AMNIOTIC MEMBRANE TRANSPLANTATION The amniotic membrane is the innermost layer of the placenta with 5 layers. It has anti-inflammatory, anti-fibrotic and antimicrobial properties making it useful for ocular surface reconstruction. Amniotic membrane transplantation involves placing the membrane on defects of the cornea, conjunctiva or ocular surface to promote healing. The membrane acts as a biological bandage to promote epithelization and inhibit fibrosis. Complications can include infection transmission or premature dissolution but techniques using fibrin glue or devices like Prokera are being developed for sutureless fixation.

1. AMNIOTIC
MEMBRANE
TRANSPLANTATIO
N

2. AMNIOTIC MEMBRANE
• it is the innermost layer of placenta with a thickness
of 0.02 – 0.05 mm.
• 5 layered membrane with 3 basic layers:
– epithelium
– thick basement membrane
– avascular hypocellular stromal matrix.
• epithelium
– single layer of cuboidal cells with microvilli on apical
surface.
• basement membrane
– thick layer with reticular fibrin network  conjunctiva.
– resistant to cryo preservation.
– collagen 1,4,5,7,fibronectin,laminin,Elastin.

3. – produce growth factors: TGF  & α, FGF, EGF, HGF,TIMP
1,2,4,PDGF A & B
– promote :epithelial proliferation,differentiation,
migration,adhesion.
• stroma
– avascular ,antiangiogenic & has antibacterial properties.
– rich in fetal hyaluronic acid,protease inhibitors, anti
inflammatory proteins .
– suppresses TGF β signaling, cytokines like IL 1a,2,8,IFN 
,FGF,PDGF.proliferation ,differentiation of myofibroblast
of cornea and limbus,apoptosis of basal epithelial cells.
– does not express histocompatability antigens.

4. PROPERTIES MAKING AMT USEFUL IN
DISEASES
• easy & unlimited availability.
• possibility of preservation for future use.
• no immunological rejection.
• antimicrobial properties
• anti fibroblastic activity
• promotor of cell migration,growth

5. INDICATIONS FOR AMT
• NON OPHTHALMIC USES
– dressing of burnt skin
– large skin wounds & c/c ulcers
– artificial vagina reconstruction
– omphalocoele repair
– post abdomen,pelvis,head sx to prevent adhesion.
• OPHTHALMIC USES
– in stem cell deficiency
–chemical injury.
– in the absence of stem cell deficiency
– Corneal epithelial defects
– Corneal/Corneoscleral ulcers
– Bullous keratopathy.
- Band keratopathy

6. • for conjunctival reconstruction
– Pterygium
– Conjunctivochalasis
– OSSN
– Limbal dermoid
– Symblepharon
– Conjunctival lesions
– Leaking blebs.
• in ocular cicatricial diseases
– Toxic epidermal necrolysis
– Ocular cicatricial pemphigoid
– Oculopalpebral and reconstructive surgery
• Other uses
– Stem cell cultures

7. FUNCTIONING OF AMT:
1. PROMOTER OF EPITHELISATION
• BM:
– facilitate epithelial cell migration
– promote epithelial celldifferentiation
– prevents epithelial cell apoptosis
– ensure basal epithelial cell adhesion
– inhibits protease

epithelisation under cover of AM as it acts like a BCL.

8. 2.INHIBITOR OF FIBROSIS
• STROMA:
–  TGF  inhibition & myofibroblast differentiation.
– antiinflammatory effect1L-α and 1L-β suppression.
– avascularityprevents growth of new vessels.

acts as an anatomical barrier to keep adhesive surfaces
apart

9. PREPARATION OF AMNIOTIC MEMBRANE
• placenta after elective cesarean delivery from a consenting donor -
for HIV, hep B,C ,syphilis.
• Under a laminar flow hood the placenta cleaned of blood clots with
sterile saline + 50 μg/ml of amphotericin B.
• separate AM from the chorion by blunt dissection
• flattened on to nitrocellulose membrane of 4X4 cm with epithelial
BM surface up.
• stored at –70°C in a sterile vial containing DMEM (Dulbecco's
Modified Eagle Medium) and sterile glycerol in 1:1 ratio, 3.3 % L-
glutamine, 25 μg/ml genta, 50 units/ml penicillin ,100 μg/ml
ciprofloxacin and 0.5 mg/ml ampho B.
• can be used upto 6 months after preparation
• The vial is thawed at room temperature x 10 min / at 4°C x 30 min b4
use.
• cellular viability  >50 % in 2 months.
• Damage of AM cells by cryopreservation growth factors.

10. MICROBIOLOGICAL SAFETY OF AMT
• DUAL SCREENING 4 HIV,HEP- B,C,SYPHILIS.
– at procurement
– 6 months later
• prions like CJD can be spread.
• PRECAUTIONS
• Donor selection
– r/o high risk behaviours
– social $ medical h/o
– consented & screened for donation
• local research & ethical committee approval
• competent labs for testing.
• serum samples from donor saved for 11 years post
transplant

11. FRESH VS PRESERVED AMT
• functions equally well.
• DISADVANTAGE OF FRESH AMT
• wastage of unused AMT
• lack of dual screening
• DISADVANTAGE OF PRESERVED AMT
• storage at -80⁰c needed.
• Damage of AM cells by cryopreservation
growth factors.

12. ORIENTATION & IDENTIFICATION OF
AM
• stains to identify AM:
– ICG
– RB
– Trypan blue
– lissamine green B(intra op)
• both stroma & BM stained
• can identify wrinkles on graft.
• Stromal side has vitreous like strands when
touched with a sponge.

13. COMMERCIAL AM
• AMBIO 2
– single layered native AM.
– biostructure of native AM retained.
• AMBIO 5
– multilayered with amnion+ chorion
– thick ,intact,more biostructure.
• AMBIO DISC
– 15 mm disc
– as bcl
– self retained

14. SURGICAL TECHNIQUES
OVERLAY TECHNIQUE:
• performed in :
– non-healing epithelial ulcer (- )stromal thinning.
– persistent epithelial defects of cornea
– acute chemical injury
• A single /multilayered AM with stromal side down is
secured over trimmed epithelial defect on cornea
with 10.0 nylon suture with buried knots.

15. INLAY/GRAFT TECHNIQUE:
• In case of epithelial defect/ulceration with stromal
thinning.
• multiple AM layers are trimmed to fit the size of the
ulcer defect.
• The underlying layers are compactly packed with
stromal side down, layer by layer, filling up the
stromal defect & left unsutured.
• The topmost layer is sutured to the cornea with
10.0 nylon with buried knots.
• Compact packing helps in good healing .
• AM acts as a scaffold for epithelial cell migration .

16. PATCH TECHNIQUE:
• for patching the cornea by placing basement
membrane side downwards and stromal side up .
• sutured to the surrounding perilimbal conjunctiva.
• acts as a biological CL to protect the healing surface
beneath it.
• falls off/removed after use.

17. INLAY WITH OVERLAY:
• compact packing of the stromal ulcer defect with
multiple layers of amniotic membrane and sealing it
with a sutured top layer of membrane,
• an overlay of multilayered amniotic membrane
patch covering the whole cornea may be placed and
sutured to the underlying conjunctiva.
• used for early ocular surface stabilization in severe
grades of ocular chemical injury

18. AMT : CONJUNCTIVAL RECONSTRUCTION
• 8-0 or, 9-0 or 10-0 Vicryl sutures are used to anchor
AM to the conjunctiva after adequate dissection
and removal of pathological subconjunctival tissue.
• anchor a sheet of AM to the fornix with two sets of
double armed 4-0 chromic gut sutures on a cutting
needle may be used. The needles are passed from
the AM surface through the inferior fornix, via the
full-thickness of the eyelid and exit through the
eyelid skin. The two needles of each of the two sets
of sutures are passed through two segments of an
encircling band and then tied.

19. CORNEAL SURFACE RECONSTRUCTION
• A Weckcel sponge or blade is used to remove all cellular
debris or exudates from the base of defect, fine forceps and a
straight crescent blade to remove Loose epithelium
surrounding an epithelial defect
• A single sheet of AM may be applied as an inlay graft or
overlay patch at least 1 mm larger than the defect.
• Non-absorbable interrupted 10-0 nylon monofilament sutures
are used.
• The sutures must be placed circumferentially or parallel to
the cut edge of the graft in an interrupted or continuous
manner. knots must be cut short and buried in corneal tissue.
• in deep defects, a multilayered approach is preferred with
Small pieces of AM layered into the defect or a single sheet
may be folded on itself twice (blanket fold) with a larger patch
anchored over the entire defect in an overlay fashion].

20. OCULAR SURFACE RECONSTRUCTION
• all fibrotic tissue is meticulously dissected and
removed from the corneal and conjunctival surfaces.
• continuous sheet of AM devoid of buttonholes used .
• The lower lid is everted with chalazion clamp. A large
sheet of AM is placed on the ocular surface and
anchored to the inner surface of lid margin using
multiple interrupted 10-0 vicryl sutures.
• multiple interrupted vicryl sutures used to attach AM
to the inner lid surface, beyond the inferior fornix and
onto the bulbar conjunctiva
• A continuous encircling 10-0 nylon suture is used to
anchor the membrane at the limbus or the peripheral
360 ⁰cornea.

21. SUSPENSION CULTURE TECHNIQUE
• Single cell suspensions of LECs from dispase II
digested limbal tissues are seeded onto AM &
allowed to adhere and cultured to obtain a
monolayer of cells.

22. EXPLANT CULTURE TECHNIQUE
• HAM can be used with or without its native epithelium,
• de-epithelialized membrane facilitates good visibility of
cultured cells and confluent growth of cells.
• intact amniotic membrane retains stem cell
characteristics, promotes growth rate with better
stratification.
• explant fragmented limbal tissues on the membrane,
• the cells are fed with 3-4 ml of medium, incubated at
37⁰C, 5 % CO2 and
• monitor for growth on alternate days.
• The culture is terminated when a monolayer of the cells
growing from the explants becomes confluent, in 10 to
14 days..

23. COMPLICATIONS
• transmit HIV, hepatitis B or C.
• Premature dissolution of the membrane,
• cutting through sutures and loss of membrane,
• residual opaque membrane
• fluid vesicles or collection of blood under the
membrane and failure of achieving the intended
outcome
• calcification

24. RECENT ADVANCES
SUTURE LESS AMNIOTIC MEMBRANE FIXATION
• with fibrin glue
• suture fixation of amniotic membrane causes:-
– time consuming and cumbersome process.
– corneal irritation, scarring
– graft loss due to membrane shrinkage
– need for subsequent suture removal
• advantages :
– Reduced surgical time;
– patient comfort post operative period due to reduced
inflammation and suture related irritation;
– no need to remove sutures
– rapid recovery.
• disadvantages are:-
– cost of tissue glue (fibrin glue)
– The glued membrane is not as secure as sutured membrane

25. • Prokera is a conformer type device made of
amniotic membrane with a rigid frame so that it can
be inserted into the conjunctival sac without any
sutures or glue, like a contact lens .
• designed by Dr Tseng.
• AM is clipped into a dual, concave, polycarbonate
ring set.
• acts as a cover to the surface and also keeps the
bulbar and palpebral conjunctival surfaces apart.
• not available in India and is also very expensive.
PROKERA