Title: The chorioallantoic membrane (CAM) assay for biomaterial testing in tissue engineering: a

short term in vivo preclinical model

Running title: A short term in vivo model for biomaterial testing: the CAM

Authors: Inés Moreno-Jiménez1, Janos M. Kanczler1, Gry Hulsart-Billstrom1, Stefanie Inglis1 and

Richard OC Oreffo*1.

Bone and Joint Research Group, 1Centre for Human Development, Stem Cells and Regeneration,

Human Development and Health, Institute of Developmental Sciences, Faculty of Medicine,

University of Southampton, Tremona Road, Southampton, SO16 6YD, UK

*Corresponding author Richard OC Oreffo; richard.oreffo@soton.ac.uk

Keywords: organ culture; chorioallantoic membrane, CAM assay, preclinical model; in vivo; tissue

engineering; biomaterials, 3Rs.

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Abstract

The fields of regenerative medicine and tissue engineering offer significant promise to address the

urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases and

tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies

needs to be assessed prior to clinical application, often necessitating animal research as a

prerequisite. The growing number of newly developed potential treatments, together with the

ethical concerns involved in the application of in vivo studies, requires the implementation of

alternative models to facilitate such screening of new treatments. The present review examines the

current in vitro and in vivo models of preclinical research with particular emphasis on the

chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative.

Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a non-

innervated rapidly growing vascular bed which can serve as a surrogate blood supply for organ

culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM

assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis.

Moreover, the application of imaging techniques (magnetic resonance imaging, micro computed

tomography, fluorescence labelling for tracking) will be discussed for the evaluation of biomaterials

cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to

facilitate the adoption of this technique across laboratories and the regenerative medicine

community, and thus aid the reduction, replacement and refinements of animal experiments in

research.

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1. Introduction to preclinical testing: in vitro and in vivo

models

Given the urgent need to provide safe and effective biomaterials for a raft of unmet clinical

applications, current research efforts are focused on developing biomimetic scaffolds which

resemble the natural properties of autologous tissue. To achieve this, researchers typically combine

scaffolds (mechanical support) with multiple biological cues (extracellular matrix proteins, growth

factors) and cells (induced pluripotent stem cells, adult stem / progenitor cells, and genetically

modified cells) to develop smart regenerative materials1. Such strategies dramatically increase the

number of biomaterial variables/combinations to test prior to translational clinical application.

Thorough and extensive preclinical testing is essential for approval from regulatory bodies, such as

the Food and Drug Administration (FDA), prior to use in the clinic. A large part of preclinical

biomaterial testing (i.e. optimisation, characterisation, safety) is conducted in vitro, however, often,

it is desirable to assess the performance of such materials within the context of whole animal

physiology. While in vivo studies serve as a critical final step/option in the research pathway, their

cost and time implications demand careful consideration, in conjunction with associated ethical

concerns. Moreover, despite the large variety of in vitro tests available, these tests, to date, do not

fully predict in vivo outcomes2. Hulsart-Billström et al. recently examined the correlation between 47

in vitro and 36 in vivo experiments, scoring the same 93 biomaterial variables across eight European

laboratories. The authors reported no significant association between the various outcomes

examined2. Importantly, only when examining two different categories of in vitro assays, involving

biocompatibility and functional assays, did the authors report a significant positive correlation2. This

report demonstrated the urgent need to improve and develop new strategies to link in vitro and in

vivo data across the continuum of research from the bench through to in vivo and subsequent

clinical translation. Current efforts within animal research are centred on producing high quality

science while decreasing the use of animals and improving the wellbeing of all laboratory animals

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used3. In this context, the chorioallantoic membrane (CAM) assay of the developing chick embryo

can serve as a short term, simple, cost-effective and, importantly, less sentient in vivo model for

biomaterial assessment. The present review will discuss the current in vitro and in vivo models for

biomaterial assessment, with particular emphasis on the background and practicalities of the CAM

model. The use of the CAM assay for organ culture as well as biomaterial evaluation will be explored,

together with a revised list of analytical techniques (µCT, MRI, and histology) applied to evaluation of

the CAM as an in vivo model.

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1.1. In vitro and organotypic culture

Biomaterial preclinical testing typically involves an initial stage in vitro using cell lines and primary

cells to screen and assess the biocompatibility (proliferation, cytotoxicity), characterisation

(gene/protein expression) and functionality of the biomaterial construct4,5. Indeed, in vitro culture

has evolved from the conventional cell monolayer culture systems and transitioned to more complex

culture conditions that include three dimensional (3D) sphere mono or co-cultures of different cell

types to enable the study of cell-cell interaction and cell-environment interactions which mimic

more closely their natural niche6–8. In addition, in vitro bioreactors have been developed for the

expansion and differentiation of progenitor cells by modifying their culture and mechanical

conditions9–12. Advances in bioreactor and microfluidic technologies have led to the development of

lab-on-a-chip devices able to simulate the physiological conditions and responses of the human

organism. These 3D in vitro biological systems have been scaled up into devices capable of culturing

multiple cell types to mimic the response of whole organs (organ-on-a-chip)13. Thus, currently, a

variety of tissues and organs (skin, cartilage, bone, lung, heart, kidney, etc.) are under development

across a number of groups and offer significant potential as replacement alternatives to in vivo

studies13–15.

In the meantime, the in vitro culture of organs (organotypic culture/organoids) provides a closer

biological approach to in vivo physiology while minimising the need to conduct procedures on living

animals. Organotypic cultures have been used to incubate a living organ in vitro at a controlled

air/liquid interface while maintaining the original 3D structure of the tissue or organ and hence the

interaction between multiple cell types and extracellular matrix. Such an approach has enabled

whole organs to be cultured statically16 or using perfusion techniques17–28.

As an example of organ culture, studies conducted implementing organotypic cultures of bone have

provided an in vitro system to study tissue regeneration and repair29 as well as insights into the

skeletal tissue development of the chick embryo30. In particular, Kanczler et al. developed a critically-

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sized chick femur defect model for organotypic culture which has been used to evaluate more than

fourteen biomaterial combinations for tissue engineering applications30–33.

Hence the organotypic model offers a number of advantages as an in vitro model for the study of

bone and cartilage repair, as well as providing a relatively high throughput system to test

biomaterials in vitro. However, while this model provides a refinement alternative for animal

research, the lack of a complete animal physiology system (vasculature, inflammatory and immune

system) remains an unaddressed, crucial aspect of the preclinical research testing pipeline. In

addition, the observation that graft vascularization is a crucial component in any tissue engineering

strategies further demands the use of a vascular bed for the evaluation of tissue regeneration 34, and

therefore the use of in vivo models in research and development.

1.2. In vivo models

Despite the plethora of in vitro models offering an alternative to animal testing, novel biomaterials

still need to be tested in the context of a full animal physiology to assess their safety and efficacy.

This is typically a mandatory requisite to address the regulatory steps/hurdles prior to clinical

evaluation35. Preclinical testing of biomaterials is generally performed encompassing a number of

distinct steps; initially a subcutaneous implant model is used to assess biocompatibility/safety,

followed by the assessment on an actual tissue wound/healing scenario (bone fracture, skin wound,

etc.). These studies are typically conducted in small animal models before evaluation in larger

animals, that provide a closer physiological context to the human response36. Thus, studies often

employ subcutaneous models to optimise (i.e. dose) and characterise (i.e. drug release profile)

construct variables prior to application into an injured tissue. Subcutaneous implants in mice are

typically used to test whether the constructs are biocompatible in the context of vascular supply

and/or immune system37–39. In vivo evaluation of a cell construct commonly requires the use of

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immunodeficient mice and a surgical procedure for subcutaneous implantation, maintained

approximately over 28-56 days40.

In contrast, the chorioallantoic membrane (CAM) assay in the developing chick embryo offers a

naturally immunocompromised host, allowing in vivo implantation of xenografts organs and

construct41. Importantly, the CAM offers a rapidly growing vascular bed which lacks a nervous

system, and hence is a less sentient alternative for animal research41. The main difference between

the subcutaneous implant and the CAM assay is the length of the incubation period, limited to 10

days in the CAM; however, due to its simplicity, high throughput and low cost , the use of the CAM

assay is progressively expanding within the research community 8,42–45.

A number of studies have evaluated the same biomaterial constructs on the CAM as well as in

rodents (subcutaneous implant), and even compared the outcome from both models46–50. Steffens et

al. used the CAM assay to examine the effect of cell-seeded bovine cancellous bone scaffolds on the

CAM for 8 days or on a mouse subcutaneous model for 21 days46. The results demonstrated that the

vascular response from the CAM was comparable, if not higher, to the mouse model 46. Ling et al.

used cellularised gelatin scaffolds and reported a similar angiogenic response when these scaffolds

were implanted into the CAM (7-9 days) and the dorsal subcutaneous SCID mouse (28 days). Hence,

the aforementioned studies provided evidence of a significant vascular response from the

extraembryonic membrane even within a much shorter incubation period. A number of publications

have already employed the CAM assay as a substitute for the subcutaneous murine model.

Martinez-Madrid et al. investigated the effect of cryopreservation as a method to preserve fertility

after aggressive chemotherapy on patients51. Cryo-stored human ovarian tissue was implanted onto

the CAM, providing equivalent results to the traditional assay using immunodeficient mice, hence,

validating the CAM model as an alternative to the murine model51. The CAM assay has already

replaced the use of the eye irritation test in rabbits, as a mandatory assay based on a scoring system

which measures hyperaemia, haemorrhage and clotting52,53. Furthermore, a number of FDA-

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approved anti-cancer drugs have been tested on the CAM and compared retrospectively with the

preclinical data obtained from mice and rat models54. Correlation analysis of these studies

demonstrated that the CAM was predictive of the results shown in the preclinical studies54, further

validating the potential of the CAM as a less-sentient replacement/refinement alternative for

currently used in vivo models.

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2. The principles of the chorioallantoic (CAM) assay

2.1. The chorioallantoic membrane of the chick embryo

From the moment of fertilisation, the chick embryo develops over 21 days before hatching, and

these stages have been named by Hamilton and Hamburger as embryo development day (EDD) 55.

The chick embryo is surrounded by four extra-embryonic membranes: i) the yolk sac ii) the amnion

iii) the allantois and, iv) the chorion, which function together to protect and nourish the embryo

during development.

The CAM is formed around EDD 4 by the fusion of the mesoderm tissue of the allantois and chorion

membranes (Figure 1) and this fused membrane becomes fully developed by EDD 14, growing

exponentially from 6 cm2 up to 65 cm2 in 10 days41. The CAM is located between the eggshell and the

allantois, surrounding the embryo structures where the allantois serves as a deposit for waste

material such as urea and uric acid (Figure 2). After EDD 14, the capillary plexus of the CAM becomes

attached to the eggshell membrane. This fused, non-innervated membrane serves as a respiratory

organ facilitating the gas exchange of O2 and CO2 between the eggshell pores and the embryo as well

as serving as a nutrient-waste interchange. In addition, the CAM contributes to ion transport,

incorporating calcium from the eggshell to allow bone mineralization56,57.

As a consequence of the rapidly developing vascular system present within the CAM, the chick

embryo is a commonly used host to perform (anti)angiogenic studies and in cancer research58–60. In

essence the CAM assay enables evaluation of a compound /material/construct that can promote or

inhibit the angiogenic response of the extraembryonic membrane (Figure 3). This is achieved by

placing the test component on the CAM surface, approximately between EDD 4 and EDD 10, to

observe the angiogenic response61. At harvest, the number of developed chick embryos is recorded,

as well as the number of integrated samples within the CAM as an indicator of construct

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biocompatibility and safety. Crucially, graft integration on the CAM is an important parameter to

assess the success of the assay (see section 4 for further details).

2.2. The primitive immune response of the CAM assay

While the chick embryo is conventionally considered an immunodeficient host for graft implantation,

a number of studies indicate that the chick embryo is able to elicit a primitive immune response62–65.

It has been shown that as early as EDD 7, the chick thymus starts the process of recruiting

lymphocyte precursors66. T lymphocytes develop in the chick thymus around EDD 11, while

lymphocyte B cells develop from the bursa of Fabricius (the equivalent organ to the bone marrow in

mammalian) around EDD 12. Both lymphocyte B and T cells start to circulate through the blood

stream from EDD 12, together with monocytes and heterophils- the latter acting as a mammalian

version of neutrophils67. Critically, T and B lymphocytes and monocytes do not become mature until

EDD 18, and hence this is why the chick is considered an immunocompromised host62. However,

Friend et al. showed that no differences in macrophage function were observed between day 14

chick embryos and 16 week old chickens68.

A number of studies have reported an immune response following the implantation of (xeno)graft

material on the CAM62–65. One study implanted lymphocytes from various animal species (pigeon,

duck, sheep, rat and guinea pigs) onto the CAM and reported swelling of the chick embryo spleen 62.

Sys et al implanted human osteosarcoma biopsies on the CAM to study their tumorigenic potential

and described an inflammatory response together with fibrous deposition and CAM hyperplasia 63. In

a similar manner, the CAM produced significant fibrous deposition after implantation of bacterial

endotoxins or cotton threads65. In the same study, Valdes et al. reported the presence of leukocytes

and macrophages histologically, a possible indication of an acute inflammatory response

comparable to that observed in mammalian systems65. Other investigations testing the similarities

between mammalian and CAM immune systems showed biocompatibility within the CAM to nylon

and silicone, commonly used surgical materials in the clinic64. The previous studies demonstrated

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that the chick embryo maintains a primitive immune response, which matures upon the end of the

gestational process. Thus, depending on the research question, the potential of an immunogenic

reaction from the chick embryo could be considered a positive or a negative feature when

performing the CAM assay. Indeed, the culture of tissues from different species (xenograft) requires

a suppressed immune response; however it is the initial inflammatory response which triggers the

healing process of the grafted tissue69. The second observation becomes particularly important in

the context of biomaterials screening, as information around the physiological immune response

would be relevant to assess construct efficacy and safety.

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3. The CAM assay for tissue engineering and biomaterial

applications

3.1. The CAM assay in biomedicine

The first use of the CAM was documented in 1911 by Rous and Murphy, who described the culture

of chicken sarcomas in the CAM70. A few years later, the authors published similar studies with

xenotransplants from rat tumours71–73. From these seminal studies, the use of the CAM has evolved

into multiple applications, predominantly related to cancer research60,74–76 and development of viral

vaccines 77,78. More recently, the CAM has been primarily used as a highly reactive vascular bed for

the study of the angiogenic properties of a variety of compounds such as vascular endothelial

growth factor (VEGF), bone morphogenetic protein (BMP), fibroblast growth factor-2 (FGF2) and

endothelin79–81. Other cytokines and growth factors such as osteogenic protein 1 (BMP7), thrombin

peptide, osteocalcin, Vitamin D and human angiotensin have also been examined82–85.

Additional applications of the CAM include evaluation of the dosage and toxicity of drug delivery

systems54,86. Other studies have examined the effect of applying X-rays on the CAM as a model to

determine the side-effects in blood vessels after radiotherapy87 or following hyperglycaemia for

diabetes research88. The CAM has also been used to evaluate novel surgical tools for retina

vascularization89,90, glucose biosensors64,91 and Doppler tomography measurements of blood flow

rate92. In summary, the aforementioned list of publications illustrate the wide range of applications

of the CAM assay, as well as the large body of data referencing the chick embryo as an in vivo model

in biomedical research over the last 30-40 years.

3.2. Organ culture on CAM: xenograft model

Given the immature immune system of the chick embryo, as detailed in section 2.2, the CAM has

been used to implant tissue (living and decellularized) for xenograft culture, with reports of

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successful engraftment and vascularisation63,93–97. Kunzi et al. described blood vessel formation and

infiltration of chick erythrocytes in the pre-existing capillaries of a human skin graft and preservation

of human specific markers after culture93. Furthermore, Carre et al. engrafted healthy mouse fetal

skin onto the CAM to then induce a laser injury, generating an in vivo model of wound healing and

tissue regeneration94. Other publications by Ribatti et al. examined the effect of decellularised brain

and aorta tissue from rats on the CAM as extracellular matrix scaffolds and demonstrated graft

vascularisation95–97. Additional studies implanting living human tissue on the CAM included

cryopreserved ovarian tissue51 and patient derived tumours63.

To date, there has been a limited number of publications in the literature on human bone tissue

cultured on the CAM63,98,99. In 2010 Holzmann et al. studied the effects of the bone banking process

on human allograft bone using the CAM assay98. To evaluate the angiogenic properties of the various

allografts, samples were collected at different banking stages and incubated on an ex ovo CAM for 48

hours, with fresh human femoral head bone chips as a control98. The vascular reaction of the CAM

was significantly higher for control bone chips compared to the allografts samples, however no

attempt was made to measure tissue repair98. Recently, our research group has developed a model

to culture human bone tissue on the CAM for regenerative medicine applications99. Moreno-Jimenez

et al. demonstrated avian vascularisation of the human bone tissue as well as a significant increase

in volume of the bone implants following 7-9 days in vivo implantation99. Thus offering an

alternative platform for biomaterial testing as well as a humanized CAM model as a short term in

vivo model99.

Hence, the aforementioned studies demonstrate the ability of the CAM to culture viable xenograft

organs, including human-derived tissue, and thus the possibility of generating humanized in vivo

models using the CAM. Moreover, the use of the CAM as an in vivo bioreactor for xenograft culture

offers an additional dimension to the standard safety and efficacy applications using the chick assay,

offering a more clinically relevant context for biomaterial evaluation.

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3.3. Biomaterial efficacy using the CAM model: assessment of the

angiogenic response

In recent years the CAM assay has come to the fore as a screening platform for biomaterials. A

diversity of constructs with growth factors and/or cells have been implanted onto the CAM over the

last 40 years (Table 1). Table 1 provides a detailed summary from a variety of studies implanting

biomaterials containing cells/growth factors on the CAM, demonstrating the large diversity in start-

points, choice of in ovo versus ex ovo approach, output measurements and incubation time. The

present section will review the most common application of the CAM for biomaterial efficacy testing,

which is the examination of the angiogenic response of the membrane as an early indicator of

construct performance in vivo. Typically, the CAM has been used to assess the angiogenic responses

of the biomaterial based on macroscopic evaluation of vessel formation at the implant site (vascular

density, vessel branching points/mm2 or blood vessels length) and/or histomorphometric analysis

such as immunohistochemistry for CD31, an endothelial cell marker. Alternative methods to quantify

angiogenesis include injection of a contrast dye for vessel perfusion and biochemical assays to

measure haemoglobin at the implant site (Table 1).

In 2001, Valdes et al. used the CAM to test the effect of a variety of biomaterials regularly used in

operating theatres, showing comparable results to the mammalian response65. Naturally-derived

materials such as small intestine submucosa, polymer derived materials such as polyglycolic acid

(PGA) and PGA modified with poly(lactic-co-glycolic acid) (PLGA) with and without growth factors

have also been tested on the CAM100,101. Covalent immobilisation of angiogenic growth factors (VEGF,

Ang-1) on collagen scaffolds for cardiac repair has been examined on the CAM, improving

performance over growth factor conjugation44. Additional collagen-based scaffolds composed of

microporous spheres proved to induce a greater angiogenic response compared to polycaprolactone

(PCL) based scaffolds102.

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The CAM assay has also been used to evaluate constructs for bone tissue engineering applications.

Composite materials such as bioactive glass nanoparticles with collagen scaffolds, and PLGA

combined with amorphous calcium phosphate, have been tested on the CAM to observe an

angiogenic response103,104. In 2009, Vargas et al. examined the biocompatibility and bone

mineralization potential of 45S5 Bioglass® using an ex ovo approach where the authors used the

embryo survival rate as an indicator of biocompatibility105. Buschmann et al. seeded adipose-derived

stem cells in electrospun nanocomposites PLGA-calcium phosphate scaffolds and achieved complete

infiltration of blood vessels throughout the scaffold104. Yang et al. used the CAM as a vascular bed

for the culture of chick femurs containing a bone wedge-defect on the diaphysis37. The implanted

chick femurs were used to examine the effect of BMP-2-PLA scaffolds seeded with patient-derived

cells, demonstrating the ability of the chick femur to heal and bridge the defect gap following CAM-

implantation37. Thus, the CAM can serve not only as an angiogenic assay, but also as a ‘bioreactor’

capable of vascular and nutrient supply for the regeneration of the grafted tissue.

3.4. The CAM assay for safety and biocompatibility evaluation

While the CAM has been traditionally used as an in vivo angiogenic assay, there is significant

potential in the use of the chick embryo in vivo model to provide additional information such as

construct biocompatibility and safety assessment. Indeed, the circulatory system of the embryo and

the CAM are connected and therefore any compound/construct applied on the CAM can affect the

normal development of the chick. Thus, in addition to the vascular response (CAM blood vessel

counting), changes in the normal development of the chick (i.e. viability rate) can serve as an

indicator of the toxicity of an implanted substance86. As a proof of concept, the CAM assay has

already been used as a sensitivity assay, replacing the invasive eye irritation test in rabbits since

200553. Following the same approach, regulatory bodies, such as the Food and Drug Administration

(FDA), are promoting the incorporation of the 3Rs (Reduction, Replacement and Refinement) in

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newly submitted research proposals. As an example, the CAM assay was recommended in 2006 as a

less sentient alternative for the testing of chronic cutaneous ulcer and burn wounds treatments35.

Documentation of the number of animals employed in each experiment (initial number of CAM eggs,

number of viable eggs at the end-point) and assessment of normal and healthy chick embryo

development can serve to aid biomaterial safety evaluation, in addition to the CAM angiogenic

response. However, there is a paucity of data available on the starting number of chick embryos

used and/or survival rate at harvest, preventing comparison across studies which omit this

information45,99,105. Similar to any in vivo study, reporting of the number of experimental animals

(mandatory for in vivo work in vast majority of peer-reviewed journals (see ARRIVE guidelines106, and

standard for good scientific practice) would aid the community.

In addition to chick embryo viability rates, a further important parameter to assess in graft

biocompatibility is the response of the CAM to the implant or integration of the graft (Figure 4). Only

after a graft becomes integrated within the avian membrane will the graft benefit from the surrogate

blood supply of the chick embryo (Figure 4 A). Interestingly, the chick embryo has developed several

mechanisms, including material isolation in fat tissue and encapsulation of the material in amniotic

fluid, to prevent the interaction with the implant (Figure 4 B).

In summary, the CAM assay can serve as a biocompatibility assessment tool enabling documentation

of chick embryo survival rate at harvest as well as the number of CAM-integrated grafts at harvest.

Thus, maintenance of chick embryo viability is important to observe differences between actual

treatments (i.e. biomaterials). The following section will review the technical aspects that can

influence the normal development of the chick embryo and the different types of CAM assay

models.

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4. Experimental and technical considerations for the

CAM assay

Although the incubation of chick eggs is a relatively simple process, there are various technical

aspects to consider in order to establish and standardise baseline embryo survival rate (ideally

approximately 90 %). Upon fertilisation the chick egg can be stored at a chilled temperature

(approximately 14°C) to prevent the initiation of embryo development. Stasis or developmental

arrest can be conducted for 10-14 days post fertilisation, however egg viability reduces in proportion

to stasis duration. Before resuming incubation at 37.5°C, eggs should be allowed to gradually

increase in temperature over a 5-6 hours period (setting eggs), as significant temperature

fluctuations can compromise chick embryo viability. Stasis is used to set two different batches of egg

incubation, common when implanting organs from donor chick embryos (i.e. EDD 18 chick femurs)

into the host CAM (EDD 10-11 chick embryos) to evaluate tissue regeneration and/or repair 37.

The incubation temperature for the chick eggs can be set between 37.7-38.3 °C, with a relative

humidity established between 52-55 %, oxygen levels above 20 % and CO 2 levels below 0.5 %. To

prevent the membranes of the chick embryo adhering to the eggshell, incubators need to have a

rotation programme which typically involves egg rotation by 90 ° every hour. Evaluation of fertility

and viability of the embryos can be undertaken by candling the eggs. Candling the eggs consist of

passing a bright light (i.e. torch) through the eggshell which helps visualise the air pocket of viable

embryos, normally located in the wide portion of the egg. Given the variability in fertility rates (55-

95%) from batch to batch of chick embryos, and unforeseeable conditions that can arise during

transportation, candling of eggs before commencing an experiment is advisable to enable

adjustment of experimental numbers if required.

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4.1. Ex ovo versus in ovo

There are two forms of the CAM assay depending on whether the chick embryo continues to

develop in the eggshell or, alternatively, in a petri dish (shell-less culture), respectively termed in ovo

and ex ovo culture. The ex ovo assay permits direct continuous visualization of the implant during

the incubation period, however ex ovo causes an increase in the chick embryo death rate of 50-70 %

during the initial days of culture, reaching 90% mortality around EDD 1460,64. The presence of the

eggshell is important since the CAM regulates the transport of calcium required for the normal

mineralization of the chicken skeleton107. As a result, in the context of an ex ovo assay, the CAM can

uptake mineral from an implant to compensate for the absence of eggshell. Vargas et al.

demonstrated the ability of the CAM to completely resorb bioglass-ceramic scaffold which resulted

in significant mineralization of the chick skeleton compared to control embryos105.

Since the embryo is allowed to develop normally prior to graft implantation, lesser numbers of

animals are required when in ovo assays are undertaken, hence adopting the 3Rs policy106. For an in

ovo approach, the shell of the egg is carefully etched to open a small window, which allows

placement of the implant on top of the CAM (Figure 2B and 3B). Following this, the eggshell window

is sealed to preserve sterility and humidity, and the chick egg is placed in the incubator to resume

incubation. The start point of the CAM (eggshell windowing) depends on the size/weight of the

implant and the desired incubation period. The size of the window created will impact on embryo

survival and development. For angiogenic assays, the CAM assay is typically started at EDD 10-11 as

this corresponds with a peak in the vascular expansion of the membrane79. For tissue engineering

applications, the CAM offers a limited period (EDD 10-EDD 18) for in vivo implantation and thus an

earlier start point can offer an extended incubation time; however the size and weight of the implant

should be compatible with the maturity of the CAM (EDD) to avoid perforation of the membrane.

The termination date and procedure of the protocol should be determined following local ethics

committee guidelines and approval. In most countries, including the UK and the US, the chick

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embryo becomes a legally ‘protected’ animal during the last third of the gestational/incubation

period (EDD 14), thus requiring a license to conduct any regulated procedures (Animals Scientific

Procedures Act (ASPA), UK 1986, amended 2012; Policy on the Humane Care and Use of Laboratory

Animals).

4.2. Imaging techniques: MRI, PET, µCT

A central imaging application for the CAM has been the quantification of vessels using microscopy

and histology as the cornerstones for assay assessment. In particular, the numbers of vessels

including the diameter, density, length of each vessel and vessel branch points have been the

parameters typically evaluated41,59,108. As fluorescence imaging has advanced, this modality has

become a basic tool in the examination of the CAM. Furthermore, fluorescence imaging has been

widely used to study cancer metastasis and drug delivery. As an example, the CAM model has been

employed to screen for fluorescent tumour markers where tumour cells or grafts of tumours have

been labelled with fluorescent markers and then seeded onto the CAM 109,110. In addition to tumour

cell labelling, recently developed quantum dots have also been used in the CAM to visualise blood

vessel development and angiogenesis111,112. Further applications of the CAM model include the

development of novel radiotracers for in vivo imaging113,114. Haller et al. provided strong evidence of

shared bio-distribution and in situ stability of the radiotracers when comparing the CAM model with

a conventional mouse model113,114. Nevertheless, it is important to consider the radiation dose for

the chick egg when employing X-ray based imaging techniques (PET and CT) as such techniques can

compromise the angiogenic readouts of the assay87,115.

Magnetic resonance imaging (MRI) has been used as a tool in the CAM model for quantification of

the perfusion capacity of scaffolds cultured on the CAM as well as for the evaluation of

mineralisation using a custom contrast agent104,116,117. Another emerging tool, which has been

implemented in the CAM model, is photo acoustic microscopy (PAM). PAM is an exciting tool that

uses generated ultrasound signals, via a laser, to image detailed aspects of tissue. PAM has been

19

used for three-dimensional morphological analysis of vascular networks as well as for analysis of

oxygen saturation118. PAM has been used in the CAM model to study dose-dependency effects of

angiogenesis inhibitors119 and for real-time monitoring of vascular changes in the CAM model during

tumour destruction120. In summary, a wide range of validated imaging techniques (CT, MRI, PAM,

PET, etc) are available (and continuously evolving) to analyse outcomes from the CAM assay and to

enable the quantification of effects following treatment on the CAM.

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5. Advantages and disadvantages of the CAM assay

The main advantage in using the CAM assay is the potential to collect information with regards to

safety (biocompatibility and integration) and efficacy (angiogenic response, tissue

regeneration/formation) of biomaterials using a minimally invasive, rapid and cost-effective in vivo

model. As detailed in section 3.3, the angiogenic response can be quantified using a variety of

methods (Table 1), however distinguishing pre-existing vessels from newly formed vessels remains a

challenge. A number of authors have proposed methods to overcome this challenge successfully,

such as counting vessel numbers at the implant site with respect to vessel numbers in a distal region

of the CAM away from the implant121, or introducing a nylon mesh or grid between the implant and

the CAM to score only the new vessels58,90,122. In addition to the angiogenic response, assessment of

construct biocompatibility is based on i) the survival rate of the chick embryos at the experimental

end-point, ii) integration of the implant within the CAM and, iii) the presence of a primitive

inflammatory response. The ability of the chick embryo to reject an implant by non-integration or

development of an inflammatory response is an important parameter to take into consideration in

the safety evaluation of a construct. Thus, the CAM assay provides additional benefits, providing a

stepping stone for subsequent in vivo studies and therefore a less sentient and high-throughput

screening platform for biomaterials.

A common limitation in the use of the CAM assay for xenograft culture (i.e. human tissue) is the

inability to differentiate between host and graft tissue due to antibody cross-reactivity between

species123,124. To circumvent this issue, genetically modified chick embryos constitutively expressing

green florescent protein (GFP) can be used for the CAM assay and thus enable differentiation

between host (GFP-CAM) and graft tissue99. GFP chick embryos were originally developed by

McGrew et al. with the central objective to generate therapeutic proteins in chick eggs 125. The

production of transgenic chick embryos was achieved by using a lentiviral vector carrying a reporter

transgene (GFP) followed by cross-mating to identify homozygous GFP+/GFP+ birds125. GFP

21

transgenic chickens have been used to examine gene expression regulation patterns in different

tissues126 as well as to substitute neural tube grafts in wild type chickens for developmental studies

of the nervous and vascular system127.

An important constraint of the CAM assay compared to other in vivo models (i.e. mouse

subcutaneous implant) is the incubation period, normally limited to 7-10 days in the chick embryo. A

potential approach to extend the incubation period on the chick model consists of re-implanting the

graft harvested onto a second CAM (double CAM). We have recently explored this concept and

implanted human bone tissue on a GFP-CAM for the first incubation period and, at harvest, re-

implanted the graft into a wild type CAM for an additional 7 days culture period (Figure 5). Chick

embryo survival on the second CAM was above 80 %, indicative of the excellent integration between

graft and host membrane. Histological analysis showed integration of the graft in both CAMs, as well

as a close interaction between both GFP + and GFP – membranes (Figure 5). Additional evidence of

the interaction between the double CAM was evidenced by the fusion of both membranes (GFP

staining within wild type CAM membrane). The implementation of a double-CAM culture approach

offers the possibility to extend the culture of the CAM assay and hence increase the in vivo

implantation period to allow for tissue regeneration, critical for preclinical testing in regenerative

medicine. In summary, while the short-term implantation period available in the CAM can limit the

extent of tissue formation compared to commonly used murine models (min. 28 days implantation),

the advantages in terms of cost, simplicity of the procedure, minimally invasiveness, access to higher

numbers and rapid angiogenic response, make the CAM an attractive model serving as a unique

bridging step between in vitro and in vivo studies in tissue engineering.

22

6. Future directions

Translatable research relies on the use of in vivo models to examine the safety and efficacy of

treatments before the research can reach the clinic. The advances within the regenerative medicine

and tissue engineering fields, necessitate the development of alternative models to evaluate the

new proposed modalities for treatment and the urgent need to refine, reduce and replace (3Rs) the

use of animals in research. In the present review, we have introduced the CAM of the developing

chick embryo as a short-term in vivo model together with multiple validated applications in different

fields of regenerative medicine research. The CAM assay provides a simple, cost-effective and high

throughput in vivo model which can serve as an excellent bridge between in vitro and in vivo models

for preclinical research. Numerous studies have compared the outcome from murine and CAM assay

models and reported comparable findings when examining similar treatments, demonstrating the

potential of the CAM to refine, or even replace, the use of murine hosts in animal experimentation.

Future directions will focus on extending the incubation time of the CAM assay by re-implantation

on subsequent chick eggs to prolong the in vivo implantation time. Additional research avenues

include the use of larger avian species with longer embryo development periods ( i.e. ostrich egg),

hence extending the incubation time available on the CAM for up to 42 days. Moreover, the

implementation of genetically modified chick embryos for the CAM assay offers a significant

potential to culture human xenograft tissue for mimicking human physiologic conditions and enable

differentiation between host and implant tissue. Thus the humble but complex chick egg and derived

CAM model, often used as a poor research surrogate tool, offer a unique research test model to

inform the transitional in vivo phase and to evaluate the plethora of technologies and therapeutic

strategies proposed of the clinic.

23

7. Acknowledgements

Work in the authors’ laboratories was supported by grants from the BBSRC (BB/L021072/1 and

BB/L00609X/1, European Community Seventh Framework Programme Grant, BioDesign (262948)

and EU FP7 (FP7/2007-2013 under grant agreement no. [318553] Skelgen) and UK Regenerative

Medicine Platform Hub Acellular Approaches for Therapeutic Delivery (MR/K026682/1) to RO. PhD

funding from the National Centre for the Replacement, Reduction and Refinement of Animals in

Research (NC3Rs) for IMJ is gratefully acknowledged. The work presented here is based on many

useful discussions with past and current members of the Bone and Joint Research Group in

Southampton, United Kingdom.

24

8. Disclosures

No competing financial interests exist.

25

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