Tissue engineering with the use and manipulation
of Mesenchymal stem cells (MSCs) is a novel treat-
ment modality targeting applications in a great
variety of pathologies. The advantages of this
approach are numerous. They include a high
quality repair with regeneration of the injured
tissue but without fibrous tissue formation. The
site morbidity is minimal compared to the
currently used bone and cartilage autografts as a
small number of cells is required with subsequent
expansion ex-vivo. The risk for immunorejection
and pathogen transmission appears to be very low.
Furthermore, MSCs have high proliferation poten-
tial, can be handled and manipulated easily
permitting differentiation prior implantation.
With the persistent objective of clinical
applications, four main strategies have been used
in tissue engineering. These consist of the use of i.
Unfractionated fresh bone marrow cells, ii.
Culture expanded MSCs, iii. Differentiated cells,
and finally iv. Genetically modified cells that
express key growth factors. 73Excluding the first
approach, all other strategies consist of a cycle of
events that is initiated by the isolation of cells,
culture until sufficient numbers are produced and
finally re-implantation to the injured site.
The aim of this review article is to illustrate the
different available methodologies that could be
used in tissue engineering as well as to analyse
their efficacy for a widespread clinical use.
The human body houses several types of
uncommitted progenitor cells capable of giving
Mesenchymal stem cell tissue engineering:
Techniques for isolation, expansion and application
Ippokratis Pountosa, Diane Corscaddenb, Paul Emeryb,
Peter V. Giannoudisa,*
aAcademic Department of Trauma & Orthopaedics, School of Medicine University of Leeds
bAcademic Unit of Musculoskeletal Disease, School of Medicine, University of Leeds
Mesenchymal stem cells (MSCs) are undifferentiated multipotent cells
which reside in various human tissues and have the potential to differentiate into
osteoblasts, chondrocytes, adipocytes, fibroblasts and other tissues of mesenchymal
origin. In the human body they could be regarded as readily available reservoirs of
reparative cells capable to mobilize, proliferate and differentiate to the appropriate
cell type in response to certain signals. These properties have triggered a variety of
MSC-based therapies for pathologies including nonunions, osteogenesis imperfecta,
cartilage damage and myocardial infarction. The outcome of these approaches is
influenced by the methodologies and materials used during the cycle from the isolation
of MSCs to their re-implantation. This review article focuses on the pathways that are
followed from the isolation of MSCs, expansion and implantation.
© 2007 Elsevier Ltd. All rights reserved
* Corresponding author. P.V. Giannoudis, Professor,
Academic Department of Trauma & Orthopaedics,
Clarendon Wing, Level A, Leeds General Infirmary,
Belmont Grove, Leeds, LS2-9NS, United Kingdom
Tel: 0044-1132433144, Fax: 0044-1132065156
Injury, Int. J. Care Injured (2007) 38S4, S23-S33
Mesenchymal stem cells;
0020-1383/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
rise to daughter cells with restricted potential.
Examples of such cells include the totipotent
zygote, the embryonic stem cells, the haemato-
poietic stem cells and the mesenchymal stem
Mesenchymal stem cells are non-haemato-
poietic stromal cells that were first isolated from
the bone marrow (BM) but subsequently from
other adult connective tissues.78,79They exhibit
multilineage differentiation capacity being
capable to give rise to diverse cells like
osteoblasts, chondrocytes, adipocytes, myocytes,
tenocytes and possibly neural cells. 74,78
Originally MSCs were isolated from the BM and
the stroma of spleen and thymus but subsequently,
trabecular bone was described as a source of
MSCs. 33,104The frequency of MSCs obtained from
BM aspirates is about 0.01% or lower.
Recently, many other tissues were found to harbor
a population of MSCs, with MSCs being isolated
from various skeletal sites including cartilage,
synovium, fat, muscle and periosteum. 79
MSCs in the human body represent reservoirs of
reparative cells lacking tissue specific character-
istics. Signals including injury, inflammation and
necrosis could trigger their activation for
regeneration. 71An example includes the ability of
these cells to migrate and colonize the injured site
after intravenous injection. 4,54Myocardial infarc-
tion, 4 fracture, 26 ischemic cerebral disease 52,112
and spinal cord injury 108 are conditions where
these beneficial properties are demonstrated.54
Similarly, suspended MSCs injected intra-
articularly into the knee joint following injury
appeared to engraft and regenerate damaged
meniscus and cartilage. 64
At present well-defined phenotypic criteria to
characterize MSCs do not exist. This is because till
today there is not a single marker that specifically
delineates the in vivo MSCs. Therefore, positive and
negative phenotypic staining is performed which
results in a loose phenotypic definition with
significant controversies in this area (Figure 1). This
task is further aggravated by the fact that MSCs share
features with other types of cells including
endothelial, epithelial and muscle cells. 61MSCs
express various surface markers including CD9, CD10,
CD13, CD44, CD54, CD55, CD90, CD105, CD166, D7-
FIB and are negative for CD14, CD34, CD45 and
CD133. A more extensive phenotypic profile of MSCs
can be found in Table 1. 11,24,25,31,44,70,75,93
To date there is no established procedure for the
isolation of MSCs but a wide number of protocols
exist providing non-comparable data. The first and
simplest method used implies the adherence
properties of MSCs which were first identified by
the pioneer work of Friedenstein et al. 33In that
study whole bone marrow was placed in plastic
culture dishes and after 4 hours the non-adherent
cells were washed out. They observed that the
cells remained dormant for 2-4 days and then they
proliferated rapidly. A modification of this
protocol includes the density centrifugation of BM.
This technique involves the use of solutions of high
density with low viscosity and low osmotic
pressure (eg. Ficoll, Percoll) to obtain the
mononucleated fraction of BM which contains
MSCs (Figure 2).50Subsequently, adherence to
plastic occurs resulting in a population of MSCs.
The initial number of MSCs can be increased as
much as 36.6% by simple collection and replating
of the initially nonadherent cell population which
is washed out during the first feeding. 111
MSCs from solid tissues like bone could be
isolated using the above mentioned philosophy. In
this method a small piece of bone is placed
directly inside the flask or plate and cells grow out
of bone explants onto the plastic substratum
during culture. 93An alternative to this technique
is the collagenase digestion process (see below).
The issue arising from the above mentioned
techniques is the purity of the MSCs sample. The
contamination of the cells with other types of cells
like haematopoietic stem cells results in a
heterogeneity of the preparation. In order to
overcome this problem two similar techniques
namely the magnetic bead sorting technique and
the fluorescence-activated cell sorting have been
developed. Magnetic bead sorting uses epitopes
positive for MSCs which are labelled with
antibody-coated magnetic beads. 43Subsequently,
an external magnetic field is applied separating
the positive from the negative labelled cells. 43
Fluorescence-activated cell sorting (FACS) is an
alternative method to isolate MSCs. In this
technique a heterogenous population of cells (eg.
Blood, BM, etc) are characterized and separated
S24I. Pountos et al.
Figure 1. Positive phenotypic selection for MSCs.
based on the intensity of fluroescence they emit
while passing through an illuminated volume
(Figure 3).5,106The cytometer will only isolate cells
whose light scatter meets the defined parameter.
5,106More precicely, one or multiple monoclonal
antibodies tagged with fluorescent dye are bound
to a specific population of cells. Cells that will be
positive or negative for these antibodies will be
included or excluded from the respective
collecting tubes. A great variety of antibodies has
been used with this technique based on the
phenotypic profile of MSCs (Table 1).
An alternative source of MSCs to BM could be solid
tissues including bone, cartilage and fat. In order
to isolate the cells from these tissues, enzymatic
treatment with collagenases is required.
Collagenases are enzymes that are able to cleave
the peptide bonds in the triple helical collagen
Mesenchymal Stem Cell Tissue engineeringS25
Phenotypic profile of MSCs. 11,24,25,31,44,70,75,93
CD14 (Lipopolysaccharide receptor)Neg
CD45 (Leukocyte common antigen) Neg
CD133 (AC133) Neg
CD31 (Platelet Endothelial Cell Adhesion Neg
CD44 (Hyaluronate receptor)Pos
CD50 (Intercellular adhesion molecule 3) Pos
CD54 (Intercellular adhesion molecule 1)Pos
CD56 (Neural cell adhesion molecule)Pos
CD58 (Lymphocyte function-associated Pos
CD62L (L-selectin) Pos
CD102 (intercellular adhesion molecule 2)Pos
CD106 (Vascular cell adhesion molecule-1) Pos
CD144 (Calherin 5)Neg
CD166 (Activated leukocyte cell Pos
CD11a (Lymphocyte function-associated
CD11b (Macrophage-1 antigen)Neg
CD11c (Complement receptor type 4 a chain)Neg
CD18 (Lymphocyte function-associated
CD29 (Very late antigen β)
CD49a (Very late antigen a1)Pos
CD49b (Very late antigen a2)Pos
CD49c (Very late antigen a3)Pos
CD49d (Very late antigen a4) Neg
CD49e (Very late antigen a5) Pos
CD49f (Very late antigen a6)Pos
CD51 (Vitronectin R a chain)
CD61 (Vitronectin R β chain)
Phenotypic profile of MSCs. 11,24,25,31,44,70,75,93
Growth factors and Cytokines
CD71 (Transferrin receptor)Pos
CD114 (Granulocyte-colony stimulating Neg
CD117 (Stem cell factor receptor)
CDw119 (Interferon γ R)
CD120 a & b (Tumor Necrosis factor-a 1&2 R) Pos
CD121 a & b (Interleukin-1R a&b chain) Pos
CD123 (Interleukin-3R) Pos
CD124 (Interleukin-4R) Pos
CD126 (Interleukin-6R) Pos
CD127 (Interleukin-7R) Pos
CD140a (Platelet derived growth factor Pos
FGFR (Fibroblast growth factor receptor)Pos
CD271 (Low affinity nerve growth factor Pos
CD3 (CD3 complex) Neg
CD13 (Aminopeptidase N) Pos
CD19 (B-lymphocyte Surface Antigen B4) Neg
CD73 (Ecto-5’-nucleotidase) Pos
CD83 (HB15a) Neg
CD86 (B7-2) Neg
CD90 (Thy-1 glycoprotein) Pos
CD146 (MUC18,Mel-CAM, S-endo) Pos
CD157 (BP-3 or Bone Marrow Stromal cell Pos
SH3 (Src homology 3) Pos
molecule. In this way cells are released from the
tissue and can be easily collected by wash and
The isolation of MSCs with collagenase digestion
from bone is a technique used for more than two
decades. 27,51,82,109,115Unfortunately, there is not a
standardised protocol for its use, resulting in a
variety of concentrations and digestion times. 36,86,105
The yield of MSCs using enzymatic digestions
appears to be higher than that of BM aspirates.
Collagenase released fraction contains 100-fold
more MSCs compared to BM aspirates.
Furthermore, several authors suggested that these
cells are identical to those isolated from BM both
in terms of differentiation potential and
phenotypic characteristics. 85,104On the contrary,
Thomas et al suggested that enzymatic isolated
MSCs possess a higher metabolic activity,
intracellular protein levels and calcium production
compared to aspirates, while ALP activity was
found to be lower in collagenase treated cells. 101
These suggest that enzymatic treatment could
potentially result in alterations of the metabolic
profile of MSCs.
The next vital step after isolation is the ex-vivo
expansion of MSCs. This stage allows the free ex-
vivo self replication of MSCs targeting sufficient
numbers for clinical use. Several factors could
influence the yield of ex-vivo expansion of MSCs.
These factors are either donor dependent or
technique dependent. Donor dependent include the
age of the donor and the sex, the presence of
trauma, and the presence of systemic disease. 72,88,99
Similarly, MSCs from patients suffering from
Hepatitis B present with a lower growth curve
although they have the same appearances, shapes
and surface markers. 72The technical issues are
mainly related to the target number of cells that
is the number of passages of MSCs, the method of
culture and media that are used for expansion.
MSCs can be expanded tremendously within a
relatively short period of time. This rapid
proliferation could result in an expansion of a
thousand-fold in two to three weeks time. 18In
addition, MSCs could proliferate for about 19
doublings in culture without losing their property
to proliferate and differentiate.63
expansion has shown to gradually reduce the
maximal differentiation potential of MSCs. 3Exten-
sive subcultivation impairs the cells’ function
resulting in cellular senescence that is associated
with growth arrest and apoptosis. 28,99However,
retroviral transduction with human telomerase
gene can extend MSCs’ lifespan to more than 260
doublings without losing multilineage capacity.94
On the other hand, there are data suggesting that
prolonged culture could result in spontaneous
transformation acquiring tumorigenic potential. 83
In addition, it was shown that particular proper-
ties of MSCs are lost during culture. The cardio-
protective effect of MSCs is reduced in cells at
passage 5 and 10 compared to those of passage 3. 22
This could be explained by the reduced vascular
endothelial growth factor release potential.22
S26 I. Pountos et al.
Figure 2. Density gradient centrifugation for the
isolation of mononucleated fraction from bone marrow.
Figure 3. Simplified diagram of the Fluorescence-
activated cell sorting.
Therefore, a compromise should be made between
the yield and the quality of the expanded cells.
Media and serum
The most important component of successful
expansion of MSCs is the media used. Culture
medium consists of a basal medium containing
glucose, amino acids and ions including calcium,
magnesium, potassium, sodium, and phosphate, as
well as fetal animal sera in concentrations of 10%
or 20%. 29Both basal media and sera can result in
a significant difference to the MSC yield after
expansion. Sotiropoulou et al compared 8 differ-
ent basal media in terms of adherence efficacy,
growth index and final number of cells obtained in
culture. 96The results showed great differences
between basal media. The alpha Modified Eagle’s
Medium (aMEM) containing Glutamax had the best
results. The same authors also presented data to
show that the quality of plastic adherence surface
of the flasks influences both the isolation and
expansion of MSCs.
Media commonly used today contain heat
inactivated Fetal Bovine or Calf sera (FBS or FCS).
There are two main issues arising from their use in
culture media. The first is their efficacy compared
to human serum and secondly their potential side
effects such as transmission of disease and
Several studies have been conducted comparing
autologous serum with fetal animal sera. Their
results were divergent. A number of studies
suggested that autologous serum is inferior or
equivalent to animal sera but can support the
expansion of MSCs. 49,55,87,100,119Koher et al studied
the effect of autologous and allogeneic human
sera together with fetal animal sera and 8 serum
free media. 49The results suggested equivalent
performance levels between human sera which
was inferior to that of fetal animal sera. Serum
free media performed poorly.
autologous serum was found to perform as well as
the animal sera for isolation and proliferation, but
it was superior when used in osteogenic
differentiation. 100On the other hand, several
studies suggested that autologous serum is
superior to fetal animal sera. 37,47,48,58,90Our results
suggested that autologous serum isolated during
the first weeks after fracture is superior to FCS
both in terms of proliferation and osteogenic
differentiation. 77Serum free media supplemented
with serum substitutes (ULTROSER)60 or platelet
instead of FCS are recently
developed as alternatives, but their suitability for
clinical scale expansion should be tested.
The second issue arising from the use of animal
sera are the potential hazards. Life-threatening
arrhythmias in clinical application of MSCs for
cardiomyoplasty were reported when cells were
cultured in FCS.
autologous serum was used instead.14Further-
14This was avoided when
more, immune reactions to fetal calf proteins
have been reported. 89,107These events could be
explained by the high amounts of fetal calf proteins
which are withheld by the cells during culture. 74
Although this risk exists it seems to be relatively
low as MSCs are low immunogenic and micro-
organisms and viral parts contained in animal sera
are destroyed by heat inactivation. 74,80Serum free
media could be beneficiary in this setting.
Proliferative growth factors
During expansion growth factors can be used to
increase the time and enhance the yield of the
cells. The concern arising from this procedure is
the potential alteration of the multilineage
capacity of MSCs. Fibroblast growth factor (FGF) is
a known mitogen that could be used in this setting.
102However, recent data suggest that FGF produces
HLA-DR and Stro-1 induction and CD44 down
regulation as well as alternation on the levels of
Other approaches could
include the use of a variety of growth factors
including platelet-derived growth factor-BB (PDGF-
BB), epidermal growth factor (EGF) and Insulin-like
growth factor (IGF), as well as approaches like
transforming growth factor beta 1 gene transfer,
use of molecules like the Plastrum Testudinis or
even low-level laser irradiation. 16,34,35,103
Monolayer (2 D) and three-dimensional (3D)
Monolayer culture is the basic and the most
economical technique for MSC expansion (Figure 4).
Monolayer cultures are commonly grown in glass or
polystyrene roller bottles, culture flasks, or Petri
dishes. Plastic vessels used in tissue culture are
specially treated to ensure good adherence of
cells to the vessels’ walls. Culture conditions
include incubation in a maintained temperature of
37oC and a humidified atmosphere with 95% O2and
supplemented with 5% CO2. Usually the culture
medium is changed every 2-3 days. When cells
reach confluency they are treated with trypsin and
seeded into two new flasks.
Three-dimensional static cultures on the other
hand are not yet fully explored. A variety of three-
dimensional systems for the culture of MSCs have
been developed during the last years. Materials
Mesenchymal Stem Cell Tissue engineeringS27
including alginate, 57hyaluronic acid, 23collagen,121
fibrin 6 and chitosan 124have been used as struc-
tural frameworks to encapsulate and support
MSCs. Markusen et al studied MSCs proliferation in
conjunction to the use of alginate beads. 57The
results showed that although the cells were
viable, they did not proliferate during a period of
two weeks. 57 Negligible or decreased cell growth
was reported by other studies too by the use of
23,111On the contrary cell
growth was observed with the use of a 3-D
chitosan-gelatin polymer. 124
Although the monolayer culture is simple to
perform it includes several disadvantages.
Mesenchymal stem cells have been shown to have
reduced differentiation capacity after been
passaged.3Furthermore, it was shown that with
this culture method MSCs reach senescence
earlier. 8In cases where differentiated cells are
needed for implantation it has been shown that
the culture method can affect their osteogenic
and chondrogenic potential.
phosphatase (ALP) activity and osteocalcin
content were significantly lower compared to 3D
cultures. 67As far as chondrogenic differentiation
is concerned, chondrocytes in monolayer culture
become fibroblast-like with decreased ability to
produce proteoglycans and with changes in the
collagen synthesis pattern (produce Collagen I
instead of Collagen II). 7
The use of bioreactors for MSC culture is an alter-
native to the static expansion in the flasks.
Bioreactors produce a unique environment
mimicking the in-vivo condition of the cells. Their
advantages include minimal shear stress, micro-
gravity, efficient nutrient supply and metabolite
removal. 117,125Although similar in concept, several
types exist including spinner flasks, 117rotating-
wall vessel bioreactors,92concentric cylinder
bioreactors 84and perfusion bioreactors. 125 Results
from the use of bioreactors suggest that they
enhance the proliferation of the cells without
producing alteration of the phenotype and the
differentiation potential of the cells. 117,125
Once we ex-vivo isolate and expand MSCs, the
next frontier for providing clinical application lies
in the successful delivery of fresh cells at the
defective site. Current approaches consist of the
harvest and application of autologous grafts e.g.
the treatment of nonunions and segmental bone
defects with autologous bone grafting. However,
this technique poses severe morbidity, limited
supply and difficulty to fabricate functional shape.
Recent advances both in our understanding of
molecular biology and in the field of tissue
engineering allowed two distinct alternatives to
bone grafting. The first is the simple injection of
bone marrow directly to the defect without prior
manipulation of the progenitor cells and the
second implies the use of scaffold materials
loaded with MSCs.
Simple BM injection
The simplest approach to deliver MSCs to a
specific site is by BM aspiration followed by
immediate injection to the site. This approach
found applications in a diversity of pathologies
including the enhancement of healing rates of
fresh fractures, 45treatment of nonunions, 40,95,114
spinal fusion, 66osteochondral defects, 13osteo-
necrosis of the femoral head, 39bone cysts, 15
meniscal injury, 1enhancement of recovery after
ischemic spinal cord injury, 91rescue of the retinal
ganglion cells in glaucoma 123and induction of
angiogenesis and improvement of cardiac function
in acute or old myocardial infarction.122Although
this approach is effective, it seems to be
influenced by the rarity of MSCs in bone marrow.
Hernigou et al studied the effectiveness of BM
injection in 60 tibial nonunions.40An overall 88%
(53 out of 60) success rate was reported. It was
also found that the BM injected in the failed cases
contained on average 2.8 times less number of
osteoprogenitors compared to the cases where
union occurred. Therefore, in order to increase
the number of MSCs in aspirated bone marrow two
techniques can be used. Firstly, small volumes of
S28I. Pountos et al.
Figure 4. Static culture of MSCs. i. MSCs appearance
during culture, ii. Colony, iii. CFU-F, iv. ALP stained
MSCs, v. ALP stained individual cell, vi. Chondrogenic
differentiation: Toluidine blue staining.
two milliliters should be aspirated from each site
as larger volumes dilute the BM aspirate with
blood decreasing the concentration of MSCs and
secondly by the concentration of BM with
A large number of matrices has been produced and
tested over the last few years both in vitro and in
vivo for their efficacy as vehicles for tissue
regeneration. The ideal scaffold should mimic the
native characteristics of the tissue, providing a
source of cells capable of promoting the
regeneration as well as a biodegradable matrix
that would act as scaffolding for vasculogenesis,
cell migration and attachment. Table 2 presents
the properties of the ideal scaffold for clinical
use. Depending upon the structural and biological
requirements of the tissue in question, various
types of scaffolds have been developed from
substances found in the body as well as synthetic
substances. Scaffolds used include carbohydrate-
based polymers such as poly-lactic-co-glycolic acid
chitosan 19and protein-based polymers like fibrin,118
collagen 32and gelatin.76Other types of scaffolds
include corals, 41 TCP (Tricalcium phosphate), 10
59 and hydroxyapatite.
manipulated MSCs or adenoviral transfected MSCs
as well as already differentiated cells have been
uploaded on scaffolds targeting the healing of
authors studied the efficacy of composites
consisting of scaffolds and growth factors as an
alternative in tissue engineering. 41,46,69Hou et al
compared the effectiveness of a coral-MSCs and
BMP-7 composite with autologous bone for the
treatment of critical sized cranial bone defects in
a rabbit model. Comparable results between the
two grafts were reported.
41Using the same
model, Kim et al achieved healing using a
composite consisting of acrylated hyaluronic acid
and BMP-2. 46Similar in nature, complete union of
femoral defects in a rat model was achieved by
implantation of a collagen-ceramic carrier loaded
with MSCs infected with a BMP-2 carrying
A growing number of research and clinical
evidence have definitely demonstrated the great
regeneration potential of MSCs. However this area
is characterized by controversial and convergent
data. The methodologies and techniques followed
in each study varied considerably, making the
comparison of the results challenging. Optimiza-
tion of the techniques will clearly provide a more
robust understanding of MSCs biology allowing
novel therapeutic approaches.
Conflict of interest
All authors declare that no benefits in any form
have been received or will be received from a
commercial party related directly or indirectly to
the subject of this article. No funds were received
in support of this study.
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