Experimental study on allografts of amniotic epithelial
cells in calcaneal tendon lesions of sheep
A. Muttini & M. Mattioli & L. Petrizzi & V. Varasano &
C. Sciarrini & V. Russo & A. Mauro & D. Cocciolone &
M. Turriani & B. Barboni
Published online: 22 May 2010
# Springer Science+Business Media B.V. 2010
Abstract An experimental protocol was designed to study the survival and behaviour of an
allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted
on three healthy adult sheep. A core lesion was created in both calcaneal tendons under
ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml
saline. The AECs were obtained from a 60–80-day-old fetus and cultured under standard
conditions. After 15 days of collagenase treatment, 2×106AECs stained with a vital
membrane fluorescent probe (PHK26) were injected under US guidance in 500 μl saline
solution into the lesion of one limb. The contralateral untreated limb was used as a control.
Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on
explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the
lesion without any adverse reactions. The damaged tissue of the treated tendons showed a
high number of reparative cells in active proliferation that were accumulating collagen within
the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized
into parallel arrays of fibers oriented along the longitudinal axis of the tendon.
amniotic epithelial cells
Herovici histological staining
Vet Res Commun (2010) 34 (Suppl 1):S117–S120
A. Muttini (*):L. Petrizzi:V. Varasano:C. Sciarrini
Department of Veterinary Clinical Sciences, University of Teramo,
Viale Crispi 212, Teramo, TE 64100, Italy
M. Mattioli:V. Russo:A. Mauro:D. Cocciolone:M. Turriani:B. Barboni
Department of Biomedical Sciences, University of Teramo, Teramo, Italy
Ligament and tendon injuries, especially of the superficial digital flexor tendons (SDFT),
are a substantial cause of wastage within the horse industry (Dowling et al. 2000). The
reported incidence reaches 42% in racing thoroughbreds (Wilson et al. 1996). Low
performance syndrome is sometimes present regardless of clinical signs. Additionally, in
humans, tendon lesions are an important problem particularly in athletes (Abate et al.
2009). Regenerative therapy with bone marrow autografts in horses dates from 2001 (Hertel
2001). In Italy, this protocol was previously described and demonstrated good results
(Muttini et al. 2006). The clinical efficacy of stem cell implantation has been reported by
many experimental studies and clinical trials. However, comparative studies focused on
stem cells from other sources and characterization of the precise cell type is lacking.
Recently, the distinguishing features of stem cells taken from amniotic membranes
(Niknejad et al. 2008) or from amniotic fluid (Siegel et al. 2008) have been described.
Briefly, the most important characteristics of these cells are the anti-inflammatory and anti-
microbial properties, and low immunogenicity. Based on these features, the use of these
cells in regenerative treatment of tendinopathies could be utilized in both veterinary and
human medicine. This preliminary report shows the results of the allotransplantation of
amniotic epithelial cells (AECs) into experimentally-induced lesions of calcaneal tendons of
adult sheep. The aims of this study were 1) to verify the feasibility of the AEC allograft
without inducing immunologic or other adverse reactions, 2) to demonstrate the survival of
AECs in the experimental defects, and 3) to verify by histological examination of the tissue
if the AECs could induce regeneration rather than scar formation.
Materials and methods
A bilateral defect was created in the middle portion (5 cm proximal to the calcaneal
tuberosity) of the Achilles tendon (calcaneal tendon) of three 50-kg adult sheep. The defect
was realized as previously described (Crovace et al. 2008) by injecting 400 UI of
collagenase 1 (Sigma tau) in 0.6 ml saline under ultrasound (US) guidance. After 7 days the
defect was detected by ultrasonographic examination in transversal and longitudinal scans.
AECs were collected from the placenta of slaughtered sheep at 60–80 days of pregnancy.
AECs were obtained from the epithelial layer of the amniotic membrane by enzymatic
digestion (EDTA-trypsin) and cultured in standard conditions (Parolini et al. 2009) for three
passages. Before grafting, AECS were stained with a vital membrane fluorescent probe
(PHK26) to identify the transferred cells in the host tissue. AECs were transferred into the
experimental defect under US guidance. A total of 2×106AECs in 500 μl saline were
grafted into the experimental defects 15 days after the collagenase injection. The contra
lateral untreated limb was used as a control. Animals were stabled and observed daily for
signs of discomfort. One sheep was euthanatized after 7 days; two animals were
euthanatized after 30 days.
Tendon samples obtained in correspondence to the experimental defect were collected.
The specimens were frozen in liquid nitrogen and cryosectioned at 10 μm. Sections were
analyzed for 1) haematoxylin-eosin and Herovici (HE) histological staining, 2) nuclear
contrast with fluorescent DAPI staining, and 3) immunoreaction with a cellular proliferation
marker Ki-67 (Santa Cruz Biotechnology) detected by a secondary anti-mouse AlexaFluor
488-conjugated antibody. All the sections were analyzed with an Axioscope 2 microscope
(Zeiss) at ×100 and ×400 magnification.
S118Vet Res Commun (2010) 34 (Suppl 1):S117–S120
The experimental protocol was tolerated well by all animals. A slight bilateral lameness was
noted a few days after collagenase administration. Markedly hypoechoic core lesions were
detected 7 days after the injection. One lesion was clearly lateral due to a technical mistake
and was used for morphological validation 7 days after the collagenase injection.
Macroscopic examination revealed some inflammatory reaction in the collagenase injection
site 7 days after allograft. On the contrary, inflammation was absent in 30-day explanted
tendons. Histological examination revealed damaged tissue 7 days post-grafting. The
extracellular matrix appeared to be composed of a few collagen fibers with an irregular
distribution as demonstrated by HE. A high number of cells was identified in the damaged
tendon. No proliferative (Ki67 negative) PKH26 positive AECs were observed within the
lesion site. The control tendon showed an altered organization similar to that observed in
the allografted tendon even with a minor cellularity. The presence of inflammatory cells
was similar in the grafted and control tendon. Tendon sections obtained from the animals
sacrificed after 30 days, displayed AECs distally and marginally to the defect. Many
proliferating cells (Ki67-positive) were identified near AECs, some of which had flat nuclei
and were enclosed within collagen fibers that started to be oriented along the longitudinal
tendon axis (HE). In control specimens, no tendon healing was observed 30 days after
collagenase injection. In fact, histological examination revealed no proliferating cells with
little non-organized collagen deposition.
The reported data supported the experimental hypothesis. The AEC allograft did not
induce any immunological reaction in the host tissue up to one month after implant.
This data confirms the low immunogenicity of amniotic-derived cells (Niknejad et al.
2008). The inflammation clinically noted subsided in a few days. Histological
examination demonstrated the survival of labeled AECs in the experimental lesions until
1 month after grafting. A relevant production of newly formed collagen fibers followed
stem cell allotransplantation that, one month later, started to assume a parallel orientation
along the axis of the tendon. The presence of several proliferating cells near inactive
AECs, localized between healthy and damaged tissue, led to the hypothesis that
transferred stem cells could not participate directly in the regenerative process. Their
role in the regenerative process, as opposed to their role in other tissues, could be
explained with the production of growth/chemotactic factors able to activate tendon
regeneration. These preliminary data need to be experimentally confirmed using
increased numbers of animals and by using specific markers to detect the type of
collagen deposited. Long-term follow-up is also required to confirm the absence of
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tendon grafted with cBMSC and BMMNCs after collagenase-induced tendonitis. Vet Comp Orthop
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