelial barrier and retrocorneal membraneformation occurred approximately 2 weeksafter transplantation. When the animals bear-ing heterologous cell-lined grafts were fol-lowed for 28 days after transplantation, thecorneal thickness approached that of a homo-graft from which the endothelium had beenremoved.

A final rotational autograft and contralat-eral heterologous bovine cell-lined homo-graft series was undertaken for the followingreasons: (1) We wished to decrease thedouble-antigenic dose represented by bilat-eral heterologous (bovine) endothelial cell-lined homografts to determine whether asingle heterologous endothelial-lined graftelicited a similar host response. (2) Wewished to have a "nonantigenic" surgical con-trol to study the keratocyte response at thegraft-host junction and its relationship to ret-rocorneal membranes. (3) The application ofa topical steroid for local anti-inflammatory/immunosuppressive effects is used clinicallyand had been employed in the other reportedbovine endothelial-lined feline corneal trans-

plant series.8 We therefore wished to de-termine whether topical steroids would in-fluence the host response to the heterologousendothelium. The results of this transplantseries are presented in Table IV.

Morphologically the grafts demonstratedthe same features as observed in the bilateralheterologous bovine endothelial cell-linedhomograft series or in the rotational autograftseries. Although there were minor differ-ences, the bovine cell-lined grafts wereagain decorated by cells of the lymphocy-toid-monocytoid series and formed retrocor-neal membranes, whereas the rotationalautografts repaired as discussed above. Ap-plication of a topical steroid appeared to re-duce the number of fibroblasts at the host-graft junction as well as decrease the numberof leukocytes associated with it. The topicalsteroid effects should be regarded as tenta-tive because of the very small sample size.

Discussion

The surgical procedure of penetrating ker-atoplasty was easily accomplished in the cat.Large areas of undisturbed host endotheliumas well as the transplanted endothelium

Fig. 15. Low-power scanning election micrograph of host cornea (H), the host-graft junction,and a heterologous cell-lined graft (G). Solitary cells and cellular aggregates adhere spe-cifically to the heterologous endothelium. (X150.)Fig. 16. Higher-power scanning electron micrograph of cells adhering to the heterologousendothelium (E). The larger cellular aggregates are responsible for the keratic precipitatesobserved with the slit lamp. The heterologous endothelium of this 9-day-old graft more closelyresembles the arrangement of native endothelium. (X325.)Fig. 17. Scanning electron micrograph of a cell aggregate adhering to heterologous cornealendothelium. The cell surfaces are highly plicated with fine cell processes extending betweenadjacent cells and to the heterologous cell surface. A broad cell process extends betweenadjacent bovine endothelial cells (arrow). (X2850.)Fig. 18. Survey transmission electron micrograph of a cellular aggregate applied to theheterologous endothelial cell surface. A cell (arrow) appears to be entering the endothelial celllayer, which in this region consists of multiple plies of endothelial cells, lymphocytoid-monocytoid cells, and other cells of undetermined types. (X1175.)Fig. 19. Transmission electron micrograph of a cell within an aggregate that has the cytologiccharacteristics of a lymphoblast. (X8300.)Fig. 20. Transmission electron micrograph of a heterologous endothelial cell 9 days aftertransplantation. The cell has laid down additional basement membrane (arrow) on the pre-existent feline Descemet's membrane. The dark line on the cell surface is a gold-palladiumcoating applied prior to scanning the specimen. (x23,700.)


86 Bahn et at.Invest, Ophthahnol. Vis. Sci.

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Fig. 21. Distribution of 3H-thymidine- labeled bovine endothelial cells subcultivated onto afeline cornea. This is the distribution of bovine cells on corneal buttons at the time of trans-plantation. (X150.)Fig. 22. Nine days after transplantation the endothelial surface demonstrates an extensivepopulation of labeled cells, indicating their origin from subcultivated bovine cells. A popula-tion of lightly labeled cells indicates that certain cells have undergone several divisions.(X150.)

could be easily assessed because of the rela-tively large size of the cat cornea. Drawbacksto the cat model included the outbred natureof the feline population and the fact that gen-eral anesthesia was required to adequatelystudy the transplanted corneas. Transplantsfrom which the native endothelium had beenmechanically removed substantiated a previ-ous study4 that demonstrated the limitedregenerative capacity of feline corneal endo-thelium.

Evaluation of the rotational autograft andhomograft (fresh or cultured) series demon-strated that these grafts were successful forthe duration of the experiment both by clini-cal and morphologic criteria. No essential dif-

ference could be attributed to the surgeon,and the culture methods were not detrimen-tal to the donor tissue. (A comprehensivespecular microscopic and scanning electronmicroscopic study of an independent cul-tured normal and denuded homograft seriesis in preparation.) As in the rabbit modelof keratoplasty,15 the cat demonstrated anexuberant fibroblastic response at the woundmargin during the 2 week postoperative pe-riod. This fibroblastic response was observedin all of the different types of transplants per-formed. Additionally, fibrin accumulationand white blood cell adherence to the host-graft junction were observed in all proce-dures even when aspirin and heparin were


Volume 22Ntimber 1 Heterologous cell-lined corneal homografts 87

administered preoperatively and the anteriorchamber was liberally irrigated with heparinduring surgery.

The procedure of transplanting a heterolo-gous bovine endothelial cell-lined homograftdid not present any unusual technical prob-lems when compared with the autograft orhomograft procedures. Tritiated-thymidinelabeling of cultured bovine corneal endothe-lial cells demonstrated insofar as was possiblethat the endothelium lining the grafts wasderived from the cells subcultivated onto thedenuded feline corneas. The observed failureof host endothelial cells to migrate across thewound margin by the fourteenth postopera-tive day in any of the transplant series wouldfurther support the position that the endo-thelial cells lining the graft were bovine cells.

Postoperatively there was always a vigor-ous host inflammatory response directed to-ward the heterologous bovine endothelium,which was characterized by white blood cellsadhering specifically to the heterologous en-dothelial cells, often forming large cellularaggregates. Clinically these cell aggregatescould be identified as keratic precipitates onthe donor endothelium. The additional find-ing of cell and flare produced a picture iden-tical to that observed in human homograftrejection.16 Ultrastructural studies demon-strated that many of the responding host cellshad morphologic characteristics of lympho-blasts and monocytes and that the inflamma-tory process was similar to that observed inrabbit homograft rejection.17"19 The hostresponse to the transplanted heterologous en-dothelium occurred in the absence of vascu-larization of the graft, suggesting that the pos-terior surface of the cornea is not an immuno-logically privileged site as has been previouslysuggested.20 We are currently investigatingthe route by which heterologous cells sensi-tize the host as well as the immunologic andmorphologic events that occur after transplan-tation of heterologous cell-lined grafts. Theseparameters are being compared with similarones occurring as a consequence of homograftrejection induced by skin grafting.

An additional finding in the heterograftseries was the formation of a "floating" type

Denuded

050

Bovine

Cot

— Cleor CorneaRetro Corneol OpacityStromal Opoeily

7 14 21DAYS POST OP

28

Fig. 23. Corneal thickness of homologous, de-nuded homologous, and heterologous cell-linedhomologous corneal transplants is depicted. Het-erologous cell-lined grafts maintain a thin, clearcornea until the second postoperative week whenthickening and retrocorneal membranes both oc-cur. Data are from a preliminary experiment andare expressed as the average thickness for two de-nuded homografts, four conventional homografts,and four heterologous cell-lined homografts.

retrocorneal membrane that appeared tooriginate from the wound margin. The vigor-ous host inflammatory response directedagainst the heterologous bovine endotheliummay have in some way promoted the forma-tion of the membrane. An alternative sourceof this formation may involve the heterolog-ous cell surfaces themselves. However, anexplanation for the nearly universal formationof retrocorneal membranes in cats bearingheterologous endothelial-lined homograftsmust await further study. Although we didnot conduct a sequential study of retrocor-neal membrane formation, we believe themembranes arose from fibroblasts at thewound margin rather than by metaplasia ofthe host or donor endothelium as has beenreported in other animal models.21 This con-clusion is based primarily on the finding of aspace separating the cellular membrane fromthe heterologous endothelial cell-lined pos-terior corneal surface and the similarity of thecells within the membrane to those cells foundat the host-graft junction from which themembrane arises.

The rotational autograft/contrilateral het-erograft series of transplants demonstratedthat the host response to the heterologous


88 Bahn et at. Inoest. Qphlhalmol, Vis. Set.January 1982

29


endothelium was nonsympathizing in thatthe rotational autografts remained unaf-fected. Topical steroids in the doses em-ployed did not prevent the host response tothe transplanted heterologous bovine endo-thelium in the few animals studied.

Analysis of corneal thickness measure-ments indicated that the heterologous bovineendothelium possessed at least some of thefunctional characteristics of the native endo-thelium in that the repopulating endotheliumwas initially able to prevent substantial swell-



ing of the graft stronia. As the host responseprogressed, the stronia of the heterologouscell-lined transplants hydrated and ap-proached the condition of grafts transplantedwithout endothelium. Even at 9 days postop-eratively, stromal edema was clinically man-ifest over those portions of the transplantsmost involved by the host response. No evi-dence of postoperative glaucoma was encoun-tered in any of the transplants, a potentialcomplication resulting from the use of cul-tured cells to repopulate corneal grafts.20

From a review of the literature and ourinitial attempts at culturing the corneal en-dothelium of various species (rabbit, bovine,

feline, and human—not shown), we believethat cultured bovine corneal endothelium isthe most promising type of cultured cornealendothelial cell that has potential for clinicalapplication in the treatment of corneal dis-ease. The results of Gospodarowicz et al.8

further substantiate this view by demonstrat-ing that cultured bovine corneal endotheliumcan be successfully transplanted into a het-erologous host. It is not clear why culturedbovine endothelium elicited a host responsein our experimental series while not doingso in the transplant series of Gospodarowiczet al.8 Perhaps early passage, subculturedcells shed greater quantities of cell surface

Fig. 24. Scanning election micrograph of the host graft junction illustrating both the posteriorcorneal surface and stronia. A layer of fibrin (F) obscures the zone of fibroblasts locatedbetween the host (on the left) and graft (out of the picture on the right) endothelia. Theinterweaving of host and graft collagenous lamellae (one group of which is indicated by thearrow) is evident in the stronia. (x80.)Fig. 25. Scanning electron micrograph of the host-graft junction of a feline homograft. Thediagnonally oriented zone of fibroblasts (individual cells are difficult to discern) are covered byadhering leukocytes, some of which are also present on both the host (upper left) and graft(lower right) endothelia. (X220.)Fig. 26. Sixty micrometer thick, unstained epoxy section of a host-graft junction 9 days aftertransplanting a heterologous cell-lined graft. The depth and lateral extent of the fibroblast-filled zone is depicted. One micrometer thick sections of the regions indicated at (a) and(b) areillustrated in Figs. 27 and 28. (X100.)Fig. 27. Junction of a corneal graft and its cut Descemet's membrane (arrow a, Fig. 26) withthe host cornea. Several layers of radially oriented fibroblasts extend over Descemet's mem-brane for a short distance (see Fig. 26). The graft and host corneal stroma contain a normalnumber of keratocytes. (Toluidine blue stain; X500.)Fig. 28. Host-graft junction in a region lacking Descemet's membrane (arrow b, Fig. 26). Theposterior surface of the junction is covered by a fibrin meshwork containing a few red bloodcells. Multiple layers of fibroblasts are interposed between the fibrin layer and the cornealstronia. The presence of a zone at the host-graft junction occupied by fibroblasts was a constantfinding in all corneal transplant series. This fibroblastic zone effectively prevented the admix-ing of graft and host endothelial cells for the duration of the experiment. The degree of surfacefibrin formation and the number of leukocytes associated with the junction was variable.(Toluidine blue stain; X525.)Fig. 29. Photomontage of'unstained, glutaraldehyde-osmium- fixed 60 /xm thick epoxy sec-tions that illustrates a "floating" type retrocorneal membrane. Retrocorneal membranes, afinding limited to heterologous cell-lined grafts, always had their origin at the host-graftjunction. The membranes were usually constructed of a posterior layer of fibrin (F) and amembrane (M) composed of several layers of fibroblasts. The cellular component of the mem-branes appeared to originate from the zone of fibroblasts located within the host graft junction(open arrow). A fluid-filled space (S) separated the membrane from the posterior cornealsurface and stronia (C). Fusion of the membrane with the endothelial cells on the posteriorsurface of the graft was not observed during the 2 week posttransplant period. The blackmaterial on the epithelial surface is silver contact paint used to prepare the specimen forscanning microscopy. (xlO.)



January 1982

antigens than do multiple-passed cells. Al-ternatively, a greater proportion of endothe-lial cells on grafts we employed may havefound their way into the general circulation,thus increasing the antigenic challenge to thehost. However, obstructional glaucoma, aconsequence of over populating grafts,20 wasnot observed in the present study. A clearexplanation for the host response differencesencountered in our studies in comparisonwith those of Gospodarowicz et al.8 mustawait further experimentation. However, thepresent set of experiments suggests that theuse of cultured heterologous cells in cornealtransplantation has significant dangers as wellas potential benefits.

We thank Angela M. Welford for technical assistancein all phases of electron microscopy and Steven McKel-vey for assistance in cell culture. We also thank Dr.Leslie J. Fisher for demonstrating the 60 /im thick sec-tioning of epoxy resin—embedded tissue.

R E F E R E N C E S

1. Stacker FW: The endothelium of the cornea and itsclinical implications, ed. 2. Springfield, 111., 1971,Charles C Thomas, Publisher.

2. Kaufman HE and Katz JI: Pathology of the cornealendothelium. INVEST OPHTHALMOL VIS SCI 16:265,

1976.3. Van Horn DD and Hyndiuk RA: Endothelial wound

repair in primate cornea. Exp Eye Res 21:113, 1975.4. Van Horn DD, Sendele DD, Seidman S, and Buco

PJ: Regenerative capacity of the corneal endothe-lium in rabbit and cat. INVEST OPHTHALMOL VIS SCI

16:597, 1977.5. Hogan MJ, Wood I, and Fine M: Fuchs' endothelial

dystrophy of the cornea. Am J Ophthalmol 78:363,1974.

6. Jumblatt MM, Maurice DM, and McCulley JP:Transplantation of tissue-cultured corneal endothe-lium. INVEST OPHTHALMOL VIS SCI 17:1135, 1978.

7. Ben Ezra D and Maftzir G: Transplantation of cor-neal endothelium. In Immunology of the Eye:Workshop I, Steinberg GM, Gery I, and Nussen-blatt RB, editors. Immunology Abstracts SpecialSupplement, 1980, pp. 257-270.

8. Gospodarowicz D, Greenburg G, and Alvarado J:

Transplantation of cultured bovine endothelial cellsto species with nonregenerative endothelium. ArchOphthalmol 97:2163, 1979.

9. Timpl R, Martin GR, Bruckner P, Wick G, andWiedmann H: Nature of the collagenous protein in atumor basement membrane. Eur J Biochem 84:43,1978.

10. Rohde H, Wick G, and Timpl R: Immunochemicalcharacterization of the basement membrane glyco-protein laminin. Eur J Biochem 102:195, 1979.

11. MacCallum DK, Lillie JH, Frederick W, ScalettaLJ, Occhino J, and Ledbetter S: Bovine corneal en-dothelial cells elaborate an organized basementmembrane in vitro. (Submitted for publication.)

12. Gospodarowicz D and Greenburg G: The coating ofbovine and rabbit corneas denuded of their en-dothelium with bovine corneal endothelial cells.Exp Eye Res 28:249, 1979.

13. Bourne W: Examination and photography of donorcorneal endothelium. Arch Ophthalmol 94:1799,1976.

14. West RW: Superficial warming of epoxy blocks forcutting 25-150 fim sections to be resectioned in the40-90 nm range. Stain Technol 47:201, 1972.

15. Inomata H, Smelser GK, and Polack RM: Finestructure of regenerating endothelium and Desce-met's membrane in normal and rejecting cornealgrafts. Am J Ophthalmol 70:48, 1970.

16. Maumenee AE: Clinical aspects of the corneal ho-mograft reaction. INVEST OPHTHALMOL 1:244, 1962.

17. Polack FM and Kanai A: Electron microscopic stud-ies of graft endothelium in corneal graft rejection.Am J Ophthalmol 73:711, 1972.

18. Renard G and Montcourrier P: The action of sen-sitized lymphocytes on the corneal endothelium ofrabbits. Albrecht Von Graefes Arch Klin ExpOphthalmol 203:201, 1977.

19. Montcourrier P, Renard G, Pouliquen Y, and FaureJP: Lymphocyte cytotoxicity against corneal endo-thelium in rabbits. In Immunology and Immuno-pathology of the Eye, Silverstein AM and O'ConnorGR, editors. New York, 1979, Masson PublishingUSA, Inc., pp. 161-166.

20. Gospodarowicz D, Greenburg G, and Alvarado J:Transplantation of cultured bovine corneal endo-thelial cells to the rabbit cornea: clinical implicationsfor human studies. Proc Natl Acad Sci USA 76:464,1979.

21. Michels RG, Kenyon KR, and Maumenee AE: Ret-rocorneal fibrous membrane. INVEST OPHTHALMOL

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complications associated with bovine corneal endothelial cell … native endothelium had been...

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