Perspectives on Animal Models Utilized for the Research and Development of Regenerative Therapies for Articular Cartilage
Curr Mol Bio Rep
Perspectives on Animal Models Utilized for the Research and Development of Regenerative Therapies for Articular Cartilage
Dan Xing 0 1 2 3
Jiaqing Chen 0 1 2 3
Jiabei Yang 0 1 2 3
Boon Chin Heng 0 1 2 3
Zigang Ge 0 1 2 3
Jianhao Lin 0 1 2 3
0 Faculty of Dentistry, The University of Hong Kong , Pokfulam , Hong Kong
1 Department of Biomedical Engineering, College of Engineering, Peking University , Beijing 100871 , China
2 Arthritis Clinic and Research Center, Peking University People's Hospital , Beijing 100044 , China
3 China Orthopaedic Regenerative Medicine Group , Hangzhou , Peoples Republic of China
Animal models are integral and indispensable for biomedical research and regenerative medicine studies, as these provide invaluable information for systemically evaluating the potential risks and efficacy of newly developed biomaterials, drugs, medical devices, and therapeutic modalities, prior to initiation of human clinical trials. Nevertheless, it is important to be aware of the unique strengths and limitations of the various different small and large animal models commonly utilized for biomedical research as well as the various challenges faced in extrapolating results acquired from animal studies and the risks of data misinterpretation. This review will thus critically examine various animal models utilized for studies on articular cartilage regeneration. Particular emphasis will be placed on comparing and analyzing the unique strengths and limitations of each animal model, with the aim of establishing principles for evaluating the suitability of different animal models for individual studies as well as for comprehensive interpretation and extrapolation of results obtained from various animal species. Additionally, this review will also discuss to evaluate animal studies with in situ imaging techniques, how the animal genome may result in variability in experimental outcomes, as well as the contribution of animal models to the development of cartilage tissue engineering.
Animal model; Articular cartilage; Regeneration; Microfracture; Systematic review
It is often challenging to emulate in vivo studies with
highthroughput and standardized in vitro screening assays in
biomedical research, which makes the use of animal models
indispensable in most cases. However, the lack of
comprehensive understanding of animal models limits our ability to
extrapolate research data acquired from animal studies to human
clinical practices, which in turn results in misinterpretation of
data and unnecessary wastage of experimental animals .
Non-human primates may serve as an alternative option.
Nevertheless, in practical terms, it is often necessary to
balance multiple aspects of the cost-benefit axis, such as in vitro
versus in vivo, small animals (rat, rabbit, and dog) versus large
animals (sheep, goat, pig, and horse), scientific gain versus
animal welfare, as well as value for money . From
innumerable ideas and prototypes, to millions of small animals and
hundreds of thousands of large animals utilized, to thousands
of animal studies and clinical trials being initiated per year
(from in vitro models to small animals, large animals,
primates, and human beings) (estimated from https://
clinicaltrials.gov/), this disparity poses significant challenges
and raises many pertinent questions: i.e., BHave we utilized
animal studies wisely and efficiently?^ BHave we extrapolated
data from animal studies correctly?^ BHow can we compare
and analyze data from varied and diverse animal species?^
With the accumulation of research knowledge and data over
the past few decades, we are now at an even stronger position
to tackle this challenge than ever.
Amongst the various different animal disease models
utilized in preclinical research, we would like to focus on the
articular cartilage defect model in this review. Articular
cartilage possesses low intrinsic regenerative capacity , which
could be a leading cause of why half of the world’s population
aged 65 years and above suffer from osteoarthritis (OA) .
Cartilage injury-induced OA is implicated in at least 12 % of
all OA cases . However, the underlying mechanisms of
cartilage regeneration are as yet not fully understood .
Cartilage regeneration is an extremely complicated biological
process involving a diverse multitude of different
mechanisms, such as inflammation, mechanical loading, recruitment
of stem/progenitor cells, as well as clinical interventions .
The advantages and limitations of individual animal
models in articular cartilage research have been extensively
reviewed elsewhere . Ahern et al. conducted a systematic
review to critically examine the advantages and limitations of
different preclinical animal models of cartilage defects .
However, further perspectives and comprehensive discussions
are required. In particular, input-output ratio and
animalhuman extrapolation need to be evaluated. Each individual
animal model could provide some meaningful information
. Small animals (rabbits, rats, and mice) are usually
utilized for the purpose of initial evaluation and biological
compatibility tests, but these only provide limited information, as
the joints of small animal tend to heal more readily and
spontaneously than the human clinical model. Due to ethical
considerations, large non-companion animals (sheep, goat, and
pigs) are preferred to companion animals (dogs and horses)
[11 ]. Moreover, new techniques such as minimally invasive
methods and biomechanical tests can be applied to large
animals. Despite having much similarity to humans, non-human
primates have been seldom utilized in cartilage regeneration
research, due to scarcity, high costs, ethical consideration, and
high profile in animal welfare and also because these are often
unable to provide additional information beyond the
aforementioned large animal models .
The purpose of this review is to systematically and
critically examine the different animal models utilized for
investigating new regenerative therapies of articular cartilage, through
evaluating selection of animal models, proportion of
sacrificed animals, animal-clinical extrapolation, results
justification, and comparison between various species and
anatomical locations. A systematic search of PubMed-listed
publications was used to identify relevant studies from October
2005 to October 2015 that were related to animal models and
regeneration of articular cartilage. The medical subject
headings (MeSH; National Library of Medicine, Bethesda, MD)
Bmodels,^ Banimal,^ Bcartilage,^ Barticular,^ Bregeneration,^
and Bcartilage^ and the free-text words Banimal models^ or
Barticular cartilage^ were combined, as presented in Appendix
Table 2. The contribution of animal models to cartilage tissue
engineering was summarized according to the statistical data
obtained from results of the systematic search.
Animal Welfare, Ethical Approval, and Minimization of Numbers of Animal Utilized
Necessity of animal studies has to be determined, before any
animal study is designed and gets approval. Estimation of the
sample size of experimental animals have to be kept minimal
according to forms and variation of data, while experimental
animals have to be well kept and maintained in accordance
with international standards . Firstly, all experimental
studies utilizing animal models should be conducted
according to the International Guiding Principles for Biomedical
Research Involving Animals that emphasize the 3R principles
(reduction, replacement, and refinement)  or Public Health
Service Policy on Humane Care and Use of Laboratory
Animals . Secondly, in vivo studies based on live animals
must get approval from the ethics committee of the relevant
local institution. Thirdly, it is recommended that surgical
implantation of a tissue engineering construct into any animal
model should be performed according to standard protocols
It is essential to minimize numbers of animal used as well
as utilize less intelligent species, avoiding the usage of dog
and non-human primates where possible. In general, using
inbred strain animals can reduce the required numbers of
individual animals because of uniformity in their genetic
background. However, higher prices of inbred strain animals will
limit their usage as surgical animal models. The number of
animals required is usually determined based on several
parameters, such as variation, species, and prior experience.
Although small animals have priority to be selected under
rational circumstances , large animals are often
irreplaceable due to more similar biomechanical functions and
physiological responses to human beings.
Animal Models Used in Articular Cartilage Research
As microfracture is the most widely used regenerative therapy
for cartilage in current clinical practice, drilling varied holes
through cartilages with an aim to recruit stem cells/progenitor
cells from bone marrow is widely utilized in animal studies
. The microfracture procedure  involves debridement
of the lesion to a stable rim to enable the lesion to be well
contained. Subsequently, the calcified cartilage layer is
removed with a curette, and the subchondral bone is then
penetrated with microfracture awls utilizing minimal force to
achieve 3 to 4 perforations/cm2. In order to allow extrusion of
fat and blood droplets containing stem cells and growth
factors from the bone marrow, sufficient depth of penetration by
the awl is required. At the base of the prepared chondral
lesion, bone marrow-derived mesenchymal stem cells (BMSCs)
and growth factors infiltrate into the fibrin clot and
subsequently generate fibrocartilaginous repair tissue .
However, fibrocartilage lacks the biomechanical features of
native cartilage and becomes fibrous, which results in wearing
out of the newly repaired cartilage tissue under physiological
loads . On the other hand, modified procedures are often
used in cartilage defect models, which involve drilling holes in
articular cartilage into subchondral bone with varying depths
. Various strategies have been used to improve quality and
quantity of regenerated cartilage .
In Vitro Model
Effective cartilage regeneration involves orchestration and
coordination of external and internal signals spatiotemporally
with partial mimicking of embryonic development. In vitro
models can be used to investigate some factors, which is
difficult to analyze in vivo. Novel 3D cell culture systems ,
biomaterial scaffolds , shear/compression bioreactors
, stem cell differentiation techniques , and analytical
procedures could recapitulate functional cartilage regeneration
in vitro [28 ]. To model exogenous intervention, articular
chondrocytes were cultured within a collagen sponge in the
presence or absence of IL-1β to generate cartilage in vitro,
which enabled researchers to study the responses of
chondrocytes to inflammatory cytokines . This in vitro
model aimed to screen the effects of compounds with
therapeutic potential in osteoarthritis. When chondrocytes
transduced with different genes were cultured in vitro, improved
cartilage regeneration would be expected to correlate with
specific chondrocyte phenotypes [30, 31].
On the other hand, in vitro models are often limited by lack
of mechanical stimuli, maturation of regenerated cartilage
tissue, and absence of inflammatory responses [28 ]. It is one of
the most critical problems that in vivo and in vitro pliability of
multi-potent stem cells and chondrocytes largely depends on
their microenvironment. Various signaling cues, cytokines,
and growth factors from the cellular microenvironment are
crucial for the differentiation, proliferation, and maintenance
of differentiated stem cells . Phenotypes of progenitor/
stem cells and chondrocytes can be altered by soluble factors
from the surrounding tissue, by paracrine signals from
neighboring cells or by direct cellular contact . Although
several co-culture systems provide valuable information
regarding molecular control in vitro that aim to mimic the
physiological conditions at the injured cartilage site [34, 35],
functional regeneration of cartilage with articular
characteristics is still a challenge. As there is still a long way to go to
comprehensively understand spatial-temporal regulation of
in vivo cartilage regeneration, promising results from in vitro
studies have to be further evaluated by in vivo studies.
In Vivo Model
As utility, total expenses, cost-benefit considerations, and
aims of studies have to be considered in selection of animal
models, it is often challenging to balance these various
conflicting parameters (Table 1). Small animal models, such as
murine, lapine, canine, and caprine, are broadly used.
Chondrogenesis has been studied with subcutaneous 
and intramuscular implantation in nude mice . The
reasonable costs of animal purchase and care together with ease
of handling and caging are the advantages of mouse models.
High reproductive capacity and short duration of the
reproductive cycle make mice more commonly utilized in genetic
studies. However, relatively small joints and the thinner
cartilage of mice would limit the usefulness of cartilage defect
models in murine models [38–40]. To date, nude mice as well
as transgenic or gene knockout mice have been utilized for
studies involving ectopic chondrogenesis [31, 38, 41]. Rats
have bigger joints and thicker cartilage than mice [40, 42],
which make them more easily utilized in cartilage defect
models than mice [43, 44]. In the meantime, transgenic rats
have demonstrated some advantages as cartilage defect
models [45, 46]. Nevertheless, caution has to be exercised in
attempting to extrapolate results from rats to both large animal
and human clinical models, as rat cartilage generally has better
and continuous healing potential, which would obviously
exaggerate the results .
Rabbits are the most widely utilized animal model in
cartilage regeneration research. Their relatively larger joint size
and thicker cartilages, compared with rats and mice, would
increase their usefulness significantly. Full- or half-thickness
cartilage defect models could be constructed in rabbits
[47–49], with the subchondral bone being involved in 90 %
of cases. The main limitation of rabbit models includes
spontaneous healing potential, with rabbit cartilage possessing
greater intrinsic healing capacity than larger animals such as
horses and humans [50, 51]. A 3 mm diameter has been
considered the critical size of cartilage defect to prevent
spontaneous healing . The higher flexion in knee joints of rabbits
makes it difficult to extrapolate results obtained from the
rabbit model to humans .
Dogs are often utilized in cartilage regeneration research
due to their advantages in having larger joints, thicker
cartilage, low intrinsic healing capacity, and similar mechanical
properties of cartilage to humans . Dog models are also
widely used in surgical research. The cartilage thickness
allows for surgical creation of defects involving the articular
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cartilage without the subchondral bone. Moreover, dogs are
easily trainable after implantation surgery according to
rehabilitation protocol, making these animals appropriate for
exercise and physiotherapy studies . Arthroscopic
evaluation of the knee joint is feasible in dog models instead of
animal sacrifice . However, usage of dog models is limited
in cartilage regeneration research due to relatively small defect
volumes compared with primates, in addition to ethical
reasons pertaining to their companion animal status .
Pig models for partial- or full-thickness cartilage defects
were also used in several studies . Thicker cartilage
(1.5–2.0 mm) , up-right (instead of squatting) knee, and
larger joint sizes are advantages of the porcine cartilage defect
model. Nevertheless, pigs are characterized by difficult
handling, large size, requirements in housing, and aggressive
demeanor, while mini-pigs could offset some of these
shortcomings . It is critical to use mature mini-pigs to diminish the
influence of spontaneous cartilage repair, as immature
minipig cartilage (less than 42–52 weeks) has a relatively high
spontaneous healing capacity .
The ovine model also has certain advantages such as large
joint size, thicker cartilage, and lower spontaneous repair
capacity, but late maturity and variability of cartilage thickness
limit their wide usage for in vivo studies [58, 59]. The variable
thickness (0.4–1.0 mm) of cartilage in sheep makes the defect
volume different between individual animals. This could in
turn confound the replicability of results of in vivo studies.
Another disadvantage of the ovine model is the very dense
and hard subchondral bone, which could restrict choices of
study design for cartilage regeneration requiring bleeding of
the subchondral bone bed.
Goats are not costly and easier to handle than other large
animals. Cartilage thickness in goats allows for creation of
partial and complete thickness defects. Similar with sheep,
the high variability of goat cartilage thickness could lead to
variations of the volume of cartilage and subchondral bone
defects within studies. On the other hand, compared to the
sheep model, the subchondral bone of goat is softer and more
compatible with normal surgical techniques for creating
osteochondral defects. Thus, the goat, being a large animal
model, is useful for in vivo studies of cartilage defect
Horses are the largest animal models used for studies of
cartilage defect regeneration [63, 64]. As an animal with a
relatively long lifespan, a long-term model of cartilage injury
can be created in the horse . As the horse is an athletic
animal, it is an ideal animal model for evaluating resurfacing
technologies in chronic defects . The most significant
advantage is that the cartilage thickness (1.75–2.00) in the horse is
similar to that in humans . However, persistent standing
position during rest will place sustained weight load on the
knee joints, which will influence the in vivo results compared
with the human body. The higher costs, difficulty of handling
and caging, and physiological condition may also restrict its use
in preclinical studies. Their weight and special biomechanical
conditions in the knee joint will also affect extrapolation to
cartilage defect regeneration in the human clinical model.
How to Validate Results Acquired from Different
Animal models are essential for the development of novel
clinical therapeutic modalities. However, the utilization of
individual animal model is limited by their unique pros and
cons. Hence, we will systemically dissect and compare some
key factors involved with an aim to comprehensively
understand animal models. Particular attention will be focused on
the creation of cartilage defects by drilling holes of 2.5–4 mm
diameter within articular cartilage (Fig. 1).
Mice are extremely useful due to the diverse array of inbred
and genetically modified mouse strains available. In recent
years, a number of mouse OA models have been described,
including interior cruciate ligament rupture in the knee joint,
cyclic tibial compression loading of articular cartilage, and
intra-articular fracture of tibial subchondral bone .
Furthermore, multi-potential stem cells with or without
scaffolds were usually implanted at the back of nude mice.
However, murine cartilage defect models are not commonly
utilized due to the smaller knee joints of mice.
Rodents are the most often utilized animal models to
provide proof-of-concept data at the very beginning stages of
research and development. The medial or lateral femoral condyle
of rats could be used in full-thickness defect models involving
the subchondral bone, but not as partial cartilage defect models
due to its thinner cartilage. Studies involving ectopic
chondrogenesis were often carried out at the back of nude mice [66, 67].
However, the lack of several key factors, such as similarity in
biochemical characteristics, mechanical environment, and
tissues interfaces, thus limits the value of results from rodent
models. The lack of immunogenicity further limits the value
of the nude mice model. The knee joints of rodents with a
higher flexion degree have a different mechanical environment
compared with the more erect position of humans.
Fig. 1 Estimation of the proportion of various animal models utilized in
cartilage tissue engineering research and relative contribution to in vivo
studies before clinical application in the past 10 years. a The proportion of
studies involving different animal models. Most studies utilized rabbit as
an animal model in cartilage tissue engineering. b The proportion of
animals sacrificed in the studies. More than half of the number of
sacrificed animals comprises of rabbits. c Average sample size of each
type of animal model. The trend of the average number of animals utilized
in individual studies decreasing with increasing body weight is
significant. d Relative contribution of each type of animal model in
cartilage tissue engineering research. The trend of improving
contribution of each type of animal model with increasing body weight
In large animals, partial- or full-thickness cartilage defects
are often made in medial, lateral, or both femoral condyles. As
it is difficult to construct partial-thickness cartilage defect
models, most of the cartilage defect models in rabbit involve
the subchondral bone, due to the variability of thickness of
rabbit cartilage . Anatomical selection includes the
femoral trochlea and the medial or lateral femoral condyle in rabbit
Dogs are better models for cartilage defects, as the thicker
cartilage thickness of dogs allows creation of full or partial
defects of articular cartilage without involvement of
subchondral bone [9, 69]. A majority of studies still utilize
the osteochondral defect model in dogs [70, 71]. Dog cartilage
defects were selectively created in the femoral trochlea, the
medial femoral condyle, and both condyles.
Sheep have articular cartilage of variable thickness, which
leads to difficulty in comparing parameters amongst
experimental groups. The particularly hard subchondral bone of
sheep limits the choices of experiments that can be performed.
In goat models, the subchondral bone is softer compared to
sheep. Furthermore, the consistent thickness of goat cartilage
allows for defect modeling in articular cartilage that more
closely resembles human cartilage . Defect location in
sheep and goat includes the femoral trochlea, the medial
femoral condyle, and both condyles.
Although pig is not regularly used as a cartilage defect
model, adult mini-pig is occasionally utilized for studies
involving the following anatomical locations: the femoral
trochlea, the medial femoral condyle, and both condyles. The
thickness of the cartilage of mini-pigs would meet the requirements
for construction of partial- or full-thickness cartilage defect
OA is the leading cause of retirement of equine athletes.
Special attention was given to the horse model recently .
Although the horse has equivalent joint size, the lateral
femoral trochlea is the most common location for cartilage defects
. Cartilage defects of more than 350 mm2 can be produced
without subchondral bone involvement. Defects have also
been created in the lateral condyle of the metacarpophalangeal
joint and the middle carpal bones. However, we should take
their body weight and static loading into consideration when
extrapolating the in vivo results.
Age and Gender: How to Justify and Compare?
It is generally agreed that young and adolescent animals have
stronger capacity for cartilage regeneration, compared with
older animals . Hence, defect models in animals usually
utilize mature adult animals to minimize spontaneous cartilage
healing. Intrinsic healing abilities of the animals have to be
seriously considered when investigating cartilage
regeneration. For dogs, it is difficult to obtain consistent skeletally
mature animals. This is probably because the age of skeletal
maturity ranges from 12 to 24 months [77 ]. Sheep and goats
exhibit similar skeletal maturity at 2–3 years of age . The
FDA states that mini-pigs reach skeletal maturity by 42–
52 weeks. It is suggested that further studies should be
conducted when skeletal maturity is reached. In practice, horses,
retiring from various athletic careers, are often used for animal
models but often require examination for coexistent joint
diseases . The skeletal maturity and timing of modeling are
presented in Table 1.
Currently, 3 and 6 months are the most frequently used time
points after surgery. Twelve and 24 months are sometimes used
to more rigorously evaluate potential degeneration of de novo
cartilage, which actually happens quite often . Nevertheless,
the very beginning stages, say hours or days, are critical but
often neglected, limited by current technology constraints. In
practice, body weight and age are used as rough estimates.
Intervals between sample harvests are often chosen according
to experiences of individual researchers. The purpose of
taking time points is to evaluate the healing progress
dynamically, usually from the beginning stage to ultimate
regeneration. In principle, cartilage regeneration in large animal
models need longer duration compared to small animal
species. Because large animals have a longer life span
and relatively slow regeneration processes, the follow-up
duration ranges from 4 to 24 weeks for ectopic
chondrogenesis in nude mice models to between 2 and 78 weeks of
follow-ups in rabbit, dog, and sheep cartilage defect
models, while up to 104 weeks of follow-ups have been
reported for the goat model and 1–52 weeks have been
reported for the pig and horse models .
Limitations of In Vivo Evaluation
When interpreting results from in vivo studies, it is important
to note possible bias in the presentation of histological and
biochemical data, i.e., (i) slides with Boptimal regeneration^
have better chances to be presented, which are not
representative of the entire tissue, (ii) the in vivo results which are
consistent with that in vitro may get more attention than data
that are inconsistent with the in vitro results, and (iii) the
in vivo histological results may exhibit variability due to
different morphologies. The best results of histological
evaluation are often chosen for presentation or publication by
investigators, a practice commonly referred to as Bcherry-picking.^
Accordingly, while the results of previous individual studies
should be considered in designing new experimental studies,
these biases in subjective evaluation should be considered
when interpreting the findings.
Integration of In Situ Imaging Technology with Animal
Currently, non-invasive evaluation technologies have
provided innovative detection tools for in vivo studies. By using
realtime and non-invasive techniques, the evaluation of live
tissues in vivo could permit a better understanding of in situ
regeneration dynamically. In vivo non-invasive evaluation
technologies include functional mechanical testing (assessing
the tensile, shear, and compressive properties of engineered
cartilage), imaging technologies, and cell and growth factor
tracking in animal models . Indentation testing is a
compressive test that offers a new method for in situ,
nondestructive mechanical analysis of cartilage, which aims to
quantify some biomechanical characteristics . Magnetic
resonance imaging (MRI) has been broadly used in clinical
diagnostics of joint diseases but is limited by inconvenient
access of experimental animals to facilities. The roughness
of cartilage surface could be evaluated by ultrasound .
Non-invasive imaging of cartilage at the micrometer-level
resolution based on detecting the equilibrium partitioning of an
ionic contrast agent via microcomputed tomography enables
in situ imaging of cartilage and bone simultaneously in three
dimensions . Fluorescent labeling has been used to track
seeded cells , while superparamagnetic iron oxide (SPIO)
magnetic nanoparticles have been used to label seeded cells in
combination with MRI .
These technologies offer real-time and dynamic evaluation.
However, several limitations of these technologies restrict
their widespread utilization in animal models. These include
the lack of appropriate resolution for large animals, the larger
stature of some animals that is not suitable for some
measurements, the potential harm from radiation exposure and
contrast agents, and penetration into hard tissues.
More Functional Parameters
Information on matrix maturation is largely neglected or
limited . The intricate structure of articular cartilage imparts it
with essential functions, including lubrication, load bearing,
and transfer. The components, concentration, structure, and
morphology are highly anisotropic in native mature articular
cartilage. However, engineered cartilage can be immature
with incomplete components and isotropic structure. The
majority of studies evaluate cartilage formation by histology
(e.g., type I/II/X collagen and glycosaminoglycan),
immunohistochemistry, and gene expression, which are inadequate.
Moreover, zonal structural data are usually omitted. Very
often, there is a lack of further testing for better evaluation of
engineered cartilage. It is necessary for us to improve on
current therapeutic modalities to enable engineered cartilage
tissue to be more similar to native cartilage. Hence, we need
to pay more attention to components and structural features of
cartilage regeneration, particularly anisotropic structural
information. The extent of cartilage maturation can be roughly
determined by tensile properties . To be more precise,
the zonal structure of engineered cartilage can be
characterized by the picrosirius polarization method , for example.
Fiber optic confocal imaging technology for 3D histology has
been developed to supplement traditional histology and
constructed confocal arthroscopy for in vivo imaging of
chondrocytes in articular cartilage . Mechanical testing,
which is also important for better evaluation, is often lacking
in many studies.
Although various methods can be selected, a
comprehensive and standardized evaluation system for engineered
cartilage can facilitate progress in the field. However, it is
challenging to select key parameters. Furthermore, regenerated
cartilage with zonal structure is still an enigma in the current
field. Paying more attention to maturation of engineered
cartilage can lead to better evaluation of cartilage regeneration as
well as provide the basis for exploring further mechanisms.
Although several clinical studies on therapy of human
cartilage defects have been conducted in the human body [88–90],
the current evidence would suggest limited efficacy of most
therapeutic modalities . The reasons may include
deficiencies in study designs, follow-up methods and duration,
inclusion/exclusion criteria, and evaluation methods amongst
these various studies. Despite the advancement of cartilage
tissue engineering with in vitro or in vivo animal studies,
human models or clinical trials are still required.
Immunology: Integral to Cartilage Regeneration but Often Neglected
The immune system is crucial in determining the quality of
cartilage repair. It has been the consensus opinion that
immunological factors are integral to tissue regeneration. However,
its role in cartilage regeneration has not been fully
characterized. Studies in diverse animal species have demonstrated an
association between the loss of regenerative capacity and
maturation of immune competence. However, other studies
reported that the immune response enhances repair and ensures
local tissue protection. Therefore, the role of the immune
response in cartilage repair is rather complex. Additionally,
mesenchymal stem cells that play a key role in modulating the
immunological response to implanted biomaterials and other
cells, either allogenic or autologous, further increase the
complexity . Inhibiting or modulating the immune response
could potentially boost cartilage regeneration. However, it is
unclear whether the immune system has a functional impact
on more complex repair processes.
Does the Genome Lead to Variability of Outcomes?
It is better that early-stage in vivo experiments are initially
conducted in smaller animal models. Biocompatibility and
bioactivity should be confirmed initially in small animals
because of the genetic similarities between animals and humans.
In the late-stage in vivo experiments, larger animal models
could be used for testing clinical hypothesis and
biomechanical quality of implants because of their more similar
anatomical features to humans. Additionally, researchers should try to
create more experimental systems in animal models because
of limitations imposed by cost economics and ethical
standards. Further animal studies are still required in tissue
engineering for cartilage regeneration with the aim of increasing
their predictive value.
Contribution to Cartilage Tissue Engineering
A total of 1392 titles and abstracts were reviewed preliminarily,
in which 976 studies involved in vivo research on cartilage
tissue engineering. It was estimated that the number of various
species of animals sacrificed was 26,667. The distribution of
studies involving different types of animal models is presented
in Fig. 1. The most frequent type of animal sacrificed in basic
research is rabbits. With an increase in animal body size, the
sample size decreased significantly. Various factors, including
ethical reasons, feeding difficulty, and economic costs, may
limit the widespread use of larger animals. More importantly, there
is an evident trend that the relative contribution of each type of
animal model is improved with increasing body size (Fig. 1).
We could therefore infer that animals with larger size close to
humans may reduce the number of laboratory animals required
and lead to the generation of more reliable results. However, for
the initial stage of in vivo studies, it is advantageous to utilize
small animals to confirm some crucial parameters.
Acknowledgments The authors would like to acknowledge support
from the National Basic Research Program of China (973 Program)
(2012CB619100) and the National Natural Science Foundation of
China (81471800, 81271722, 81501919).
Compliance with Ethical Standards
Conflict of Interest Dan Xing, Jiaqing Chen, Jiabei Yang, Boon Chin
Heng, Zigang Ge, and Jianhao Lin declare that they have no conflict of
Human and Animal Rights and Informed Consent This article does
not contain any studies with human subjects performed by any of the
authors. With regard to the authors’ research cited in this paper, all
institutional and national guidelines for the care and use of laboratory animals
Papers of particular interest, published recently, have been
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