Abstract The purpose of this study was to demonstratethe histological effects of amniotic fluid on bone healingin the rabbit fetus. Fetal femurs (n=34) were fractured inthe diaphysis and two experimental groups were estab-lished. In group A fetuses (n=18), the fracture ends werebathed in amniotic fluid through their unsutured skinwounds. In group B fetuses (n=16), the fracture siteswere isolated from amniotic fluid by suturing the woundedges. Nonoperated fetuses were used as control group(n=9). The fetuses were sacrificed on the first, third andseventh postoperative days for histological studies. His-tological examination of the specimens was performed todetect inflammation, fibroblastic proliferation, bone cel-lularity, callus and cartilage formation. The results werecompared, using the Fisher exact chi square test criteria,where no statistically significant differences were detect-ed between group A and group B ( P>0.05).

Key words Fetal bone healing · Amniotic fluid Histology

Introduction

Recent developments in fetal diagnosis have initiated anew era in medicine and the fetus is now regarded as anew patient group [2,3,20]. Various research studies havebeen performed on various tissues of fetal animals

[5,8,13,14,17,31,34–38,41]. Although fetal animals havebeen subject to a considerable number of experimentalstudies, the human fetus has been operated on only overthe last 20 years and until now, only lethal diseases havebeen considered ethically appropriate for fetal surgicalmanipulation [1,9,15,16,21,24]. It is likely that at somepoint, elective surgery on the human fetus will be per-formed to harness its scarless wound healing and its highregenerative capacity [6,9,16,41].

A number of studies have proved that amniotic fluidmay have significant effects on scarless fetal woundhealing, not only due to its physical properties, but alsoto the high hyaluronic acid content and hyaluronic acidstimulating activity, growth factors, enzymes, hor-mones, and immunological components [3,7,12,21,23].Unlike skin, fetal diaphragmatic wounds are known toheal by scar formation. Longaker et al. [22] unsuccess-fully bathed diaphragmatic incisions with amniotic fluidin the hope that scar formation would be prevented orreduced. However, they failed to demonstrate any sucheffects.

Although bone is a mesothelial tissue, in adults it hasan ideal remodeling capacity under favorable circum-stances [10,19,32]. It was decided to investigate whetheror not this specialized tissue could be affected by thepresence of amniotic fluid. An experimental study wasperformed on fetal rabbits, in order to find out whether,by bathing fractured bone ends with amniotic fluid, cel-lular differentiation could be induced or not. To the bestof our knowledge, this was the first research project toexamine the effects of amniotic fluid on the early cellu-lar changes of fetal bone tissue.

Materials and methods

This study was performed on 74 fetuses of 30 pregnant albinoNew Zealand rabbits of 3–5 kg. Sixty-five fetuses were operatedon, but 37 fetuses survived, three fetuses were excluded from thestudy and the remaining 34 formed the experimental group, whilenine nonoperated fetuses were the control group.

A. Kayıkçıoglu (✉ )38 Sokak 4/4, 06500 Bahçelievler, Ankara, Turkeye-mail: akayikci@hacettepe.edu.trTel.: +90-312-215-1577, Fax: +90-312-309-0445

A. Kayıkçıoglu · A. KeçikDepartment of Plastic and Reconstructive Surgery, Hacettepe University School of Medicine, Ankara, Turkey

H. ÇelikDepartment of Morphology, Hacettepe University School of Medicine, Ankara, Turkey

Ö. ÖzkayaDepartment of Pathology, Hacettepe University School of Medicine, Ankara, Turkey

Eur J Plast Surg (2000) 23:272–277 © Springer-Verlag 2000

O R I G I N A L

A. Kayıkçıoglu · A. Keçik · H. Çelik · Ö. Özkaya

Histologic effects of amniotic fluid in the early phases of bone healing:an experimental study on rabbits

Received: 24 March 1999 / Accepted: 15 September 1999

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Experimental group

The rabbits were prepared for surgery at the 23rd–25th day of preg-nancy (term 31 days). After a 12-h fasting period, the rabbits wereanesthetized with an injection of intramuscular ketamine hydro-chloride 50 mg/kg and xylazine hydrochloride 50 mg/kg. Antibiot-ic prophylaxis was provided by a single dose of intramuscular 250 mg sulbactam plus ampicillin. The rabbits were placed in asupine position and 2–3% halothane and nitrous oxide-oxygenmixture was administered by mask. Abdominal fur was shavedand the area was cleaned with polyvinyl povidone solution. Aftersterile draping, the uterus was explored through a midline abdomi-nal incision. A maximum number of three fetuses was selectedfrom the bicornuate uterus, taking care to avoid the fetuses nearthe cervix and ovaries. A #6–0 Ti-Cron (Davis-Geck, Pearl River)pursestring suture was placed on the antiplacental site and theuterus was incised; either the left or the right lower extremity ofthe fetus was taken out of the uterus. A 3-mm incision was per-formed on the posterolateral aspect of the thigh over the midpointof the femur. After incising the tiny mass of muscle, the femoralbone was reached. The femur was cut transversely into two piecesusing iris scissors. Following this, two experimental groups wereconstructed:

Group A: In this group of fetuses, 3-mm skin ellipses were ex-cised from the wound edges and the thigh incisions were left openin order to allow contact of amniotic fluid with the fracture sites.Group B: The fetal skin incisions were closed with interrupted#6–0 Ti-Cron sutures to prevent amniotic fluid penetration.

After replacing the amniotic fluid loss with physiologic serumsolution, the purse string sutures were tied. The abdominal fasciaand skin were closed with 4–0 Ti-Cron Each rabbit was returnedto its individual cage, where it was fed by standard diet and water.The fetuses were sacrificed at the first, third and seventh postoper-ative days. Following a thorough macroscopic examination, theywere embedded in 10% formaldehyde solution for histological ex-amination.

Control group: Nine fetuses of 25, 27 and 31 days of gestation,three in each subgroup, were sacrificed as non operated controls.The gestational ages of each subgroup corresponded with those ofthe experimental fetuses. Age matched control animals were usedto determine the normal histological architecture (Figs 1,2).

Macroscopic evaluation

The extremity development of the fetus and the incisions on thefetal thighs were evaluated. Fetuses with marked differences in theextremity development were excluded from the study, on the as-sumption that the extremity circulation could have been hampered.The fetuses were examined in order that all of the incisions ofGroup A fetuses were open, while the converse was true for thefetuses of Group B.

Histologic evaluation

Light microscopy

The fetal thighs were fixed in 10% formaldehyde solution andblocked in paraffin. Five micron specimens were prepared andstained with haematoxylin and eosin. The control group femurswere evaluated first, in order to provide experience of fetal tissuesunder the light microscope (Olympus BH-2) at 40–800× magnifi-cation. Fetal fracture sites were then evaluated and inflammationin the soft tissue, fibroblast proliferation at the fracture site, acel-lular bone formation, callus formation, and the presence of carti-laginous tissue at fracture ends were marked as positive wheneverobserved, and negative when not seen.

Scanning electron microscopy

The paraffin blocks for light microscopy were further contrastedwith uranyl acetate and lead citrate. Specimens were dehydratedby bathing them in a series of acetone. Dried examples were coat-ed with gold and inspected by scanning electron microscope(JEOL JEM 1200 SEM).

Statistical analysis

Statistical differences between Groups A and B were compared ondays 1, 3, and 7 separately for inflammation, fibroblast prolifera-tion, callus and cartilage formation. The Fisher exact chi squaretest was used and P<0.05 was accepted as statistically significant.

Results

Three of the 37 alive fetuses were excluded from thestudy; two because of an atrophic extremity, and the oth-er for technical problems. The overall fetal survival was57%. Group A consisted of eight first day fetuses, fivethird day fetuses and five seventh day fetuses (n=18).Group B consisted of five first day fetuses, six third dayfetuses and five seventh day fetuses (n==16).

Macroscopic evaluation

With the exception of the fetuses excluded from the ex-periment, there were no obvious disparities in the ex-tremity sizes in either group and fracture ends were dis-orientated, displaying no sign of rigidity.

In all group A fetuses, wound closure was not evi-dent, and the defects on the thighs were consistent. Therewas no crust formation but a fibrin layer on the openwounds. Group B fetuses had complete wound coverageand minimal linear fibrinoid material on the incision site.

Histologic evaluation

Light microscopy

Group A fetuses had fibrinoid coverage on their openwounds. Group B fetuses had intact surface coverage ren-dering amniotic fluid penetration impossible (Table 1).

First day group: Some of the fetuses displayed a markedinflammatory response, which was widespread through-out the soft tissue covering the fracture ends. Amongthese, polymorphonuclear leucocytes predominated (Fig.3). Areas of fibroblastic proliferation and sequestratedbone segments could be detected in a number of fetuses,while in none was callus formation and cartilage forma-tion present.Third day group: Third day fetuses revealed that the in-flammatory response was almost nonexistent except forone fetus in each group. Areas of fibroblastic prolifera-tion were noted. Sequestrated bone was seen in one fetusof Group A. There was no sign of callus and cartilageformation (Figs. 4,5).

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Seventh day: Two fetuses, one from each group had aninflammatory response of predominantly mononuclearcells. Fibroblastic proliferation was seen. Sequestratedbone segments were detected in a considerably highnumber of Group B fetuses. Neither callus nor cartilageformation could be detected (Fig. 6).

Scanning electron microscopy

Clear images of fetal lamellar and cortical bone were ob-tained by SEM. Cartilage cells and matrix were easilyseen and were distinguished from bony architecture inthe control group. However new cartilage formation wasnot seen at the fracture ends of any group (Figs. 7–9).

Fig. 1 Femurs of 24 and 31 day old rabbit fetuses

Fig. 2 Histology of fetal femur (haematoxylin & eosin, ×40)A Bone, B cartilage, C epiphyseal zone

Fig. 3 Group A fetus, first day. Note marked, diffuse inflammato-ry response (haematoxylin & eosin, ×40)

Fig. 4 Group A fetus, third day, direct penetration of amniotic flu-id to the fracture site, except for a flimsy fibrinoid layer (haema-toxylin & eosin, ×40). a Fracture end, b fibrinoid layer, c incision

Fig. 5 Group B fetus, third day. Arrow indicates periosteum. Pro-liferation is not observed (haematoxylin & eosin, ×40). a Fractureend

Fig. 6 Group A fetus, seventh day. Fracture end has been coveredwith a periosteum like layer (haematoxylin & eosin, ×40). a Frac-ture end, arrow periosteum

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Statistical analysis

There were no statistically significant differences be-tween groups A and B (P>0.05).

Discussion

Fetal wound matrix is rich in hyaluronic acid (HA) con-tent; the latter is thought to have a high regenerative ca-pacity [3,4,6,29,39,43]. The presence of HA in the fetalmatrix for longer periods is considered to be one of themain factors in scarless fetal tissue repair [20,30]. Amni-otic fluid is known to have a high content of HA and adistinctive factor called hyaluronic acid stimulating ac-tivity (HASA). Amniotic fluid also contains variousgrowth factors and hormones [12,20].

A number of studies in rabbit fetuses revealed thatunless the skin wound was isolated from the amnioticfluid, secondary wound contraction did not occur and thedefect tended to remain open [2,18,42]. In parallel withthe literature findings, it was observed that if the thighincision was not sutured and an ellipse of skin was re-sected instead, the gap created enlarged in parallel withthe fetal growth. As a result, femoral fracture ends couldbe bathed in amniotic fluid through a defect created onthe thigh. Instead of a crust, only a thin fibrinoid layerformed. Longaker et al. described similar findings in theabdomen [22].

Fetal wound healing is characterized by minimal in-flammatory response; there are conflicting comments asto the effect of amniotic fluid on the inflammatory re-sponse [20,33,42]. Silicone sheeting was used to coverskin wounds and to isolate them from amniotic fluid inthese studies. Authors who utilized the same model re-ported various results as to whether amniotic fluid dis-played a suppressive or an augmentative effect on fetaltissue inflammation [25,27,33]. As no significant differ-ence was detected between the groups, it was concludedthat amniotic fluid had no potential effects on soft tissueinflammation.

Table 1 Results of the lightmicroscopic examination; totalnumber of scores for eachgroup

First day Third day Seventh day

Grp A Grp B Grp A Grp B Grp A Grp B (n=8) (n=5) (n=5) (n=6) (n=5) (n=5)

Inflammation 4 3 1 1 1 1Fibroblastic proliferation 3 4 2 3 1 2Acellular bone 1 2 1 0 1 3Callus formation 0 0 0 0 0 0Cartilage formation 0 0 0 0 0 0

Fig. 7 A 25-day-old fetus, fetal lamellar bone (SEM, ×500)

Fig. 8 A 25-day-old fetus, fetal epiphysis (SEM, ×800). a Chon-drocyte, b lacuna, c matrix

Fig. 9 Group A fetus, seventh day. Fracture end (SEM, ×500)

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Fibroblastic proliferation and collagen synthesis hasbeen attributed to the proliferative phase of wound heal-ing [28]. In fetal tissue, fibroblasts are known to be bet-ter regulated and excess collagen production is avoided[11,20,23]. The effects of amniotic fluid on fetal fibro-blasts was studied in a number of experiments[7,18,25,27]. In the present study, we only attempted todetect the presence of fibroblastic cells. Our observa-tions showed that there were no marked differences inthe fibroblastic proliferation histologically. Not only sur-gical trauma, but also adverse vascular and environmen-tal circumstances may cause nonviable bone formation[32,45]. Areas of bone at the fracture ends were ob-served to contain no osteoblastic cells. This findingmight be the result of surgical manipulation. Althoughstatistically insignificant, the seventh day group B had agreater number of fetuses showing these acellular areas.Amniotic fluid could be expected to prevent callus for-mation by washing out the haematoma and by interferingwith secondary bone healing.

Fetal bone heals with callus formation and cartilageformation as in the adult fracture repair process[26,35,40,44]. When a gap was created in the fetal boneand/or the fracture ends were not fixed, the callus wasmore prominent; this contrasted with rigid fixation sys-tems without any gaps which could induce primary bonehealing without any evidence of callus formation[26,40,44]. Fetal fracture healing was evaluated up to an8-week period [44]. The only difference from adult bonehealing was that fetal bones were known to have a higherregenerative capacity and minimal callus formation[26,44]. Ris and Wray studied fracture healing in the rab-bit femur and observed endochondral ossification withina week [35]. In lamb fetuses, Slate and co-workers notedthe presence of cartilage formation in the same time peri-od [40] and Longaker et al. reported similar findings in a10 day period [26]. Scanning electron microscopy wasspecially utilized to differentiate cartilage nests on frac-ture ends and although the system was capable of delin-eating bone and cartilage perfectly, we could not detectthe presence of cartilage at the fracture ends. Amnioticfluid also did not induce early cartilaginous transforma-tion.

The short gestation period of rabbits was unsuitablefor making delayed observations on fracture healing. Thesheep model is superior in this regard, but fetal healinggreatly differs among species and each class should beindividually evaluated [2,21]. Although amniotic fluidwas found not to affect early bone healing of rabbit fe-tuses, the results of this research cannot be applied toother animal species or to humans.

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