[1]

Establishment of Amniotic Membrane Bank for Orthopaedic

Surgery in dogs: Clinical and Radiographic Assessment.

Amer, M.S.; Imam, E.A; Shamaa, A.A. & Mostafa, A.A.

Surgery, Anaesthesiology & Radiology Department, Faculty of Vet. Medicine, Cairo University.

Abstract:

There are many types of biological and bioengineered bone substitutes are

presented for treatment of different bone defects Amniotic membrane (AM) has

been used in surgical transplantation as a biomaterial since 100 years ago. The

presence of hyaluronic acid and collagen, fibronectin, elastin, laminin, nidogen

and proteoglycans makes the AM as a scaffold for proliferation and

differentiation and delivery of stem cells. The aim of this study was to establish

AM bank for bone surgery in a canine experimental model through clinical and

radiographic evaluation. Nine bitches were used as AM donors, after harvesting

under strict sterilization, then the membrane was preserved in temperature

below freezing -80 c till use (one month preservation period). Ten apparently

healthy mongrel dogs were used in the study as AM recipient animals (5 dogs)

after induction of 2cm femoral bone defect and control animals (5 dogs). The

operated dogs were evaluated clinically and radiographically and randomly

allocated and observed period for 6 months. The results clinically and

radiographically were promising in recipient group than control group. The

results concluded that AM bank can be used as useful graft substitute material

for bone defect management and other non-healing bone lesions in developing

countries considering its properties and the easy availability, low cost of

procurement and cheap storage.

Key Words:

Amniotic Membrane, Orthopaedic, Surgery, Clinical and Radiographic

Assessment

Introduction:

The bone substitutes play an important starring role in the management and

treatment of major bone defects and become continuously progressing. There

is a very promising improvements in the chances of survival for patients with

a critical bone defects by wide production and use of various types of bone

substitutes. There are many types of biological and bioengineered bone

substitutes are presented for treatment of different bone defects (Kumar et al.,

2006).

[2]

An ideal graft substitute should have good bioresorbability (Dell,

Burchardet and Glewowczshi, 1984) and osteoinductive and osteoconductive

capacity (Bob and Peter, 2001). It also should be nontoxic and non-

immunogenic to the organism (Donald & Gretchen, 1997), easy to sterilize

(Dreaenert & Delius, 2007) and not compromise mechanical stability

(Gadallah, 1998). Although the utilization of autografts results in significant

success in the bone healing process, there are some disadvantages, such as

requiring the patient to undergo a second operation and increasing therapeutic

costs due to prolonged hospital stay and extended medicare. It is also non

convenient in cases of severe bone loss due to extensive destruction or in cases

of bone tumors (Schena et al., 1985). Therefore, the employment of the

allograft and xenograft and bone substitutes as an alternative to the autograft

has become common in orthopedic surgery (Beaman et al., 2006 & Kumar et

al., 2006).

Amniotic membrane (AM) is the innermost layer of fetal membranes that

composes of three layers including epithelial cells, basement membrane and

avascular stroma. (Parry and Strauss, 1998; Fukada, Chikama and

Nakamura, 1999; Toda, Okabe, Yoshida et al., 2007 and Ismail, Marcos,

Sherif et al., 2009). The AM has been used in surgical transplantation as a

biomaterial since 100 years ago. The AM may help in decrease the pain,

electrolyte abnormalities and bacterial infection and increase the rate of re-

epithelialization in patients. The presence of hyaluronic acid and collagen types

I, III, IV, V and VI, fibronectin, elastin, laminin, nidogen and proteoglycans

makes the AM as a scaffold for proliferation and differentiation and delivery of

stem cells. Amniotic cells also express both of the mesenchymal and epithelial

stem cell markers.( Gekas, Dieterlen-Lievre, Orkin et al 2005; Miki,

Lehmann, Cai et al 2005). In addition, the AM has anti-inflammatory, low

immunogenicity and angiogenic modulatory properties as well as antibacterial

activity. (Hao, Ma, Hwang et al 2000). Amniotic membranes maintain a

physiologically moist microenvironment that promotes healing. Since the fresh

AM is not available regularly, the preservation is essential to reach a ready-to-

use source of the AM for clinical applications. Fresh amniotic membranes have

a short life span as compared to the preserved membranes. There are some

procedures to preserve the AM for long time. However, preservation methods

affect cellular structure and extracellular matrix and might lead to changes in

the antibacterial properties of the AM. There is also possibility of bacterial,

fungal or viral disease transmission of donor origin (Parolini and Soncini, 2006

and Ilancheran, Michalska, Peh et al., 2007).

The aim of this study was to evaluate Amniotic membrane for bone defects

management in a canine experimental model.

Materials and Methods:

The present study was approved by the ethical committee of Faculty of

Veterinary medicine, Cairo university (EAURC) by code (Cu F

Vet/F/SUR/2013/15).The experimental study was carried out on 18 apparently

[3]

healthy mongrel dogs weighing 15-20 kg. body weight with age 2-5 years old

were. All the dogs were vaccinated and treated from internal and external

parasites. The study was designed in different stages:

Amniotic membrane Harvesting and Preservation:

The AM was harvest from caesarean section of nine apparently healthy

vaccinated bitches presented to the clinic of Surgery, Anaesthesiology and

Radiology Department, Faculty of Vet. Medicine, Cairo Univ. after placenta

collection the AM was stripped off under complete aseptic procedures. AMs

were immersed in 1L sterile normal saline, contained 100 U/ml penicillin and

0.2 mg/ml streptomycin and 0.025mg/ml amphotericin according to (Lee and

Tseng, 1997; Sharifiaghdas et al., 2007 and Vongsakul et al., 2009). The

epithelial covering was marked by external knot of silk stitch which laid on the

surface of the amniotic membrane. Under strict sterilization, 10 times serial

washing of the membranes in sterile Petri-dishes contained 20ml normal saline

with the former additives were applied. During washing the individual

membrane was finger rubbed and squeezed of the blood vessels which were

done gently to remove excessive blood clots, then kept in container contained

normal saline with antibiotics and antifungal additives for a period of two hours

cooling in a refrigerator.

After that AM was spread uniformly without folds or tears on individually

sterilized 0.22m nitrocellulose membranes of the required size (47mm or 25

mm, commercially available-Millipore or Sartorius (Biobasic Canada ®) with

the epithelial/basement layer surface up the molded membrane sheet cut to

4cm×5cm pieces each piece put in plastic sterile dish contain The preservative

medium (1:1vol/vol) ratio of sterile glycerol (sterilized by autoclave) and

(Dulbcus Modified Eagles media (DMEM) with 3.3 % L glutamine, 25 µg/ml

gentamicin, 50 units/ml penicillin-100 µg/ml ciprofloxacin and 0.5mg/ ml

Amphotericin B. then the membrane was preserved in temperature below

freezing -80 °C till use (one month preservation period). Bacteriological and

mycological evaluations were performed on the plates before preservation and

also before implantation through swapping and culture on the specific media

(Fig.1).

[4]

Fig.1: AM after collection and immersed in saline solution, AM after washing and free from

blood with identification of epithelial layer (a), AM after spreading on nitrocellulose membrane

and poring DMEM media (b).

Animals’ preparation and bone defect induction.

Ten adult apparently healthy mongrel dogs of both sexes, 2-5 years and

weighing 10-20 kg were selected with no history or clinical signs of orthopedic

disease and normal hind limb radiographs were used in this experiment. The

animals were vaccinated with Defensor -3® and Vanguard plus® and orally

dewormed by 50 mg Praziquantel, 150 mg Febantel and 144 mg Pyrantel-

Embonat/10kg.b.wt (Drontal tablets® Bayer HealthCare, Germany) and

external parasites controlled monthly by Selamectin pour on (Revolution®,

Pfizer Animal Health –New York) then randomly allocated and observed for 6

months post-operatively.

The pelvic limbs of all dogs were radiographed to document normal femoral

bone anatomy and size and exclude any skeletal abnormality. A Prophylactic

antibiotic course of Cefipim (Maxipim® Smith-Kline Beecham Co., A.R.E) at a

dose of 4.5 mg/kg body weight was administered i.v immediately

preoperatively and continued every 12 hours intramuscularly for 5 days post-

operatively.

The right pelvic limb was prepared for aseptic surgery. Skin preparation

consisted of clipping and shaving of hair, followed by surgical scrubs for at

least 10 minutes using cotton with soap and water. Then the surgical field was

degreased by alcohol 70% followed by application of Povidone iodine

(Betadine®, El-nil Co., ARE) surgical spray that was allowed to dry for 2 to 3

minutes (Fig.2A). Routine orthopaedic operative draping and gowning

procedures using towels stockinet and double surgical gloving were used.

All dogs limb was premedicated with i.v injection of a mixture of atropine

sulfate 0.05 mg/kg. (Atropine sulfate® 1mg/m1) and diazepam 1mg/kg

(Valpam®, Amoun Co., A.R.E), Anaesthesia was induced immediately through

[5]

I.V injection of a mixture of Ketamine 10 mg/kg (Ketamar® 5% Sol. Amoun

Co. A.R.E) and Xylazine 1mg/kg (Xyla-Ject® 2% ADWIA Co., A.R.E.). The

anesthetic depth was maintained with 2.5% thiopental sodium (Thiopental®

EPICO, A.R.E) administered intravenously. The anesthetic regimen was

conjugated with lumbosacral spinal epidural analgesia by Lidocaine

(Depocaine® 2%, Eldebiky Co., A.R.E).The recipient dogs were restrained in

lateral recumbence with the operated limb uppermost (Schmidt-Oechtering

and Alef, 1995).

According to the Association for the Studying of Internal Fixation, (ASIF) a

standard orthopedic bone set and implants will be used. The lateral approach

for femur bone exposure was performed. A skin incision extending from just

behind the trochanteric major to the distal metaphyseal region of the femur,

then blunt dissection of the sub-cutaneous tissue. After that cutting through

tensor facia latae for exposure of the groove between vastus lateralis and biceps

femoris muscles, Deep dissection between the two muscles to expose the

femoral shaft, the femoral shaft after exposure and stripping of the periosteum

for the intended removed segment using periosteal elevator.

Modulation of the bone plate (DCP) to simulate the contour of the femur.

Induction of an artificial bone defect 2 cm length in the mid- shaft of the femur

i.e. ostectomy.Then Fixation of the bone segments was performed primarily by

insertion of intramedullary bone pin (Synthes, Wayne, Pa) either 3 or 4 mm Ø

according to the medullary cavity diameter using Pneumatic drill by retrograde

method. After that the steps for fixation of the modulated DCP Plate was

performed as follow; drilling the plate holes with pneumatic drill and drill

guide (3.5 mmØ) and drill bit (2.7 mm Ø), Measuring the length of the drilled

hole with small depth gauge to select the screw of suitable length, tapping the

holes with cortical tap (3.5 mmØ) and T-handle and finally Driving of cortical

bone screw using screw driver 3.5 mmØ. The holes of the plate were loaded

with screws except the hole above the fracture gap. After the bone segments

fixation the defect was managed by implantation of AM in recipient animals

(Fig.2) and left free without scaffold in control group. The implantation site

was then flushed several times with normal saline solution and irrigated with

Gentamycin solution (Gentamycin® 10%, Alexandria Co., A.R.E.). The

surgical wound was closed as usual using polyglactin 910 (Vicryl®, Ethicon Lts

U.K) size (0) for close approximation of the adjacent muscles by continuous

suture pattern, then S/C tissue with the same material and pattern, finally skin

was closed by interrupted suture pattern using suture material Vicryl® size (1).

All dogs were confined to individual cages along the designed duration of

the study. The dogs were given course of antibiotic every twelve hours for five

days Maxipim® and given milk and bread for the first 3 days post-operatively

and then returned to its normal diet within the first week.

The skin wound was daily dressed and the sutures were removed 10 days

post-operatively. All animals in the groups were evaluated post-operatively as

[6]

follow: Clinically; all animals were subjected to regular clinical examination

daily, which including evidence of infection which assessed via rectal

temperature, limb function, appetite, wound drainage and popliteal lymph node

size and weight bearing capacity in standing and motion positions. And

Radiographically; Immediate post-operative radiographs of the operated limb

two views were performed using a mobile x-ray machine (Ficher Machine,

Eureka X-ray tube/ Model E-Merald-125, 1985, U.S.A); Medial lateral M/L

(50-52 kV/32 mAs) and anterior posterior and A/P (52-54 kV/15 mAs).

Sequential Radiography; Serial radiographs of the operated femora of living

animals were performed monthly till the end of 6 month. The fracture gap was

examined for visibility and its filling by new bone formation (Osteophytes).

Fig.2: Photographs showing induction of bone defect, fracture segments fixation by

intramedullary bone pin (A) and fixation of DCP bone plate fixation, and Implantation (B) of

AM inside the fracture gap (arrow).

Results:

Clinical Evaluation:

In case of control group; the animals partially bear their weight within 5-7

days P.O while partial to full weight bearing was observed with 3-4 weeks P.O.

Moderate degree of lameness was noticed in walking while sever lameness

(non-weight bearing) was observed in fast movement which disappeared at 6-8

weeks P.O. There were no signs of infection which represented by the rectal

temperature, which was slightly increased (0.5-1°C) during the first 3 days P.O

[7]

then back to normal limit all over the observation period. Slight popliteal

lymph node enlargement which subsided during a week P.O.

While in case of AM group; the dogs in this group were bearing the weight

partially 7-10 days P.O and have full bearing at 3-5 weeks P.O. The animals’

gait was ranged from observable moderate lameness in slow motion to non-

weight bearing in fast motion during 3 weeks P.O. The animals’ motion

returned to normal at 4-6 weeks P.O the rectal temperatures were slightly

elevated by 1-1.5°C in some dogs which return to normal within 3-4 days P.O

There was increase in the size of popliteal lymph node 1-2 weeks P.O while

back to its normal size at 3-4 weeks P.O. Moderate degree of seromal reaction

was detected in all operated dogs 2-3 days post-operatively. This reaction was

decreased spontaneously 4 weeks post-operatively (Fig.3).

Radiographic Evaluation:

The results of sequential radiographic evaluation were summarized as:

Post-operative radiography (Fig.4-1); in all animals immediate post-

operative radiographs revealed adequate metal-implant stability i.e. the bone

was fixed with 6 cortical bone screws, 3 placed on proximal bone segment and

3 in lower one while the holes above the gap was left free. Bone alignment was

very good.

At 2-4 weeks P.O period; in all groups there were no observable

radiographic changes except some rounding of the fracture ends. Slight

periosteal reactivity at the proximal segment was detected at 4 weeks P.O in

AM group. While At 4 weeks P.O period there were increasing in the periosteal

reactivity on both proximal and distal host segments. Slight osteophytic

formation in the gap was noticed in AM group represented by increasing in gap

radiodensity. While in control group, there was a slight osteoperiosteal

reactivity in the proximal segment and the gap was clear (radiolucent) i.e. non-

detectable radiographic detectable changes. At 8 weeks P.O period; in all

groups there was increasing in the osteoperiosteal reactivity while the gap has

more osteophytic reactivity by increasing in radiopacity in AM group while in

control group the gap still clear. At 12 weeks P.O period; the osteoperiosteal

reactivity was still present in both proximal and distal bone segments.

Concerning the fracture gap; there was increasing in osteophytic formation in

case of AM group and in control group there was slight increase in gap

radiodensity.

At 16-20 weeks P.O period; there was noticeable decreasing in the

osteoperiosteal reactivity in all groups (remodeling) especially at the proximal

bone segment. The fracture gap was nearly occupied with newly formed

osteophyte that represented by high radiodensity and decreasing defect length

all over that period in case of AM group while in case of control group there

were non-observable radiographic changes. At 24 weeks P.O period (Fig.4-2);

[8]

the osteoperiosteal reactivity was almost disappeared in both proximal and

distal bone segment in all groups’ animals. The fracture gap was nearly filled

with new callus (60-70%) in case of AM group. In case of control group, the

gap was slightly increased in radiopacity especially at the proximal part.

Fig (3): Clinical photographs at 6 weeks P.O showing partial weight bearing on the operated

limb in standing position in case of control group (A) while full weight bearing in case of AM

group (B). The same was noticed in case of hind limb stress position (C&D respectively)

[9]

Fig. (4): Anterioposterior (A, C) and Lateromedial (B, D) radiographic views of the operated

femora of control group (left side) and AM group (right side) at different observation periods;

at immediate P.O (1) showing adequate metal-implant stability in both groups, and at 24 weeks

P.O (2) showing rounding of the bone segments ends in case of control group while in case of

AM group the gap was more radiopaque and increasing the formed callus length.

Discussion:

The first reported use of fetal membrane as skin substitute was by Davis in 1910. In

1913, Salbella presented the first clinical report of successful use of amniotic

membrane in the treatment of burns and skin ulcerations. In 1940, DeRoth reported

the use of amniotic membrane in the repair of conjunctival defects. From 1940 to

1965 a number of clinical trials of successful use of amniotic membrane for use in

acute skin injuries appear in the literature. However no practical methods of

preparation, sterilization and storage were suggested and this fact seems to have

limited the use of this modality prior to 1965. In 1965 Dino et al. demonstrated that

amniotic membrane from routine deliveries could be sterilized and kept for six weeks

at 4°C and safely used on acute second degree burns and on skin donor sites. This

encouraging report stimulated great interest amongst clinicians and has resulted in

numerous reports in the world literature documenting thousands of patients with

successful healing of all kinds of skin lesions.

The AM showed good promising results in the fracture gap (critical defect 2 cm)

healing as appeared clinically and radiographically more than the control group.

[10]

These results in agreement with those reported by Gadallah, (1998) and Amer,

(2009).

There were no signs of implant rejection or immune reaction. That results was

in contrary with those reported by McIntyre and Faulk, (1979) they said that AM

has low or no antigenicity, a fact that might be related apparently to a distinct

collagen present in the amniotic membrane. They had isolated a glycoprotein from

amnion and credited it to be responsible for suppressing any "foreign body" type

reaction by acting on lymphocytes and preventing lymphoblastogenesis.

Amniotic membrane when used as an allograft in peritoneal cavity or buried

under skin has shown long term survival with no evidence of any immune reaction.

Likewise, when used as xenograft from human to animals or cattle to humans no

significant antigenicity is revealed (Robson and Krizek, 1973 and Rao and

Chandrasekhram, 1981).

AM also has bacteriostatic function; this function is said to be due to the presence

of antibodies, possibly allantoin, a bactericidal product of purine metabolism and

lysozyme, a bacteriolytic protein (Walker, Cooney and Allen, 1977). Furthermore,

the amniotic membrane has a high thrombin activity which allows a very rapid and

efficient attachment to living tissue. This close adherence allows restoration of

lymphatic integrity, protects circulating phagocytes from exposure and allows

removal of surface debris and bacteria (Walker, 1988). That explains the clinical

results of non-infection outcome.

There is great controversy as to which side of the membrane, amniotic or

chorionic, should be applied next to the wound. Trelford et.al. (1972) and Robson,

(1981) reported that if amnion and chorion are separated and the amnion's

mesenchymal side is applied to the host tissue, then vascularization and rejection

phenomenon are not seen. So the epithelial (mesenchymal) side of the membrane

should be applied to the outside.

AM It is readily available at no cost if fresh, sterilization, storage and application

are simple, prevents fluids, protein and energy loss, combats infection, promotes

healing, becomes firmly adherent to the wound and relieves pain. While its

disadvantages; it is highly fragil (Robson and Krizek, 1974 Piserchia and Akenzua

1981).

Conclusion; considering the properties of amniotic membrane and the easy

availability, low cost of procurement and cheap storage makes it appear to be a useful

graft substitute material for bone defect management and other non-healing bone

lesions in developing countries.

References: 1. Amer, M.S (2009): Studies on segmental cortical bone xenograft in dogs. MVSc.

Thesis, Surg., Fac. Vet. Med. Cairo Univ., Giza.

[11]

2. Beaman, F. D.; Bancroft, L.W.; Peterson, J. J.; and Kransdorf, M. J. (2006).

Bone graft materials and synthetic substitutes. Radiol. Clin. North Am.,

44(3): 451-461. 3. Bob Soin; and Peter J. Friend; (2001): Physiological aspects of

xenotransplantation. Transplantation Rev., 15(4): 200-209.

4. Dell, P. C.; Burchardet, H.; and Glewowczski, F. P.; (1984): A

roentgenographic, mechanical and histological evaluation of vascularized

and non-vascularized segmental fibular canine autografts. J. Bone Joint

Surg., 67: 105-112.

5. Donald, L. P.; and Gretchen, L. F.; (1997): Bone grafting in: Small animal

orthopaedics and fracture repair, (3rded).W. B. Saunders Co.,

Philadelphia,Pennsylvania. PP 147-149.

6. Draenert, G. E.; and Delius, M. ;( 2007): The mechanically stable steam

sterilization of bone grafts. Biomaterials 28(8):1531-1538.

7. Fukada, K.; Chikama, T. and Nakamura, M. (1999). Differential distribution

of subchains of the basement membrane components type IV collagen and

laminin among the amniotic membrane, cornea, and conjunctiva. Cornea;

18: 73–79 8. Gadallah, S. M.; (1998): Studies on entire segment cortical bone allografts in dogs.

Ph. D. Thesis, Surg., Fac. Vet. Med. Cairo Univ., Giza.

9. Gekas, C.; Dieterlen-Lievre, F.; Orkin, S. H. and Mikkola, H. K. (2005):

The placenta is a Niche for Hematopoietic Stem Cells. Dev Cell 8:365–

375.

10. Hao, Y.; Ma, D. H. K.; Hwang, D. G.; Kim, W. S. and Zhang, F. (2000).

Identification of antiangiogenic and anti-inflammatory proteins in human

amniotic membrane. Cornea 19:348–352.

11. Ilancheran, S.; Moodley, Y. and Manuelpillai U. (2009). Human Fetal

Membranes, A Source of Stem Cells for Tissue Regeneration and Repair?

Placenta, 30: 2-10.

12. Ismail, A.; Marcos, R. R.; Sherif, A. A. ;Thabet, A.; El ghor, H.; Ishac, A.

E.; Barsoun, B. S.; Chowdhury, A. and Selim, A. (2009). Amniotic stem

cells repair ureteric defect: a study to evaluate the feasibility of amniotic

membrane as a graft in surgical reconstruction. Indian J Surg 71:121-127.

13. Kumar, P.; Shrestha, D.; and Bajracharya, S.; (2006): Replacement of an

extruded segment of radius after autoclaving and sterilizing with

gentamycin. J. Hand Surg., 31-B (6): 616-618.

14. Lee, S. H. and Tseng, S. C. G. (1997). Amniotic membrane transplantation

for persistent epithelial defects with ulceration. Am J Ophthalmol 123:303

312.

15. McIntyre JA and Faulk WP. Antigens of human trophoblasts: effect of

heterologous and anti-trophoblast sera on lymphocyte response in utero. J

Exp Med 1979; 149: 824.

16. Miki, T.; Lehmann, T.; Cai, H.; Stolz, D. B. and Strom, S. C. (2005). Stem

cell characteristics of amniotic epithelial cells. Stem Cells 23: 1549-1559.

17. Parolini, O. and Soncini, M.: Human placenta: a source of progenitor/stem

cells? J Reproduktionsmed Endokrinol 2006; 3: 117-126.

18. Parry, S. and Strauss, J. F.: Premature rupture of the fetal membranes. N

Engl J Med 1998; 338: 663-670.

[12]

19. Piserchia NE, Akenzua GI. Amniotic membrane dressing for burns in

children: a cheap method of treatment for developing countries. Trop

Geogr Med 1981; 33: 235.

20. Rao TV and Chandrasekhram V. Use of dry human and bovine amnion as a

biological dressing. Arch Surg 1981; 116: 891.

21. Robson MC and Krizek TJ. The effect of human amniotic membranes on

the bacterial population of infected rat burns. Ann Surg 1973; 177:144.

22. Robson MC, Krizek TJ. Clinical experiences with amniotic membranes as

a temporary biological dressing. Conn Med 1974; 38:449.

23. Robson MC. Invited editorial comment. In: Rao TV and Chandrasekharam

V. Use of human and bovine amnion as a biological dressing. Arch Surg

1981; 116: 891.

24. Schena, C. J.; Gerham, D. L.; and Hoefle, W. D.: Segmental freeze-dried

and fresh cortical allografts in the canine femur. II: A sequential histologic

comparison over a one year time interval. Am. Anim. Hosp. Assoc., 1985;

21: 193-204.

25. Sharifiaghdas, F.; Hamzehiesfahani, N.; Moghadasali, R.; Ghaemimanesh,

F. and Baharvand, H. (2007). Human amniotic membrane as a suitable

matrix for growth mouse urothelial cells in comparison with human

peritoneal and omentum membranes. Urol J. spring; 4(2):71-8.

26. Shmidet-Oechteing, G. and Alef, M. (1995). Neue aspekte der veterinrana

sthesie und intensivtherpie. Blackwell Wiessenschafts-verlag.Berlin.

27. Toda, A.; Okabe, M.; Yoshida, T. and Nikaido, T. (2007). The Potential of

Amniotic Membrane/Amnion-derived cells for Regeneration of Various

Tissues. J Pharmacol Sci 105(3):215–228.

28. Trelford JD, Anderson DG, Hanson FW, et al. Consideration of the amnion

as an autograft and as an allograft in sheep. A preliminary report. J Med

1972; 3:231.

29. Vongsakul, S.; Tuntivanich, P.; Sirivaidyapong, S. and Kalpravidh, M..

Canine amniotic membrane transplantation for ocular surface

reconstruction of created deep corneal ulcers in dogs. Vet. Med. 2009;

39(2): 135-144.

30. Walker AB, Cooney DR and Allen JE. Use of fresh amnion as a burn

dressing. J Pediatr Surg 1977; 12:391.