Rats as Animal Models in Tissue Engineering

Figure 1. Rat. [2]

Background

Animal models provide simplified versions and instructive representations of the biological functions of a human. The Office of Technology Assessment approximates that 17-23 million animals are used each year in the United States for research purposes (1). All products, drugs, and inventions must be thoroughly investigated before their use on humans. Animal models allow for studies that are not feasible or ethical to produce on humans. Factors that need to be considered in choosing an animal model include the viability of implanting the tissue, how readily available the animal is, cost, and ethics. Approximately 97% of all lab animals in the United States are mice and rats (2).

Motivation

Why Rats?

Rats are one of the most commonly used animal models since they are physiologically more similar to humans than other organisms. Some of their advantages include that they are inexpensive, require low maintenance, and do not carry the same ethical issues as larger animals (3). Rats can cost anywhere from $30 to $100 depending on the weight, age, breed, and the supplier (4). Maintenance costs approximately $5 to $10 a month for food and living, which is much less than other, larger animals. The short lifespan of rats (approximately two years) also allows researchers to study the effects of aging and other biological factors in a shorter amount of time. For example, rat’s bones stop growing around 6 to 9 months leaving a considerable amount of lifespan to be dedicated for studies of biocompability, fracture, and bone defect repair for tissue engineering and orthopedic research. Rats also serve as reproducible and practical model due to their minimal genetic variance across several generations of breeding (5). Despite the many advantages of rat models, many biomechanical and anatomical differences still exist between rats and humans.

Rats vs. Mice

While the primary model of choice has traditionally been the mouse, recent advances of technology have led to the use of rats as animal models in tissue engineering. The choice of model is situational to the purpose and goal of the current study. In comparison to mice, rats differ in size, behavior, genetics, and physiological features.

Size

Rats (weighing on the order of hundreds of grams) typically weigh ten times greater in size compared to mice (weigh on the order of tens of grams). Rats are costlier to maintain because they require more food and living space than mice. However, their size also makes surgical procedures easier to perform leading to fewer errors and higher efficiency of money, time, and animal life. In addition, the larger surface area means larger tissues and samples can be taken from the rat. Smaller samples are more vulnerable to variability and error and require sensitive assays. Larger organs also provide greater detail into experimental studies on how much is involved and the distance a drug is travels.

Figure 3. Size of rats compared to mice. [2]

Genetics

Although rats and mice have very similar features, there are still many genomic differences between them. The main advantage of mice over rats the ability to genetically modify mice and provide models for diseases in humans. However, with recent advances this can now be done with rats. In 2004, the Brown Norway rat, or Rattus norvegicus, was sequenced and revealed that some genes, including those involved in immunity, production of pheromones, and the breakdown of proteins and detection of chemicals, are expressed in rats, but are absent in mice (6). Almost all the genes found in humans known to be linked with disease have counterparts in the rat genome. For these reasons, rats make a suitable model for disease (6).

Behavior

Although the size can create a challenge in handling rats, rats are typically easier to handle over mice due to their docile behavior. Furthermore, mice are often skittish and inconsistent. For experiments where the variability in behavior would result in unreliable results larger cohorts of mice would be needed. Rats are also more social than mice (6).

Brain

Rats have larger brains than mice, and thus are more similar to humans in terms of complexity. Due to their complexity, so rats are more likely to express diseases such as Parkinson’s disease, Alzeihmer’s disease, and autism. Rats have higher cognitive abilities than mice and perform far more reliably on learning and memory tasks.

History on Laboratory Rats

Animal models, especially rats and mice, have been extensively studied for centuries. Rats, however, were the first animal domesticated solely for scientific research (28).

Figure 2. Rat-Baiting. [2]

• 1728-1730 – Wild brown Norwegian rats, known as Rattus norvegicus, were very prevalent and thought to have migrated to England from Asia via ship. Rat-catchers would capture these rats and sell them for food and rat-baiting. Rat-baiting was a sport that involved filling a pit with rats and betting on the amount of time a dog took to kill them. Rats were eventually bred specifically for the sport. Albino rats were smaller, more docile, tolerated overcrowding in cages, and better breeders, so these rats were often not included in rat-baiting and instead used in rat shows.
• 1856 – Philipeaux was the first to be recognized for using albino rats to study adrenalectomy, the removal of one or both adrenal glands, in France (29)
• 1863 – Savory, an English surgeon, studied nutritional quality of proteins in mammals using mixed coat colors of black, brown, and white (29)
• 1877-1885 – In Germany, Hugo Crampe was the first to confirm that genes are inherited using over 15,000 white, grey, and black rats by studying the effects of coat color (28)
• 1894 – Stewart was one of the first to use rats in laboratory setting in the United States. At Clark University, Stewart studied the effects of alcohol, diet, and barometric phenomena on activity of wild and albino rats (29)
• 1906 – Henry Herbert Donaldson, from the Wistar Institute in Philadelphia, carried out selection experiments on albino rats. Donaldson aimed to standardize the rat to create reproducible studies on the growth and development of the rat’s nervous system. The breed, known as Wistar rats, have more than half of all laboratory rats have descended from (20).
• 1908 – First rat colony in America for nutritional studies by Elmer Verner McCollum (18)
• 1922 – Herbert McLean Evans and Joseph A. Long at the Institute for Experimental Biology at University of California, Berkeley studied the reproductive function of rats and developed the Long-Evans strain (19)
• 1925 – Sprague-Dawley strain was bred by Robert Worthington Dawley, a physical chemist at the University of Wisconsin (29)
• 1947 – William Ernest Castle identified a total of 23 mutations in the rat
• 2004 - Brown Norway rat genome was sequenced (27)

Laboratory Use

Gender

In lab, often important to note the gender of the rat. Females have hormones that effect adipogenesis, or cell differentiation where preadipocytes become adiposyctes (10). Furthermore, estrogens will act as proadipogenic hormones and ovarian factors affect preadipocyte differentiation (11,12). Test angiogenesis in new tissue, immune reaction that lead to rejection and inflammation, innervation, and kinetic properties of engine intestinal tissue. Male rats grow larger than female counterparts (10).

Rat Strains

Brown rats are valuavle, grow quickly to sexual maturity and easy to keep in captivity The major strains are Wistar, Sprague-Dawley, Osborne0Mendel, Long-Evans, Holtzman, Slonaker, and Albany

1. Wistar

The Wistar rat is also an outbred albino rat. These rats tend to grow larger than SD and LEW rats (10). The original strain of Wistar rats was developed at Wistar Institute in 1906 for biological and medical research by Donaldson. These rats are characterized by their wide head, long ears, and tail is less in length to is body

2. Sprague-Dawley

The Sprague Dawley rat is an outbred breed of albino rat. The advantage of using SDs are their calm demeanor and ease of handling (15). Ideally for general multipurpose model, safety and efficacy testing, aging, nutrition, diet-induced obesity, oncology, and surgical model. They were used in studies for adipose tissue engineering. Cheapest cost $15.30 (smallest weight). Also, has excellent reproductive performance.

3. The Lewis (LEW)

The Lewis (LEW) rats were derived from WIS stock by Dr. Lewis in the early 1950s (wiki). These albino rats possess high insulin and growth hormone levels that contribute to their obesity (10). They also possess docile behavior and low fertility (21). This rat also can suffer from high incidences of neoplasms. Adenomas of he pituitary and adrenal cortex, mammary gland tumors and endometrial carcinomas in females, and C-cell adenomas/adenocarcinomas of the thyroid gland and tumors of the haemopoietic system in males. Also, more prone to develop spontaneous transplantable lymphatic leukaemia. With age, they can develop spontaneous glomerular scleoris (21). They are ideal for transplantation research, induced arthritis/inflammation, experimental allergic encephalitis, STZ-induced diabetes (21).

4. Ratnude

Ratnude (RNU) rats are immunodeficient and permit transplantation of xenografts, but the decreased T cell populations and increased natural killer and macrophage cell populations results in wound healing kinetics different from nonimmunodeficient LEW, SD, and WIS rats. (10)

Tissue Engineering Applications

Cardiovascular Tissue

Cardiovascular tissue is specialized muscle tissue that allows blood to pump throughout the body. The benefits of using rats for cardiovascular research includes their organ size, blood volume, and Rats are often preferred for for cardiovascular research in part because of the larger heart and vessel size (27).

Tissue-engineered vessels (TEV)

Increasing need for smaller diameter blood vessels as replacement grafts, advantage is to study integration and developmental aspects of TEV implantations. Biomaterials: polysaccharide gel and decellularized rat aortas have been tested in rats (22). Rats used to study small diameter (1-2mm) and length (1 cm) TEVs.

Tissue-engineered Heart

Figure 3. Cadaveric rat hearts using PEG (a), Triton-X-100 (b), or SDS (c) over 12 hours. Ao, aorta; LA, left atrium. LV, left ventricle. RA, right atrium; RA, right atrium; RV, right ventricle. (d,e,f) staining of then thin sections from the LV of hearts perfused with PEG (d), Triton-X-100 (e), and SDS (f). Scale bars, 200 micrometers. . [2]

In the United States, approximately three thousand people are waiting for a heart donor (23). Currently, the only treatment for end-stage heart failure is heart transplantation. However, donor organs are scarce and transplants often run the risk of rejection from the recipient’s immune system. Hypertension, diabetes, and renal failure are the most common risks that come with heart transplantation and could potentially be avoided using a bioartificial heart. In 2008, Dr. Doris A. Taylor and her team engineered a rat heart by decellularization of a cadaveric rat heart and recellularization with neonatal rat cardiac cells (31). Decellularization was done by perfusion of detergents within the coronary, or the arteries that surround and supply the heart. The three detergents used were polyethylene glycol (PEG), Triton-X-100, and sodium dodecyl sulfate (SDS). The detergents washed away lipids, sugars, soluble proteins, DNA, and most other cellular material within the heart. As seen in Figure 2, SDS demonstrated the best results with complete decellularization leaving only the extracellular matrix. Collagen, laminin, and fibronectin were only left after the cellular material washed out from first the right ventricle, then the atria, and finally the left ventricle.

The scaffolds were then reseeded with cardiac cells from newborn rats and cultured under controlled physiological conditions to promote organ growth. Epithelium lining is important for prevention of clotting or leakage A bioreactor housed the whole rat heart to provide coronary perfusion with oxygenated culture medium wsystolid and diastolic medium through pulsatile anegrade. Bioreactors would also mimic beating by have electrical Two weeks later the new cells formed a beating heart that conducted electrical impulses and pumped small amount of blood. Tested viability of new hearts by implanting them into live rats which did not immediately reject the transplanted hearts. Reseeding by injection of cardiac cells and perfusion of endothelial cells into vascular conduits. After 8 days, the construct was drug responsive.

Figure 4. Process of decellularization of the heart. [2]

The researchers also bioengineered lungs and kidneys and successfully transplanted them into rats. Although the lung demonstrated proof of gas exchange, the lung soon filled with fluid (30). The kidney survived without clotting, but was not sufficient in filtering urine (32). Researchers also had previous success in transplanting a heart, but were unable to see blood pump through the organ. As researchers demonstrate higher functioning of organs the closer to producing bioengineered before transplanting into a larger animal or human.

Tracheal Tissue

Many tracheal disorders can be treated by resection and the cross-connection of the two separated segments, also known as end-to-end anastomosis (24). Treatment is not applicable for adults where the effected area is >50% in adults and >30% in children of the trachea due to anastomic failure. Patients with these conditions often are limited because of the lack of tissue available for reconstruction, deleterious effects of previous treatments to the trachea, anatomic challenges and underlying pathophysiology. In a study conducted in 2014, researchers developed both biological and synthetic tracheal scaffolds for transplantation into a rat model. The model to identify crucial properties of an ideal graft and investigate the pathways and relevant mechanisms of tissue-engineered trachea.

The first step in transplanting in transplanting the trachea Transplanted trachea were either from donor rodent or decellularized using detergent enzymatic method or artificial scaffold were created with electrospinning Electrospinning technology was used to improve existing artificial scaffolds that developed increased granulation (particles adhere to form larger, muliparticle entities) Used heterotopic (particular tissue type at a physiological site, but co-existing with orginal tissue in correct anatomical site) and orthotopic (occurring at same place). The scaffolds were then evaluated and seeded by mesenchymal stem cells (why??). Step 4: Investigated cell viability and metabolic activity using LIVE/DEAD assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assayStep 5: Transplantation

Feasibility of allogeneic tracheal transplantation in a rat model to study the development of obliterative airway disease. Which detected lymphocytic trachetis and epithelial ulcerations meaning there was adverse immune response to fresh allogeneic tracheal graft (3). Newer protocol does not provoke adverse immune response but maintains functional and mechanical integrity We use a rat model rather than a mouse model owing to drawbacks to transplantation models in mice that include the need for intraoperative microscopy and other specific infrastructure (microinstruments and microsutures), which make the use of the mouse protocol more complex, surgically challenging and costly.

Their study was useful in determining the efficiency of tracheal replacement with new materials before using larger animal models as well as can model underlying pathways of tissue regeneration and cellular contribution.

Limitations

Two main limitations of small animal models is that researchers cannot observe the animal model long term and cannot assess “clinically sized manipulations (e.g. defects and implants)” (10). In addition, there are still many remaining anatomical and genetic differences even between strains of rats. For instance, WIS rats grow larger than LEW and SD rats (10). Thus, when studying differences in mass and growth rate of tissue, such as adipose tissue, these values will vary.

1. As rats grow older, they become more limited in the amount of “basic multicellular unit-based remodeling” (2). 2. As they grow older, they also do not have impaired osteoblast function when there is estrogen deficiency during osteoporosis (2). 3. Small animals are not permissive of longer lasting experiments that may require numerous biopsies or large blood samples (2). 4. Properties of bone (torsional prop of long bones) (2) 5. Not all rodent models can be compared directly because of different strains (6) 6.Rats do not fully capture the whole scope Surgery is hard to perform due to size, so results are not easily transferred to humans

Ethics

Animal models have remained a controversial issue for many years. The majority of individuals are concerned with the humane treatment of animals (2). Ideally, animal models will be replaced by better, more accurate models. Legislation in many countries regulates humane treatment for research animals. Animals must be maintained in clean, uncrowded housing, asequately fed, and maintained in good health.

Conclusion and Future Work

Tissue engineering involves the use of biomaterials, so animal models are useful for experimental preclinical settings. Biomechanics, Experimental and clinical advantages. There is a need for new tissues…. Integrations/interspecies will be further discussed in the next chapter on chimeras.

Figure 4. Process of decellularization of the heart. [2]