Fisher John P. e.a. (ed.) Tissue Engineering
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16-2 Tissue Engineering
in small animals, then a well-controlled study in large animals such as primates may be considered before
human clinical trials.
In this chapter, we first discuss the general considerations when selecting an animal model and the most
commonly used animals in orthopedic research. Then, specific animal models for testing the biocompat-
ibility and biodegradation of tissue-engineered constructs are described. Well-established animal models
for the evaluation of osteogeneic or chondrogenic potential of these constructs are introduced. Finally,
the experimental design and evaluation methods involved in the animal studies are reviewed.
16.2 Animal Model Selection
Studies using animal models may be deemed necessary if there are no other in vitro alternatives, the
knowledge gained can be applied for the benefit of humans or animals, and the procedure does not cause
extreme pain or disability to the animals. In an ideal animal model, the anatomy and physiology should
be suitable for the specific study design, the pathogenesis and disease progression should parallel that of
humans, and there should be a similar histopathologic response to that seen in humans [1]. The preclinical
animal models in which the tissue-engineered constructs are tested should mimic the clinical situation as
closely as possible.
The use of animals in tissue engineering research has become a scientific as well as an ethical issue.
Generally, the use of invertebrates is preferred over vertebrates and the use of rats, rabbits, goats, and
sheep are preferred over dogs and cats due to their pet status. All animal protocols should be approved
by the researcher’s Institutional Animal Care and Use Committee to ensure that the experiments are
appropriately designed, the number of animals is justified, and the animal procedures comply with the
Animal Welfare Act.
From a practical perspective, animals of a particular species, strain, type, age, or weight should be easily
available for the entire experimental study [1]. It is preferable that the local animal research facility has
the capacity to house these animals. The animals should also be easy to handle in terms of transportation,
housing, peri-operative care, specimen handling, and disposal. The susceptibility of animals to disease
is also an important consideration especially for long-term survival studies. Finally, the costs of animal
purchasing, transportation, time for quarantine, housing, surgical supply, and special equipment should
be carefully calculated before the project begins. Generally, larger animals impose more housing and
handling difficulties than small animals such as rats and rabbits, and are more expensive.
16.3 Commonly Used Animals
Although a lower level vertebrate, the rat is among the most popular animal subjects in tissue engineering
orthopedic research and often the first to consider for a new study due to its low cost and easy care and
handling. Rats have a mean healthy life span of 21 to 24 months. After bone elongation ceases by the age
of 6 to 9 months, considerable useful lifespan remains for experimental evaluation [1]. Rats have been
used extensively in studies of biocompatibility [2], fracture [3], and bone defect repair [4]. The mouse,
also a small rodent, has gained popularity in tissue engineering research because its genome can be easily
manipulated and investigated. Both regular and immunocompromised nude mice have been widely used
for osteogenesis [5] and chondrogenesis [6] in subcutaneous tissue.
Rabbits are another commonly used animal in tissue engineering research. They are a relatively higher
vertebrate, with an appropriate size for surgical operation and analysis. Rabbits are suitable for studies of
bone defect repair [7], articular cartilage repair [8], ligament reconstruction [9], and spine fusion [10].
The dog, a higher-level vertebrate, has also played a dominant role in orthopedic research and has been
extensively used in studies of bone defect repair [11], cartilage repair [12], ligament reconstruction [9],
and meniscus repair [13].
Goats have become increasingly popular in tissue engineering research. They have been used for
studies of biocompatibility [14], bone repair [15], cartilage repair [16], meniscus repair [17], ligament
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Animal Models for Orthopedic Implants 16-3
reconstruction [9], and spine fusion [18]. Sheep are large animals similar to goats, but less literature is
available for their application in tissue engineering research. They have been used in studies of bone defect
repair [19], cartilage defect repair [20], meniscus repair [13], and ligament reconstruction [9].
Pigs have been used in studies such as osteonecrosis of the femoral head [21] and fractures of cartilage
and bone [22]. A miniature pig articular cartilage defect model has also been established [23]. Horses have
been used mainly for the studies of cartilage and joint conditions due to their rich cartilage tissue [24].
Primates would be ideal for tissue engineering research because they are the closest to humans; however,
due to lack of availability and high cost, they are only chosen when absolutely necessary as a step before
human clinical trials. They have been used in studies of bone repair [25], cartilage repair [26], and spinal
conditions [27].
Other animals in orthopedic research, though less frequently used, include hamsters for implant
infection, chickens for scoliosis and tendon repair, turkeys for bone remodeling, emus for osteonecrosis
of the femoral head, cats for osteoarticular transplantation, and guinea pigs for osteoarthritis [1].
16.4 Specific Animal Models
16.4.1 Biocompatibility
The biocompatibility study, as the first in vivo step of biomaterial evaluation, is typically performed by
subcutaneous or intramuscular implantation in rats or rabbits. The rat is moreeconomical and offers about
a 15 month observation period, while the rabbit is used for longer-term evaluations of up to 6 months.
Discs of 10 mm in diameter and 1 mm in thickness are commonly inserted in the dorsal subcutaneous
tissue or back muscles (up to six implants per rat and eight to ten per rabbit) [28]. Alternatively, porous
discs, biomaterials with seeded or encapsulated cells, and biomaterials contained in a stainless steel wire
mesh cage have been studied [29,30].
The factors that determine the biocompatibility of a biomaterial include its chemistry, structure, mor-
phology, and degradation. Biomaterial implantation often induces an inflammatory response through
the activation of macrophages. The degree of fibrosis and vascularization of the tissue reaction dictate
the nature of the response [29]. For instance, a mature fibrous capsule was observed after 12 weeks sur-
rounding the implanted poly(propylene fumarate)/beta-tricalcium phosphate (PPF/beta-TCP) scaffolds
in rats. Photocrosslinked PPF scaffolds also elicited a mild inflammatory response in rabbits [31]. The soft
tissue response to oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels was affected by the block length
of poly(ethylene glycol) in a rabbit model [32]. In a granular tissue reaction, fibrovascular tissue ingrowth
into porous poly(l-lactic acid) (PLLA) scaffolds was found to depend on the pore size [33].
A wide range of other biodegradable materials have also been assessed for their biocompatibility includ-
ing commonly used poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers [34], as well
as natural materials such as crosslinked gelatin [35] and hyaluronic acid [36].
16.4.2 Biodegradation
Biodegradable materials should be fully characterized for their degradation properties in vitro, but these
studies cannot substitute for in vivo evaluation of degradation. Often, the rate of material degradation is
significantly different in vivo compared to the in vitro condition. For example, poly(dl-lactic-co-glycolic
acid) (PLGA) foams [37] and injectable PPF/beta-TCP composites [2] were both found to degrade more
rapidly in vivo than in vitro. Differences in degradation rate in vivo vs. in vitro may be due to the extent
of perfusion, pH levels, enzymatic or inflammatory mechanisms, or mechanical loading. Moreover, the
apparent biocompatibility of a material may be confounded by greater toxicity of degradation products
or material fragments.
Initial in vivo biodegradation studies may be undertaken in models similar or identical to those
addressed in Section 16.4.1. Typically, discs, rods, foams, or other constructs are implanted in mice
or rabbits and evaluated at multiple time intervals for changes in dry mass, molecular weight, mechanical
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16-4 Tissue Engineering
properties, dimensions, and geometry. Standardization of sample geometry and dimensions is desirable
to allow for comparison among research groups [28]. Samples may be implanted in various soft tissue
locations, including the subdermal or subcutaneous space, intramuscular regions, or in the mesentery or
intraperitoneal cavity.
Tests under loading conditions similar to the target tissue are ideal to include effects of mechanical
loading on material degradation. If the material in question will have contact with bone, soft tissue
evaluation must be followed or replaced by evaluation in bony tissue. Bone defect models are numerous
[28], and generally involve drilled or otherwise excised bone segments. For example, self-reinforced PLLA
and poly(dl-lactic acid) (PDLLA) [38] have been evaluated by implantation in mandibular bone or a
femoral cavity of Sprague-Dawley rats. Because bone naturally remodels, it exhibits great healing capacity,
and for this reason, critical sized defects are often necessary for long-term evaluation. These are defects
that will predictably not heal spontaneously.
The choice of animal model to evaluate biodegradation in vivo also depends on the duration of the bio-
degradation study. For 1 to 6-month implantation studies, mice, rats, or rabbits can be used. Subdermal
or subcutaneous implantation for up to 90 days in Sprague-Dawley rats has been used to evaluate bio-
degradation of poly(carbonate urethane), poly(ether urethane) [39], poly(ester amides) [40], poly(ether
carbonates) [30], and poly(tetrahydrofuran) [41]. However, for long-term implantation studies, larger
animals such as goats, sheep, or dogs should be used. As an example, sheep have been used as a model to
evaluate biodegradation of self-reinforced poly(l-lactide) screws for up to 3 years in vivo [42,43].
16.4.3 Osteogenesis
In bone tissue engineering applications, after promising in vitro cell culture results are revealed, it is
often a first step to use a heterotopic animal model for testing in vivo osteogenesis of the constructs. The
major heterotopic models include the subcutaneous model, intramuscular model, intraperitoneal model,
and mesentery model. Tissue-engineered constructs, either alone or placed in diffusion chambers can be
implanted in mice, rats, rabbits, dogs, pigs, goats, or primates [44]. The rat subcutaneous model is the most
frequently used. For example, bone formation was observed by marrow stromal osteoblast transplantation
in a porous ceramic [45]. Ectopic bone formation was also demonstrated by transplantation of PLGA
foams to the rat mesentery [46].
Although the heterotopic models are useful to provide some information helping to bridge the gap of
in vitro and in vivo studies, the results obtained may not be the same once the constructs are implanted
in actual bone defects. Therefore, further studies must be performed in bone defect models. Among
them, the rabbit calvarial defect model is a very popular and reproducible bone defect model [44]. The
calvarial bone is a plate which allows creation of a uniform defect that enables convenient radiographic
and histological analysis. The calvarial bone has an appropriate size for easier surgical procedures and
specimen handling. Because of the support by the dura and the overlying skin, no fixation is needed. This
model has recently been used to evaluate the osteogenicity of a composite scaffold of PPF and PLGA [47],
and growth-factor-coated PPF scaffolds [48].
Common long bone defect models include the rabbit radial model (most popular), rat femoral model,
and the dog radial model. A composite bone scaffold consisting of nano-hydroxyapatite, collagen, and PLA
was found to integrate the rabbit radial defect after 12 weeks [49]. The effect of bone morphogenetic protein
was evaluated in the rat femoral defect model [4]. It should be noted that the rat model requires either
internal or external fixation. In the dog model, ulnae fractures may occur, resulting in unexpected loss.
Due to the regenerative capability of bone defects, it is typical in tissue engineering research to consider
critical sized defects. The critical size of the defect (CSD), defined as the smallest size that does not heal
by itself if left untreated over a certain period of time, is 15 mm in diameter for adult New Zealand white
rabbit calvarial defect model. The rat calvarial model is also popular with a CSD of 8 mm in diameter.
A bone biomimetic device consisting of a porous biodegradable scaffold of poly(dl-lactide) and type I
collagen, human osteoblast precursor cells, and rhBMP-2 was shown to promote bone regeneration in
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this model [50]. The CSD of long bones is at least two times the bone diameter (e.g., 12 to 15 mm for
rabbit radial model and 5 to 10 mm for rat femoral model).
16.4.4 Chondrogenesis
Similar to bone tissue engineering, the first step to test the chondrogenic potential of tissue-engineered
cartilage constructs in vivo is to use heterotopic models by subcutaneous, intramuscular, or intraperitoneal
implantation in nude mice, syngeneic mice, rats, or rabbits. Among them, implantation of the constructs
in dorsal subcutaneous pouches of nude mice is the most popular. For example, constructs containing
genetically engineered chondrocytes were implanted into nude mice to demonstrate transgene expression
and synthesis of glycosaminoglycan [51].
Commonly used cartilage defect models include partial-thickness (chondral) and full-thickness (osteo-
chondral) defects in rabbits and dogs [52]. The rabbit distal femoral defect model is a well-established,
reproducible model. Creating a partial-thickness defect can be challenging because the cartilage thickness
in the rabbit femoral condyles is only 0.25 to 0.75 mm (compared to 2.2 to 2.4 mm in humans) [53]. The
dog distal femur defect model, by contrast, offers a larger defect size with which to work. Besides species,
the age of the animal is also an important consideration since the potential for cartilage repair as well as
the response to various treatments varies with different animals [53]. Skeletal maturity of the commonly
used New Zealand white rabbits typically occurs between 4 and 6 months.
Tissue-engineered articular cartilage is generally anchored to a defect by press fitting, suture, fibrin
glue, or by using a periosteal flap. The local mechanical environment is well known to influence chon-
drogenesis and tissue healing. In the rabbit knee model, a defect created on the distal femoral surface
is considered weight bearing, while a defect in the intercondylar groove is partial weight bearing [52].
The type of postoperative treatment should also be considered; the use of continuous passive motion
can enhance cartilage healing [54] whereas joint immobilization may lead to decreased articular cartilage
regeneration [55].
Many naturally derived and synthetic polymers are currently used as scaffolds for regeneration of
articular cartilage [56]. They function not only as a vehicle for the delivery and retention of chondrogenic
cells, but also as a substrate for cell attachment and matrix production. Natural polymeric materials
such as collagen, hyaluronic acid, alginate, and fibrin glue have been extensively studied, as well as many
synthetic polymers including PLA, PGA, and poly(ethylene oxide) (PEO) [53]. More recently, a new
synthetic hydrogel based on oligopolyethylene glycol fumarate (OPF) was developed for cartilage tissue
engineering [57].
16.5 Experimental Studies
16.5.1 Experimental Design
The experimental design includes the experimental groups to be used, sample size, sampling error, and
control groups. The number of animals depends on the intrinsic variability among the animals, the
consistency of the surgical procedure, the accuracy of the evaluation methods, and the choice of statistical
method for data analysis. The number of animals needed in a study can often be determined from the
results of a preliminary pilot study. For example, six to eight animals can be used in a preliminary study
with histomorphometry or mechanical testing as the evaluation methods [58,59]. From this preliminary
study, the standard deviation of the mean, coefficient of variation, and mean difference among the groups
are determined. The sample size is affected by the desired power, the acceptable significance level, and
the expected effect size. To reduce the interanimal variance, the animals should be of the same strain, sex,
age, and weight. Some of the experimental animal models such as Sprague-Dawley rats and New Zealand
white rabbits have identical genetic traits, but dogs and cats are relatively heterogeneous [60]. Therefore
larger numbers of animals are often required for studies employing dogs or cats. The number of animals
also depends on the evaluation methods.
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Sampling error reflects the inherent uncertainty of results about a population based on information
gained from sample data, which is a subset of the population. Two sampling procedures used in biomedical
research are random and stratified sampling [61,62]. In simple random sampling, each element of a
population has an equal probability of being included. In stratified sampling, the elements are divided
into nonoverlapping blocks, and specimens are chosen from each block by simple random sampling.
Stratified sampling provides greater control over the distribution of specimens.
Controls in an experimental study include normal controls, treatment controls, and time controls.
Unilateral, bilateral, unicortical, and bicortical bone models are being used in bone tissue engineering
studies. Because of more efficient comparison between control and experimental groups, bilateral models
are extensively used for evaluating biocompatibility and function of foreign materials in bones [63,64].
When major procedures are performed involving a joint, a unilateral model should be designed to allow
the animal to heal over time. The bilateral cortical model is a design that doubles the number of specimens
for testing if it is appropriate from animal use perspective [65,66].
16.5.2 Evaluation Methods
The evaluation method should be valid and capable of measuring the parameter of interest. The devel-
opment of new evaluation methods requires assessment of its accuracy. Sources of error in an evaluation
method are inadequate surgical procedures or specimen preparation, systemic error of testing systems,
and data collection error. Clinical evaluation, necroscopy, morphological or structural analysis, biochem-
ical evaluation, mechanical testing, and the use of specialized devices are used in orthopedic research for
evaluation. The most commonly used methods in orthopedic animal research are clinical observation,
radiography, histological evaluation, and mechanical testing [60].
One method of biocompatibility evaluation is the implantation of disk-shaped samples of the mater-
ial into the right and left epididymal fat pads or the right and left rear haunch subcutaneous tissue
[67]. The following are removed at autopsy: the implant with its surrounding capsule, skin and
subcutaneous tissue, the axillary and popliteal lymph nodes, heart, kidneys, lungs, liver, spleen,
brain, and aorta, as well as any macroscopically unusual findings in the stomach, bowels, and mes-
enteric lymph nodes [68,69]. For evaluation of bone growth in explanted skeletal specimens, the
total cross-sectional area of newly formed trabecular bone is determined on sequential longitud-
inal sections [70–72]. Confocal microscopy is utilized for cell visualization and morphology [73].
Alkaline phosphatase activity and calcium content are measured to assess the extent of mineraliza-
tion in newly formed bone [74,75]. Western analyses of the newly synthesized collagen types I, II,
and IX from the explanted samples are done using monoclonal antibodies and chemiluminescent
substrate [76,77].
The explanted bone samples should be tested intact to establish reference mechanical properties [78].
Loads should be applied in a manner that minimizes focal loading of the specimen, so that accurate,
representative material properties are measured. Preconditioning the specimens by cyclic loading, over
several complete load-unload cycles, to a peak compressive force of approximately one average body weight
(70 kg) is reasonable to do. This technique tends to smooth out variability in the data, and increases the
likelihood of obtaining reproducible data.
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17
The Regulation of
Engineered Tissues:
Emerging Approaches
Kiki B. Hellman
The Hellman Group, LLC
David Smith
Teregenics, LLC
17.1 Introduction.............................................. 17-1
17.2 FDA Regulation .......................................... 17-2
Classification of Medical Products • Special Product
Designations • Human Cellular and Tissue-Based Products
• Marketing Review and Approval Pathways
17.3 Regulation of Pharmaceutical/Medical Human
Tissue ProductsinEurope .............................. 17-10
17.4 Regulation of Pharmaceutical/Medical Human
Tissue Products in Japan ................................ 17-11
17.5 Other Considerations Relevant to Engineered
Tissues .................................................... 17-12
FDA Regulation and Product Liability • Ownership of
Human Tissues
17.6 Conclusion ............................................... 17-14
References ....................................................... 17-14
17.1 Introduction
A critical step in translating tissue engineering research into product applications for the clinic and
marketplace is understanding the strategies developed by government agencies for providing appropriate
regulatory oversight. Since the eventual goal is the establishment of a global industry with the ability
of companies to market products across national boundaries, a harmonized international regulatory
approach would be ideal. However, while groups actively work toward that end, and, recognizing that
market acceptance can be influenced by local cultural, ethical, and legal concerns, it is essential to
recognize and appreciate the approaches of the regulatory agencies of those countries where engineered
tissue research is already moving into product development and clinical application. While the science in
the field is now worldwide in scope [1], we will limit our discussion to the regulatory approaches of the
United States, with attention to emerging trends in Europe and Japan.
The U.S. Food and Drug Administration (FDA) has recognized that an important segment of the
products it regulates arises from applications of new technology such as tissue engineering, and that,
17-1