Cell Tissue Bank (2017) 18:369–381
DOI 10.1007/s10561-017-9635-4
Co-culture of dedifferentiated and primary human
chondrocytes obtained from cadaveric donor enhance
the histological quality of repair tissue: an in-vivo animal
study
Anell Olivos-Meza . Cristina Velasquillo Martı́nez . Brenda Olivos Dı́az .
Carlos Landa-Solı́s . Mats Brittberg . Raul Pichardo Bahena . Carmina Ortega Sanchez .
Valentin Martı́nez . Enrique Alvarez Lara . José Clemente Ibarra-Ponce de León
Received: 21 December 2016 / Accepted: 26 May 2017 / Published online: 5 June 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract To compare the quality of the repair tissue
in three-dimensional co-culture of human chondro-
cytes implanted in an in vivo model. Six cadaveric and
five live human donors were included. Osteochondral
biopsies from the donor knees were harvested for
chondrocyte isolation. Fifty percent of cadaveric
chondrocytes were expanded until passage-2 (P2)
while the remaining cells were cryopreserved in
passage-0 (P0). Fresh primary chondrocytes (P0f)
obtained from live human donors were co-cultured.
Three-dimensional constructs were prepared with a
monolayer of passage-2 chondrocytes, collagen mem-
brane (Geistlich Bio-GideÒ), and pellet of non-co-
cultured (P2) or co-cultured chondrocytes (P2 ? P0c,
P2 ? P0f). Constructs were implanted in the
A. Olivos-Meza
Orthopedic Sports Medicine and Arthroscopy Service,
Instituto Nacional de Rehabilitación, Mexico City,
Mexico
C. Landa-Solı́s
C. Ortega Sanchez
V. Martı́nez
Tissue Engineering, Cell Therapy and Regenerative
Medicine Unit, Instituto Nacional de Rehabilitación,
Mexico City, Mexico
B. Olivos Dı́az
Universidad Nacional Autónoma de México,
Mexico City, Mexico
B. Olivos Dı́az
Instituto Nacional de Rehabilitación, Mexico City,
Mexico
subcutaneous tissue of athymic mice and left for
3 months growth. Safranin-O and Alcian blue staining
were used to glycosaminoglycan content assessment.
Aggrecan and type-II collagen were evaluated by
immunohistochemistry. New-formed tissue quality
was evaluated with an adaptation of the modified
O’Driscoll score. Histological quality of non-co-
cultured group was 4.37 (SD ±4.71), while co-
cultured groups had a mean score of 8.71 (SD
±3.98) for the fresh primary chondrocytes and 9.57
(SD ±1.27) in the cryopreserved chondrocytes. In
immunohistochemistry, Co-culture groups were
strongly stained for type-II and aggrecan not seen in
the non-co-cultured group. It is possible to isolate
viable chondrocytes from cadaveric human donors in
M. Brittberg
Region Halland Orthopaedics, Kungsbacka Hospital,
Kungsbacka, Sweden
R. Pichardo Bahena
E. Alvarez Lara
Pathology Service, Instituto Nacional de Rehabilitación,
Mexico City, Mexico
J. C. Ibarra-Ponce de León (&)
Instituto Nacional de Rehabilitación, Av México-
Xochimilco No. 289, Arenal de Guadalupe, ZC,
14389 Mexico City, Mexico
e-mail: cibarra.corresponding@hotmail.com
C. Velasquillo Martı́nez
Subdirection of technological research, Instituto Nacional
de Rehabilitación, Mexico City, Mexico
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samples processed in the first 48-h of dead. There is
non-significant difference between the numbers of
chondrocytes isolated from live or cadaveric donors.
Cryopreservation of cadaveric primary chondrocytes
does not alter the capability to form cartilage like
tissue. Co-culture of primary and passaged chondro-
cytes enhances the histological quality of new-formed
tissue compared to non-co-cultured cells.
Keywords Chondrocyte dedifferentiation
Co-
culture
Cartilage repair
Tissue engineering

Cadaveric chondrocytes
Introduction
Articular cartilage lesions have been reported in more
than 60% of knee arthroscopic procedures in young
patients (Curl et al. 1997; Farr et al. 2011; Hjelle et al.
2002; Johnstone et al. 2013; McNickle et al. 2008;
Widuchowski et al. 2007). Chondral lesions generally
do not heal or heal partially under biological conditions
if the defects penetrate subchondrally into bone
marrow area. The physiological process of sponta-
neous repair plays a key role in a number of surgically
based techniques (Hunziker 2002). The treatment of
cartilage defects is a major challenge in the orthopedic
field because articular cartilage has a low potential of
intrinsic repair (Ahmed et al. 2009; Giovannini et al.
2010; Hamada et al. 2013; Kolettas et al. 2001; Negri
et al. 2007; Olivos-Meza et al. 2010; Wei et al. 2012).
Surgical strategies available to treat cartilage defects
are in general two. The first approach is to enhance the
intrinsic healing capacity of subchondral bone and
cartilage through a migration of mesenchymal stem
cells (bone marrow stimulation techniques) into the
defect area leading to the formation of fibrocartilage
which does not have the resistance of hyaline cartilage
due to its biochemical composition (Ibarra et al. 2014;
Kolettas et al. 2001; Negri et al. 2007). The second
alternative is to elicit a biological repair by tissue
engineering methods that provide a formation of
hyaline like cartilage. The last approach has resulted
in long-term durability and functionality compared
with the bone marrow stimulation techniques. How-
ever, with chondrocytes contributing to less than 5% of
the total tissue volume, and the limited biopsy size that
can be collected from a minor weight-bearing area,
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only a small number of cells can be retrieved from a
patient. This issue is one major problem in the field of
cartilage repair (Giovannini et al. 2010). Since cell-
based therapy requires a large quantity of cells, it seems
that any attempt to ‘‘regenerate’’ hyaline like cartilage
requires in vitro chondrocyte expansion to increase cell
number by passaging and to be able to fill a cartilage
defect with chondrogenic cells in high density (Bagha-
ban Eslaminejad et al. 2009; Bian et al. 2011; Casper
et al. 2010; Oseni et al. 2013).
Chondrocytes possess a high proliferative capacity
when being cultured in monolayers; however, the
propensity of chondrocytes to dedifferentiate into
fibroblasts over repeated passage is a major problem
(Baghaban Eslaminejad et al. 2009; Gartland et al.
2005; Oseni et al. 2013; Qing et al. 2011).
Dedifferentiated chondrocytes changed their
morphology from rounded to fibroblastic and
steadily lose the expression of the cartilage-specific
genes such as those encoding collagen type II and
proteoglycans (Ahmed et al. 2009; Baghaban
Eslaminejad et al. 2009; Hamada et al. 2013;
Kolettas et al. 2001). It has been strongly suggested
that dedifferentiation of chondrocytes affects the
quality of the cartilage regenerated by Autologous
Chondrocyte Implantation (ACI) (Kolettas et al.
2001). Reports have shown that co-culture in
in vitro systems enhance the quality of expanded
dedifferentiated chondrocytes (Juhasz et al. 2014).
Two types of co-cultures have been described:
direct (cell–cell contact) and indirect (soluble
mediators). Some studies suggest that soluble
mediators such as TGF-B1, IGF-I, and BM-P2
may play a role in cell–cell interaction in co-
cultures both in vivo and in vitro (Juhasz et al. 2014;
Tew et al. 2008). However other studies have shown
that direct cell–cell contacts are preferentially
responsible for improved chondrogenesis in co-
culture systems (Bian et al. 2011; Juhasz et al.
2014). In a rabbit study, Chang et al. reported that
mature cartilage cells secrete growth factors such as
transforming growth factor-B (TGF-B) and insulin-
like growth factor-1 (IGF-1), which induce chon-
drocyte differentiation of Bone Marrow Stem Cells
(BMSCs) in co-cultures. Although BMSCs could be
induced into cartilage cell-like cell, they did not
determine whether the co-culture of chondrocytes
and BMSCs was more effective than single culture
of each cell type. The same effect could promote
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reversible morphological and physiological changes
in dedifferentiated chondrocytes co-cultured with
primary chondrocytes (Ahmed et al. 2009).
Limited donor tissue could be solved with the use of
allogenic cartilage as a source for the acquisition of
sufficient number of chondrocytes of the appropriate
phenotype for cryopreservation, expansion, or co-
culture models. However, the use of cadaveric carti-
lage for chondrocyte isolation, its chondrogenic
capability, and cell viability through the time has not
been described in the literature.
In this study, we compared the quality of the
repaired tissue in three-dimensional co-culture (por-
cine collagen type III and type I scaffold) of human
chondrocytes implanted in an in vivo model.
Materials and methods
Cadaveric donor cartilage harvesting
Cadaveric human donors from Musculoskeletal Tissue
and Skin Bank (Biograft of Mexico) with age from 15
to 50 years were enrolled in this study. Osteochondral
cylinders of 8–10 mm (COR; DePuy Mitek, Rayn-
ham, MA) diameter were harvested from any healthy,
smooth and white articular cartilage area from both
knees. Biopsies were harvested in sterile conditions by
the procuration staff of Biograft tissue banking after
the donor arrangements were solved. Osteochondral
samples were placed in a sterile container with culture
medium (Dulbecco’s Modified Eagle Medium F12
GIBCO, Grand Island, NY) containing 10% antibi-
otics/antimycotic. Samples were transported at 4 °C
from the hospital were the patient died to the National
Institute of Rehabilitation where they were processed.
One osteochondral plug in every donor was taken as a
control for histology and immunohistochemistry.
Live donor cartilage harvesting
After institutional review board evaluation and
approval of this study from local ethics committee,
live donors were invited to participate in this study. If
subjects accepted to participate informed consent was
signed. Patients scheduled for arthroscopic anterior
cruciate ligament (ACL) reconstruction between 18 and
50 years of age were included as donors for recently
isolated chondrocytes biopsies. We enrolled only
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patients who were scheduled to trans-tibial ACL
reconstruction due to this procedure requires taking a
cartilage fragment of the lateral wall of the notch
(notchplasty) to see perfectly the background of the
notch. All patients underwent regional anesthesia, after
diagnostic arthroscopy and confirmation of the ACL
rupture, cleaning of the footprint and notchplasty of the
lateral femoral condyle were performed. Instead of
traditional notchplasty with bone burr, two cylinders of
4 mm diameter were obtained from a non-weight-
bearing area adjacent to the intercondylar notch (COR;
DePuy Mitek, Raynham, MA) (Fig. 1). The osteochon-
dral cylinders were placed in a sterile container with
transport media (Dulbecco’s Modified Eagle Medium
F12 GIBCO, Grand Island, NY) containing 10%
antibiotics/antimycotic agents and sent out to the tissue
engineering laboratory for chondrocyte isolation, co-
culturing, and cell-collagen scaffold formation (con-
struct). After osteochondral cylinder harvesting,
remodeling of lateral wall of the notch was finished
with an arthroscopic 5 mm bone burr; then ACL
reconstruction was performed in a regular manner.
Chondrocyte isolation
The cadaveric and live donor’s osteochondral cylin-
ders were processed in the Tissue Engineering and
Regenerative Medicine Laboratory at the National
Institute of Rehabilitation. Under sterile conditions in
a laminar flow hood (100-class), transported medium
was drained, samples were washed three times with
PBS 10% antibiotic/antimycotic, and cartilage was
separated from bone by sharp dissection. Chondral
fragments were chopped in small pieces with a scalpel
and then fragments were digested with type-I-colla-
genase at 0.3% in DMEM-F12 medium for around 5-h.
Isolated cells were counted in Neubauer chamber and
viability was assessed by trypan-blue stain by two
blind observers. Eighty percent of primary isolated
chondrocytes from cadaveric donors were seeded in
T25 culture flask at a density of 250,000 cells and were
expanded until Passage-2 (P2). The remaining twenty
percent of cadaveric donor chondrocytes were stored
and cryopreserved in cryo-vials with 10% DMSO and
Bovine Fetal Serum (BFS) at a density of 5 9 105
cells per vial. Temperature was reduced gradually and
cells were stored in liquid nitrogen at -280 °C.
Primary fresh chondrocytes (P0f) were obtained from
live donors until the cadaveric chondrocytes reached
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Fig. 1 a Two osteochondral biopsies with a diameter of 4 m
were harvested in the lateral notch, then the notchplasty was
finished with a burr. b Samples were handled carefully, taking
passage-2. Isolation was performed as described
above and used immediately for co-culturing and
construct formation (P2 ? P0f group).
Expansion and chondrocyte dedifferentiation
Primary isolated chondrocytes from cadaveric donors
were seeded into T-25 culture flasks at a density of
250,000 cells with 5 ml of culture medium (Dul-
becco’s Modified Eagle Medium F12 GIBCO, Grand
Island, NY), 1% antibiotic–antimycotic agents, and
supplemented with 10% human serum. Cells were
expanded in monolayer culture until 90–100% con-
fluence was reached. Cultures were trypsinized and re-
seeded for cell expansion until passage-2. At the
beginning of the second passage, fifty percent of cells
were seeded in T-25 flasks for pellet formation. To
obtain a handle monolayer, the remain chondrocytes
were seeded in 60 9 15 mm Petri dishes. Culture
medium for these cells was supplemented with
ascorbic acid (150 mcg/50 ml medium). Medium
changes were performed twice a week.
Cryopreservation of primary chondrocytes
The remaining cadaveric donor primary chondrocytes
were cryopreserved in 2 ml-cryovials with 10%
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care do not touch and damage the articular cartilage. c Ten
millimeters of bone were preserved to manipulate and process
the cartilage without causing damage of the tissue
DMSO and Bovine Fetal Serum (BFS) at a density
of 5 9 105 cells per vial. Temperature was reduced
gradually. First received samples were stored in a
refrigerator at minus 20 °C for 20 min, then vials were
moved to -80 °C for 24-h. Finally cells were stored in
liquid nitrogen at -280 °C. Primary cryopreserved
chondrocytes (P0c) were stored until the cultured cells
reached the Passage-2 for co-culture and cell-collagen
scaffold formation (P2 ? P0c group).
3D cell-collagen scaffold preparation
Once the cadaveric chondrocytes expanded until
passage-2 reached a confluency of 90–100% the
constructs were prepared from a monolayer of chon-
drocytes in passage-2 together with a collagen type I
and III membrane (Geistlich Bio-GideÒ) as scaffold,
and a pellet of either passaged-2 chondrocytes (non-
cocultured) or co-cultured cells (P2 ? P0c, P2 ? P0f)
was added (Figs. 2, 3).
First, the peripheral borders of the monolayer were
peeled-off from the bottom of the petri dish (Fig. 2a)
followed by a circle of 8 mm of collagen membrane
that was placed over the center of monolayer with the
porous-absorbable side facing up (Fig. 2b). Finally, a
pellet of co-cultured or non-co-cultured cells was
added with a 200 ll micropipette over the scaffold
Cell Tissue Bank (2017) 18:369–381
(Fig. 2c). The pellet in every construct contained a
total of 2 9 106 chondrocytes as follow: (a) No-
cocultured group: 2 9 106 Passage-2 cadaveric chon-
drocytes (P2); (b) Co-Cultured Cadaveric cells:
1.6 9 106 Passage-2 chondrocytes ? 4 9 105 Pas-
sage-0 chondrocytes (P2 ? P0c); and (c) Co-cultured
Cadaveric Chondrocytes/Fresh Chondrocytes from
living donors: 1.6 9 106 Passage-2 cadaveric chon-
drocytes ? 4 9 105 Fresh Passage-0 Chondrocytes
(P2 ? P0f) (Fig. 4). Co-cultured cells were mixed
with a ratio of 4:1 (4 dedifferentiated chondrocytes,
P2: 1 differentiated chondrocytes, P0) in every
construct. After the pellet was absorbed the borders
of the monolayer were taken up to cover the unit
scaffold-pellet (Fig. 2d). In this way, a construct like
crepe was formed as described by Masri et al. (2007)
(Fig. 2e). To gain consistency, the construct was
placed in the incubator without medium during
30 min. After that, 5 ml of medium (Dulbecco’s
Fig. 2 Construct formation. a Peripheral borders of the
monolayer were peel from the bottom of the petri dish. b The
collagen scaffold was placed over the center of monolayer with
the porous-absorbable side facing up. c A pellet of co-cultured
or no co-cultured cells was added over the scaffold. d The
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Modified Eagle Medium F12 GIBCO, Grand Island,
NY) containing 1% antibiotics/antimycotic supple-
mented with 10% human serum, and ascorbic acid was
added (Fig. 2f). The construct was left in the incubator
for 3 days to enhance the consistency.
In-vivo animal model implantation
All work in this study was conducted with the approval
of local Institutional Animal care and use committee.
Five days after the construct was formed, it was
implanted in male athymic mouse (Nu/Nu) with age of
4-weeks. Surgeries were done in a laminar flow hood
in the animal facility of the National Institute of
Rehabilitation. Animals underwent general anesthesia
with Isoflurane. Incision length of 6–8 mm was
performed in the dorsal area (Fig. 5a). Subcutaneous
tissue was released (Fig. 5b) and the cell construct was
implanted into this area (Fig. 5c). One or two 3-0
borders of the monolayer were taken up to cover the unit
scaffold-pellet. e, f The crepe was formed and placed in the
incubator without medium during 5 min, then medium and
ascorbic acid was added. Implantation is done after 5-days
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Fig. 3 Schematic
representation of ‘‘the crepe
technique’’. (1) A
monolayer of Passage-2
cadaveric chondrocytes was
formed adding ascorbic acid
to cultures. (2) the
monolayer was peel-off
from the bottom of the
culture plate. (3) An 8-mm
diameter collagen type III
and I scaffold was placed
over the monolayer. (4) A
pellet of 2 9 106 cells was
added to the scaffold (co-
cultured or no co-cultured
chondrocytes). (5) The
monolayer was folding to
cover the scaffold-pellet unit
in a crepe manner. (6) The
3D construct is formed and
supplemented with culture
medium and leave in the
incubator during 3 days
before implantation

nylon simple stitches were used to close the skin
(Fig. 5d). After 3-months post-implantation, animals
were sacrificed (based in the animal regulation NOM-
062-ZOO-1999). New-formed tissue was harvested
and used for histology (Fig. 6).
Carl Zeiss microscope and AxioVision 4.8.2. Imaging
system. Histological parameters were assessed and
scored.
Immunohistochemical evaluation

Histological evaluation Tissue sections were fixed on a slide if de-waxed,

The cartilage and new-formed tissue were fixed in 4%
phosphate-buffered with formaldehyde, after being
dehydrated and embedded in paraffin: Three-micron
thickness sections were taken from the center and
stained with Hematoxylin–Eosin (H&E) to assess
morphology, Safranin-O (SO), and Alcian Blue (AB)
to localize the amount of glycosaminoglycan content.
We performed visualization and photography using a
followed by treatment with hyaluronidase (Sigma;
4800 U/ml in 0.025 M NaCl 0.05 M sodium acetate
pH 5.0, 2 h at RT for frozen sections) to unmask the
collagen antigens. Sections were then incubated for
1 h at room temperature with primary antibodies
against type II collagen. Endogenous peroxidase was
blocked with 0.3% hydrogen peroxide in methanol
before sections were incubated with the biotinylated
secondary antibody anti-mouse. The signal was

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Cell Tissue Bank (2017) 18:369–381 375

Fig. 4 Cell groups

distribution. Two groups of
donors were used to isolate
chondrocytes: cadaveric and
living donors. Fifty percent
of chondrocytes isolated
form cadaveric donors were
expanded until passage-2 to
form a monolayer culture
while the remain 50% was
cryopreserved in passage-0
for further use in
cryopreserved chondrocytes
co-culture. The
chondrocytes isolated from
living donors were not
expanded, they were use in
passage-0 for co-culture of
fresh chondrocytes

Fig. 5 After mouse was anesthetized, an incision of 8–10 mm was performed in the dorsal area (a), subcutaneous tissue under the skin
incision was released (b), and the construct was implanted (c). One 3-0 nylon simple stitches were used to close the skin (d)

amplified using an avidin–biotin–peroxidase reagent
(Vectastain Elite ABC kit; Vector Laboratories,
Peterborough, UK) and labelling was visualized with
diaminobenzidine as substrate. Sections were then
washed, dehydrated, and mounted in pertex. ‘Control’
sections of biopsy samples were treated either with
normal mouse IgG, normal rabbit serum or phosphate
buffered saline alone, in place of the primary antibod-
ies and then treatment continued in the same way as all
the other slides.
The degree of immunohistochemical labelling was
assessed semi-quantitatively by measuring the areas of

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Fig. 6 a New-formed tissue (black arrow) in the subcutaneous
area of atomic mouse. b After 3 months of construct implan-
tation mice were sacrificed and new formed tissue was harvested
the repair cartilage, on adjacent sections where
possible, which were immunostained for collagen
type II. Images were captured using an Optivision
Image Capture system. The area of positive staining
was delineated and this was calculated as a percentage
of the total area.
Statistical analysis
Data are reported as means (±) SD and were analyzed
statistically using the SPSS version 20.0 software
(LEAD Technologies, Inc., USA). The statistical
significance of the differences between groups was
determined by one-way analyses of variance
(ANOVA). p \ 0.05 was considered to be statistically
significant.
Results
Cadaveric osteochondral biopsies
We received a total of thirty osteochondral samples
from six cadaveric donors. However, two of those
were excluded due to positive serological testing to
Human T-Lymphotropic Virus (HTLV) and Syphilis;
eight biopsies from these positive donors were
excluded.
Mean donor’s age was 30.5 years (15–41). All
donors were men; the mean number of biopsies
obtained per donor was 5.5 (±1.91) with a mean
diameter of 8.5 mm (±1.0). Mean cartilage weight per
donor was 1560 mg (±587.88) with a mean of
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Cell Tissue Bank (2017) 18:369–381
and measured; macroscopic characteristics were recorded and
tissue was processed for histology and immunohistochemistry
3,044,908 (±1,161,090) viable isolated cells. Mean
number of chondrocytes per milligram of cartilage
was 1951 (±70.3). Mean time from death to cartilage
processing was 21.75 h (±6.13). Causes of death were
liver failure, lung thromboembolism, brain death and
cranioencephalic trauma. All osteochondral biopsies
were transparent, pearly bluish in color with firm
consistency. No crystal deposition or chondral damage
was identified in any sample.
Live donor osteochondral biopsies
Five live donors were included in this study. Three
subjects were men (60%) and two were women (40%).
All patients had diagnosis of ACL rupture. Mean
donor age was 32 (±11.33), we obtained an average of
1.4 (±0.54) biopsies from every patient with a
diameter of 4 mm. Mean cartilage weight per donor
was 97.54 mg (±69.28) with a mean of 605,600
(±514,451) viable isolated cells. The mean number of
chondrocytes per milligram of cartilage was 4396
(±2506). Mean time from osteochondral biopsy
harvesting to cartilage processing was 1.9 h (±0.54).
As in the cadaveric donor all biopsies were transpar-
ent, pearly bluish in color with firm consistency. No
crystal deposition or chondral damage was identified
in any sample.
Live donor versus cadaveric donor isolated
chondrocytes
Significant statistical difference was found in process-
ing time (biopsy to chondrocyte isolation) between
Cell Tissue Bank (2017) 18:369–381
cadaveric donor (mean 21.7-h) and live donor samples
(mean 1.9-h) (p = 0.007). However, we did not found
significant difference between the number of primary
chondrocytes per milligram of tissue isolated from
cadaveric versus live donor cartilage (p = 0.12).
Chondrocyte viability
After isolation, chondrocyte viability was assessed in
the 18-cadaveric and five-live donor processed sam-
ples. Total surface area of cartilage from cadaveric
samples was 184 mm with a weight of 6240 mg, and
12,179,632 viable chondrocytes while in live donors
total cartilage surface was 28 mm with a total weight
of 487.7 mg, and 3,028,000 viable chondrocytes.
Chondrocytes expansion
Mean number of seeded chondrocytes at Passage-1
was 1,250,160 (±353,402) cells obtaining a mean of
12,138,000 (±3,744,224) chondrocytes at the end of
Passage-2 with a mean expansion rate of 10.03
(±3.82).
In this study, we observed a close relation between
age and expansion rate. The younger the patient the
greater the number of chondrocytes at the final culture.
The youngest donor was 15 years-old and had the
greatest expansion rate (15.2 fold) while the oldest
donor was 41 years-old and showed the lowest
number of expanded chondrocytes (6.67 fold).
Gross observations of new-formed tissue
To minimize manipulation of the implanted samples
during harvesting, precise measurements of the size of
new-formed tissue were not taken. However, we
observed a reduction of the original size. After 3 months
of implantation, the consistency of the constructs
changed from soft and viscous tissue to firm consistency
like a cartilage tissue. Surface was regular and smooth,
but the color kept yellow as in the beginning.
Cell morphology
Chondrocyte morphology changed through the sub-
cultures from round or polyedrich in recently isolated
cells to fibroblastic-like when cells were passaged in
monolayer cultures. Dedifferentiation process
increased with passage numbers.
377
Non-co-culture chondrocytes seeded in 3D-cell-
collagen-constructs had fibroblastic-like cells, while
co-cultured chondrocytes either cryopreserved or
freshly isolated retained the rounded shape character-
istic from chondrocytes.
Tissue morphology and glycosaminoglycan
content evaluation
Hyaline-like cartilage observed in the co-cultured
constructs differed clearly from the fibrous-like tissue
formed by non-co-cultured passaged cells because of
its homogenous appearance of the matrix and the
round or oval shape of cells, often surrounded by
lacunae (Fig. 7b, c). Fibrous-like cartilage in contrast,
had obvious bundles of collagen fibers lying in a
random, irregular manner and often elliptical shaped
cells (Fig. 7d).
Glycosaminoglycan (GAGs) content was assessed
by Safranin-O and Alcian blue staining. There was
more intense matrix around lacunae in new-formed
tissue from co-cultured cryopreserved chondrocytes
(Fig. 7b) than in the tissue obtained from co-cultured
fresh chondrocytes (Fig. 7c) but in both cell types
there were extensive stained areas.
Quality of the repair tissue
Although in different amount, as illustrated in histo-
logical images, neocartilage was formed in either non-
co-cultured and co-cultured groups. However, the
structural characteristics in the repair tissue appeared
to be better in the cryopreserved and fresh primary
chondrocytes group compared to Passage-2 con-
structs. These observations are reflected in the histo-
logical scores. Three items of the modified O’Driscoll
score (Table 1) were used to quantify the structural
characteristics of the new-formed tissue, which
includes cellular morphology, Safranin-O staining,
and hypo cellularity. Additional to those three scor-
ings, we enclosed how easy it was to find the new-
formed tissue with different microscope objectives
(49, 109, and 209). The maximum score of 14
corresponded to normal articular cartilage. In this
study, we found the best mean score (14, SD ±0.0) in
the control samples (hyaline cartilage), while the worst
histologic results were observed in the non-co-cul-
tured group (4.37, SD ±4.71). Although co-cultured
chondrocytes did not get as good score as control
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Fig. 7 a Alcian blue stained histological sections of human
knee articular cartilage obtained from cadaveric donor used as a
control to compare the presence of GAGs content with the
repaired tissue in experimental groups. b, c Alcian blue staining
was more intense and stained more extensive areas in the new-
formed tissue obtained from co-cultured cryopreserved chon-
drocytes than in the tissue from co-cultured fresh chondrocytes.
d Small zones of light staining were observed in tissue formed
by no–co-cultured cells (P2)

Table 1 Modified O’Driscoll histological score (modified)

O’Driscoll score with
detailed breakdown of the Cellular morphology
histological items to
Hyaline articular cartilage 4
consider in the evaluation of
the neo-formed cartilage Incompletely differentiated mesenchyme 2
Fibrous tissue or bone 0
Safranin-O staining
Normal or nearly normal 3
Moderate 2
Slight 1
None 0
Hypocellularity
Considered characteristics
to evaluate in histology are: Normal cellularity 3
cell morphology, intensity Slight hypocellularity 2
of Safranin-O staining, Moderate hypocellularity 1
hypocellularity, and
feasibility to identify Severe hypocellularity 0
cartilage-like tissue by Microscopic identification
microscope. Highest score Easy identification at 49 4
is fourteen, those results
Easy identification at 109 3
means that repaired tissue is
more like cartilage. New- Easy identification at 209 2
formed tissue with more Difficult identification at 209 1
fibrous characteristics has No identification of cartilage-like structure 0
lowest scores closed to zero

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Cell Tissue Bank (2017) 18:369–381 379

Fig. 8 Safranin-O stained

histological sections of
human knee articular
cartilage (control) and
repaired tissue from co-
cultured and no co-cultured
chondrocytes. a Cadaveric
biopsy of articular cartilage
was taken as a control (14,
SD ±0.0) in the histological
evaluation with the
O’Driscoll score system;
good quality cartilage is
observed. b, c Moderate
quality cartilage is observed
in repair tissue obtained
from co-culture of
cryopreserved chondrocytes
(9.57, ±1.27) and co-culture
of fresh chondrocytes (8.71,
±3.98). d Low quality
cartilage is observed in no
co-cultured cells (4.37, SD
±4.71) with presence of
fibrous tissue characteristics
that demonstrates few
matrix formation

samples, those groups showed better results than tissue
originated from de-differentiated chondrocytes with
mean score of 8.71 (±3.98) for the fresh primary
chondrocytes co-cultured group and 9.57 (±1.27) in
the cryopreserved primary co-cultured chondrocytes.
No statistical difference was found between non-co-
cultured chondrocytes and fresh primary co-cultured
group (p = 0.623); cryopreserved co-cultured chon-
drocytes and fresh co-cultured cells neither showed
significant difference. However, the histological score
was significantly higher (p = 0.053) in the cryopre-
served co-cultured group compared to non-co-cultured
group (Fig. 8).
Immunohistochemistry analysis
Staining for type-II collagen and aggrecan were more
intense on chondrocytes and the matrix around
lacunae in normal cartilage (control). Regenerated
cartilage from co-cultured chondrocytes (P2 ? P0c
and P2 ? P0f) had strong immunopositivity for type II
collagen and aggrecan to varying extents in all
samples of those groups. However, only light if any
immunopositivity for type-II collagen and aggrecan
were present in no-co-cultured group (P2). Unspecific
immunopositive was observed in collagen type I–III
membrane used as scaffold (Geistlich Bio-GideÒ) in
all experimental groups.
Discussion
Among all the cell sources considered for cartilage
repair, chondrocytes remain the cells of choice. Most
of the available studies reports the use of human
articular chondrocytes recently isolated in live donor,
specialty for ACI technique. The use of cadaveric
donor articular chondrocytes either fresh or

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380
cryopreserved has been not reported in the literature.
This strategy would help to cover one of the major
limitations for cell-based therapeutic approaches for
articular cartilage regeneration, the lack of sufficient
number of chondrocytes because of the donor site
morbidity and dedifferentiation process because of
expansion methods.
In this study, we demonstrate the feasibility to
obtain viable cadaveric chondrocytes despite the time
between donor dead and chondrocyte isolation in the
laboratory. Our findings suggest the possibility to
eliminate the necessity of autologous osteochondral
biopsy in ACI technique. This strategy could reduce
patient morbidities, time, costs, and removing one
surgery for cartilage biopsy.
By exploiting the paracrine signals from primary
chondrocytes (P0) over the passaged cells (P2), we
demonstrated by histology that cartilage like-tissue
formation can be achieved by in a 3D cell-collagen
scaffold co-cultured chondrocytes. In contrast, non-
co-cultured cells showed predominately fibrous repair
tissue despite their culture in three dimensional
scaffolds.
This study has limitations in that we have used
immunohistochemistry to assess the number of char-
acteristic components of hyaline cartilage as type-II
collagen and aggrecan. This method does not permit a
truly quantitative assessment to compare the results
between groups. To have more specific results, DNA
quantification to determine cartilage-specific matrix
content between groups is needed.
In order to translate our results to the clinic, further
investigations should be performed regarding the
chondrocyte immune responses and the potential
disease transmission to mitigate such concerns.
Conclusions
It is possible to isolate viable chondrocytes from
cadaveric human donors articular cartilage in samples
processed within the first 48-h. There is not significant
difference between the numbers of chondrocytes
isolated from live or cadaveric human donors. Cryop-
reservation of cadaveric primary chondrocytes does
not alter the capability to form cartilage like tissue.
Co-culture of primary and passaged chondrocytes
enhances the histological quality of new-formed tissue
compared to non-co-cultured cells.
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Cell Tissue Bank (2017) 18:369–381
Acknowledgements This work is supported by the Mexican
Council of Science (CONACyT) Grant SALUD-PDCPN-2013-
01-215138; Technology, Science and Innovation Secretary
Grants: SECITI 079 BIS/2013 and SECITI/INR/GOB-25/
2013. The authors wish specialty to thank to Biograft of
México Musculoskeletal Tissue and Skin Bank; Blood Bank &
Pathology Service at the National Institute of Rehabilitation;
and Autonomous National University of Mexico (UNAM). We
also appreciate the support and collaboration of Veterinary
Facility at the National Institute of Rehabilitation: Hugo-Lecona
DMV and Javier-Perez DMV.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
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