T. Brune et al. Fibroblasts for tissue engineering
European Cells and Materials Vol. 14. 2007 (pages 78-91) ISSN 1473-2262
The present study compares fibroblasts extracted from intact
and ruptured human anterior cruciate ligaments (ACL) for
creation of a tissue engineered ACL-construct, made of
porcine small intestinal submucosal extracellular matrix
(SIS-ECM) seeded with these ACL cells. The comparison
is based on histological, immunohistochemical and RT-PCR
analyses. Differences were observed between cells in a
ruptured ACL (rACL) and cells in an intact ACL (iACL),
particularly with regard to the expression of integrin
subunits and smooth muscle actin (SMA). Despite these
differences in the cell source, both cell populations behaved
similarly when seeded on an SIS-ECM scaffold, with
similar cell morphology, connective tissue organization and
composition, SMA and integrin expression. This study
shows the usefulness of naturally occurring scaffolds such
as SIS-ECM for the study of cell behaviour in vitro, and
illustrates the possibility to use autologous cells extracted
from ruptured ACL biopsies as a source for tissue
engineered ACL constructs.
Key Words: Tissue engineering, anterior cruciate ligament,
extracted fibroblasts, small intestinal submucosa scaffold,
integrins, elastic network, in vitro models.
*Address for correspondence:
Institut de Biologie et Chimie des Protéines
Equipe Protéines matricielles et assemblage tissulaire
UMR 5086 CNRS – UCBL1
7, passage du Vercors
69367 Lyon cedex 07, France
Telephone Number: +33 (0)4 72 72 26 66
FAX Number: +33 (0)4 72 72 26 04
A common approach to create a tissue engineered
construct for replacement of injured tissue involves
seeding a scaffold with a population of specific cells in
vitro, followed by implantation of the constructs to the
site of interest (Altman et al., 2002; Bellincampi et al.,
1998; Laurencin and Freeman, 2005). The source of cells
for creating such a construct is crucial for a successful
outcome. Allogenic stem cells and primary differentiated
cells have been investigated, and have shown some
efficacy in clinical practice; however, immunotolerance
of the cells is a concern. Autologous cells are widely
studied because they pose no immunologic risk, but
availability of these cells depends upon a source of healthy
tissue, there is donor site morbidity risk, and the necessity
for multiple procedures to harvest sufficient cells for
culture and implantation. Few comparative studies have
been conducted to determine the efficacy of cells from
different sources for a particular application.
An example of a clinical application that may benefit
from a cell seeded tissue engineered product is
reconstruction of the anterior cruciate ligament (ACL).
The ACL is important for anterior-posterior and rotatory
stability of the knee (Gabriel et al., 2004; Loh et al., 2003),
and does not spontaneously heal after a complete rupture.
Without reconstruction of the ACL, many patients suffer
from progressive deterioration of the articular cartilage
of the knee, ultimately resulting in the need for a total
knee arthroplasty. The present gold standard for ACL
reconstruction is the use of an autologous tendon graft.
Although the clinical results are generally good, the use
of autologous tissue still has limitations, including
different tissue morphology and innervation, and
morbidity at the site of tissue harvest (Adachi et al., 2003;
Aune et al., 1996; Kartus et al., 2001).
Possible cell sources for a cell seeded tissue engineered
construct for the ACL would include autologous
fibroblasts from either the ruptured ACL or from an intact
contralateral ligament. Fibroblasts from the ruptured ACL
remain viable and express collagen for at least 1 year after
rupture (Spindler et al., 1996), although cell phenotype
is altered due to the changes in the local biochemical and
mechanical environment (Lo et al., 1998; Neurath et al.,
1994). Fibroblasts are also able to migrate from ruptured
ACL explants towards a collagen-GAG scaffold (Murray
and Spector, 2001).
The goal of the present study was to characterize and
compare fibroblasts from intact (iACL) and ruptured
IN VITRO COMPARISON OF HUMAN FIBROBLASTS FROM INTACT AND
RUPTURED ACL FOR USE IN TISSUE ENGINEERING
T. Brune1,2*, A. Borel1, T.W. Gilbert3, J.P. Franceschi4, S.F. Badylak3 and P. Sommer1
1 Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique - Université Claude
Bernard Lyon 1, Lyon, France
2 Natural Implant, Brest, France
3 McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA,
4 Hôpital de la Conception, Centre Hospitalier Universitaire de Marseille, Marseille, France
T. Brune et al. Fibroblasts for tissue engineering
(rACL) ACL biopsies before and after seeding on porcine
small intestinal submucosa (SIS-ECM). SIS-ECM has been
used clinically for augmentation of the injured rotator cuff,
Achilles tendon and other musculo-tendinous tissues. SIS-
ECM has been studied in the ACL location in a previous
study and showed a decrease in tensile strength during the
first 2-3 months, followed by an increase in strength to a
value approximating that of the normal ACL at 12 months
(Badylak et al., 1999). While such results are promising,
SIS-ECM did not improve the strength of the remodelled
ACL when compared to an autologous patellar tendon
graft. Several studies have shown that SIS-ECM is a
favourable substrate for attachment and proliferation of
various cells types, including human endothelial, epidermal
and fibroblastic cells (Badylak et al., 1998; Hodde et al.,
2002; Lindberg and Badylak, 2001). However, to our
knowledge, there are no studies in which fibroblasts
derived from a ruptured ACL were seeded on SIS-ECM to
create a tissue engineered ACL construct.
Traditional paraffin histology was performed to make
comparisons of the cell and tissue organization.
Comparisons of the cells were based on morphology,
immunostaining, and gene expression of fibroblasts by RT-
PCR. Immunostaining and RT-PCR results were combined
to study smooth muscle actin (SMA), the composition of
the endogenous extracellular matrix (eECM), in particular
collagen and elastic fibre organization. The interactions
between the ACL fibroblasts and the surrounding eECM
were evaluated by fibronectin and integrin expression
Materials and Methods
Biopsy Sourcing and Treatment
Human iACL and rACL biopsies were harvested from
twelve patients during standard orthopaedic surgeries (total
knee arthroplasty and ACL replacement, respectively) in
order to complete a homogenous tissue bank (Table 1): 6
women and 6 men, 6 iACL and 6 rACL. Generally, total
knee arthroplasty is performed on relatively old patients;
therefore iACL came from patients with an average age of
66.8 ± 15.9 years. Conversely, rACL came from patients
whose average age was 33.2 ± 12.5 years. This difference
was considered during data analysis.
Immediately upon harvest, each biopsy was divided in
3 equal parts. The first part was fixed using 4%
paraformaldehyde or picroformol Bouin aqueous solution
for histological and immunohistochemical analysis. The
second part of the biopsy was preserved in an RNA
stabilization reagent (RNAlaterTM, Qiagen, Courtaboeuf,
France) for semi-quantitative RT-PCR. The third part was
subjected to a validated decontamination protocol followed
by comminution into small fragments and incubation in
collagenase-1 overnight at 37°C for cell extraction by
enzymatic digestion. Extracted cells were grown to
confluence in Dulbecco’s minimal essential medium
(DMEM) supplemented with 10% new born calf serum
(NBCS), 0.5 mM ascorbic acid 2 phosphate (AA2P), 1000
UI/ml penicillin, 20 µg/ml gentamicin, 1 µg/ml
amphotericin B. Cells were removed with trypsin-EDTA
for subculture and used within passage 3.
Preparation of SIS-ECM
Porcine small intestine was harvested from market weight
pigs (~110-130 kg) immediately after euthanasia. The
tissue was rinsed and mechanically delaminated to remove
the tunica muscularis externa (abluminal side) and the
majority of the tunica mucosa above the muscularis
mucosae (luminal side). The remaining tunica submucosa
and basilar portion of the tunica mucosa consisted of ECM
and the constituent cells. The SIS-ECM was then
disinfected and decellularized in a 0.1% peracetic acid/
4% ethanol solution followed by two rinses each in
phosphate buffered saline (PBS) and deionised water. This
process yielded a sheet of acellular SIS-ECM material that
was then freeze-dried and terminally sterilized with
ethylene oxide. The thickness of the sheets was
approximately 50-100 µm.
ACL-Biopsy Patient’s age
Table 1: Tissue bank completed for the comparative study.
T. Brune et al. Fibroblasts for tissue engineering
Immunohistochemical localization of beta 1-integrins in
anterior cruciate and medial collateral ligaments of human
and rabbit. J Orthop Res 10: 96-599.
Gilbert TW, Stewart-Akers AM, Sydeski J, Nguyen TD,
Badylak SF, Woo SL (2007) Gene expression by fibroblasts
seeded on small intestinal submucosa and subjected to
cyclic stretching. Tissue Eng 13: 1313-1323.
Hannafin JA, Attia EA, Henshaw R, Warren RF,
Bhargava MM (2006) Effect of cyclic strain and plating
matrix on cell proliferation and integrin expression by
ligament fibroblasts. J Orthop Res 24: 149-158.
Henshaw DR, Attia E, Bhargava M, Hannafin JA
(2006) Canine ACL fibroblast integrin expression and cell
alignment in response to cyclic tensile strain in three-
dimensional collagen gels. J Orthop Res 24: 481-490.
Hodde JP, Record RD, Tullius RS, Badylak SF (2002)
Retention of endothelial cell adherence to porcine-derived
extracellular matrix after disinfection and sterilization.
Tissue Eng 8: 225-234.
Hsieh AH, Sah RL, Paul Sung KL (2002)
Biomechanical regulation of type I collagen gene
expression in ACLs in organ culture. J Orthop Res 20:
Kartus J, Movin T, Karlsson J (2001) Donor-site
morbidity and anterior knee problems after anterior cruciate
ligament reconstruction using autografts. Arthroscopy 17:
Kim SG, Akaike T, Sasagaw T, Atomi Y, Kurosawa H
(2002) Gene expression of type I and type III collagen by
mechanical stretch in anterior cruciate ligament cells. Cell
Struct Funct 27: 139-144.
Laurencin CT, Freeman JW (2005) Ligament tissue
engineering: an evolutionary materials science approach.
Biomaterials 26: 7530-7536.
Lindberg K, Badylak SF (2001) Porcine small intestinal
submucosa (SIS): a bioscaffold supporting in vitro primary
human epidermal cell differentiation and synthesis of
basement membrane proteins. Burns 27: 254-266.
Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, Spencer
JA, Yanagisawa H, Zuo J, Li T (2004) Elastic fiber
homeostasis requires lysyl oxidase-like 1 protein. Nat
Genet 36: 178-182.
Lo IK, Marchuk LL, Hart DA, Frank CB (1998)
Comparison of mRNA levels for matrix molecules in
normal and disrupted human anterior cruciate ligaments
using reverse transcription-polymerase chain reaction. J
Orthop Res 16: 421-428.
Loh JC, Fukuda Y, Tsuda E, Steadman RJ, Fu FH, Woo
SL (2003) Knee stability and graft function following
anterior cruciate ligament reconstruction: Comparison
between 11 o’clock and 10 o’clock femoral tunnel
placement. 2002 Richard O’Connor Award paper.
Arthroscopy 19: 297-304.
Lowrie AG, Salter DM, Ross JA (2004) Latent effects
of fibronectin, alpha5beta1 integrin, alphaVbeta5 integrin
and the cytoskeleton regulate pancreatic carcinoma cell
IL-8 secretion. Br J Cancer 91: 1327-1334.
McKean JM, Hsieh AH, Sung KL (2004) Epidermal
growth factor differentially affects integrin-mediated
adhesion and proliferation of ACL and MCL fibroblasts.
Biorheology 41: 139-152.
Murray MM, Martin SD, Martin TL, Spector M (2000)
Histological changes in the human anterior cruciate
ligament after rupture. J Bone Joint Surg Am 82-A: 1387-
Murray MM, Spector M (2001) The migration of cells
from the ruptured human anterior cruciate ligament into
collagen-glycosaminoglycan regeneration templates in
vitro. Biomaterials 22: 2393-2402.
Murray MM, Spector M (1999) Fibroblast distribution
in the anteromedial bundle of the human anterior cruciate
ligament: the presence of alpha-smooth muscle actin-
positive cells. J Orthop Res 17: 18-27.
Neurath MF, Printz H, Stofft E (1994) Cellular
ultrastructure of the ruptured anterior cruciate ligament. A
transmission electron microscopic and immuno-
histochemical study in 55 cases. Acta Orthop Scand 65:
Palaiologou AA, Yukna RA, Moses R, Lallier TE
(2001) Gingival, dermal, and periodontal ligament
fibroblasts express different extracellular matrix receptors.
J Periodontol 72: 798-807.
Spector M (2001) Musculoskeletal connective tissue
cells with muscle: expression of muscle actin in and
contraction of fibroblasts, chondrocytes, and osteoblasts.
Wound Repair Regen 9: 11-18.
Spindler KP, Clark SW, Nanney LB, Davidson JM
(1996) Expression of collagen and matrix
metalloproteinases in ruptured human anterior cruciate
ligament: an in situ hybridization study. J Orthop Res 14:
Thomassin L, Werneck CC, Broekelmann TJ, Gleyzal
C, Hornstra IK, Mecham RP, Sommer P (2005) The Pro-
regions of lysyl oxidase and lysyl oxidase-like 1 are
required for deposition onto elastic fibers. J Biol Chem
Yanagisawa H, Davis EC, Starcher BC, Ouchi T,
Yanagisawa M, Richardson JA, Olson EN (2002) Fibulin-
5 is an elastin-binding protein essential for elastic fibre
development in vivo. Nature 415:168-171.
Yokosaki Y, Matsuura N, Higashiyama S, Murakami
I, Obara M, Yamakido M, Shigeto N, Chen J, Sheppard D
(1998) Identification of the ligand binding site for the
integrin alpha9 beta1 in the third fibronectin type III repeat
of tenascin-C. J Biol Chem 273: 11423-11428.
Discussion with Reviewers
John Hunt: The study was fundamentally challenged by
the disparity in patient profiles and therefore tissue
physiology for the two groups with “normal” and ruptured
Authors: We have included information related to the
pathology of the knee necessitating arthroplasty. In the
discussion we did also bring up the point that the fibroblasts
from the unruptured ACLs may not represent the normal
Hanns Plenk: You and everybody else starts cell seeding
experiments with a solution containing cells per ml, and
T. Brune et al. Fibroblasts for tissue engineering
not per cm2. Therefore, you should first give this number,
and then, if you feel it so important, you could calculate
once this theoretical cell “density” per cm2, but keep in
mind that you can never overcome the problem of
distributing cells in solution uniform over a certain surface!
Authors: You did not appreciate the fact that the seeding
densities were formulated in cells per cm2. You thought
that it was a mistake and suggested cells per cm3, or cells
per ml. Actually, we really wanted to mean cells per cm2,
and cm2 refers to the surface area (expansion) of SIS-ECM
seeded with cells. When we wrote “1,000,000 cell per cm2”,
we meant that the volume of the seeding solution had been
adapted in order to have 1,000,000 cells on each cm2 of
Brian Johnstone: It cannot be concluded from
immunostaining that cellular constructs ‘consisted
primarily of collagen type I and fibronectin’. It can be stated
that these two ECM molecules are widely distributed
throughout the newly formed matrix but not that they are
the primary molecules. The age-related differences for
elastin should be retained and a cellular construct added,
indicating the lack of staining. This would also serve as a
negative control for the other staining images, since no
negative control is included. The vague statements that
‘nearly all cells’, ‘most cells’ and ‘few cells’ stained for
the various integrins need to be given some better
Authors: Immunostaining is not a method which brings
quantitative data. However, evaluating a large amount of
sections, we were able to determine numerical values to
describe our observations. For example, the authors
observed that the amount of SMA positive cells in the cell
constructs was notably higher after 2 weeks in culture, as
compared to after 1 week, and then not different anymore
after 2 and 3 weeks.