Breast Tissue Engineering

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Breast Tissue Engineering: Reconstruction Implants and Three-Dimensional Tissue Test Systems Karen J.L. Burg1, 2, 3, Beau Inskeep1, 2 and Timothy C. Burg1, 3 1

Institute for Biological Interfaces of Engineering, Clemson University, Clemson, South Carolina 2 Department of Bioengineering, Clemson University, Clemson, South Carolina 3 Department of Electrical & Computer Engineering, Clemson University, Clemson, South Carolina

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INTRODUCTION The chance of a woman developing breast cancer in her life time is approximately 12% [1]. It was estimated that in 2012 there would be approximately 230,000 new breast cancer cases and 40,000 breast cancer related deaths in the United States [1]. The chance of breast cancer related death in women is approximately 3%, second only to lung cancer. Fear of disfigurement due to mastectomy or lumpectomy and limitations of breast conserving options have precipitated interest in tissue-engineering breast reconstruction options. Similarly, the impact of the disease, both psychological and physical [2,3], and the limitations of current two-dimensional (2D) bioassays used to understand, combat, or prevent the disease have driven the interest in engineering 3D tissue test systems. This chapter summarizes the various cell types that may be useful for breast reconstruction, the polymers that are being used or that are being explored for use in breast reconstruction, and the limitations/advantages of specific animal models that allow one to test new tissueengineering approaches. Avenues to potentially promote the vascularization of engineered tissues are discussed, as the major limitation in engineering large tissue volumes is the inability to deliver nutrients and remove waste products once the tissue is implanted. Finally, the chapter overviews the concept of tissue engineering to create benchtop breast tissue test systems that may be able to aid in developing breast cancer therapies and, eventually, vaccines.

BREAST ANATOMY AND DEVELOPMENT The breast is a dynamic organ that evolves constantly throughout a woman’s lifetime. This tissue comprises multiple cell types that actively interact with each other [4]. Microenvironmental signals are key to the developmental processes of the breast throughout maturation [1]. Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00036-7 Copyright Ó 2014 Elsevier Inc. All rights reserved.

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FIGURE 36.1 Breast physiology.

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The complexity of the breast must be considered when investigating strategies for engineering breast tissue; however, understanding of the many interactions that occur between the stromal cells and epithelial cells in the breast tissue remains limited. Evidence indicates that paracrine molecules produced by stromal cells likely determine the success or failure of tissue-engineered solutions for breast tissue repair [5]. To better understand the development of breast cancer, it is important to first review the anatomy of the breast and the structures and functions of the tissues (Fig. 36.1). The breast structure is located on top of the pectoralis muscle, which is located on top of the rib cage [6]. Each breast contains 15 to 20 lobes which comprise 20 to 40 lobules. It is inside these lobules that the mammary glands responsible for milk production are found [7]. The lobules are connected together through ducts; the milk is collected in the ducts and then flows out through the nipple. The space between these structures is filled with fat and fibrous tissue, the ratio of which determines breast density. A high concentration of lymphatic vessels and lymph nodes is found throughout breast tissue; these structures facilitate the flow of lymph, which comprises white blood vessels called lymphocytes and a fluid from the intestines called chyle, which contains proteins and fats [8]. The lymph flows to the nearby lymph nodes located in the underarm, above the collar bone, and behind the breast bone [7]. Blood vessels are also present to carry blood around the tissues to provide nutrients to the cells. The size and shape of the breast is determined by the skin envelope and the adipose tissue surrounding the connective and glandular tissues. The firmness of the breast mound is dependent on the number of adipose clusters located within the breast, with higher adipose content resulting in a softer breast mound. The deep fascia and a thin layer of loose connective tissue are located between the breast and the pectoralis muscle; the connective tissue allows the breast to move freely over the deep fascia. The breast is attached to the skin through suspensory ligaments, also termed Cooper’s Ligaments, which provide additional support and contribute to the shape of the breast mound [9]. Several changes occur in the breast as a women goes through puberty and menopause. During puberty, hormones released by the ovaries and pituitary gland cause the tissue to grow and the ducts to expand, forming mature ductal structures. While the structures are completely formed, they do not become fully active until pregnancy, when the lobules grow and begin producing milk. During menopause, when hormones are no longer produced by the ovaries, the lobule count in the breast decreases and those lobules that remain shrink in size. This change leads to a lower breast density, since the ratio of dense, fibrous tissue to adipose tissue decreases. For this reason, a woman’s breast is typically denser before menopause than after [9].

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

BREAST CANCER DIAGNOSIS AND TREATMENTS Historically, breast cancer was not detected at a very early stage and therefore was treated with radical mastectomy, or removal of the entire breast, underlying pectoral muscles, and axillary lymph nodes. As breast cancer detection methods improved, however, breast cancer was discovered at earlier stages, allowing modified radical mastectomies or removal of small tumors. Randomized prospective clinical trials, conducted over 18 years comparing less deformative techniques, demonstrated equivalent survival rates to modified radical mastectomy [10]. These studies demonstrated that, for most women with small breast tumors, simple excision (lumpectomy) of the breast cancer, sampling of the axillary lymph nodes, followed by radiation provides a similar outcome to a radical mastectomy. Different treatment options can be used in combination to maximize the desired outcome. Breast cancer patients have therapeutic options, such as chemotherapy, radiation, and hormone therapy, and/or surgical options. Most often the treatment will be a combination of surgical and therapeutic options; treatment is determined on a patient by patient basis since individual cases of breast cancer have distinct characteristics. The treatment option is chosen based on stage of the cancer (e.g., in situ or invasive), size of the tumor, health condition of the patient, and several other factors. There are two main surgical procedures, mastectomy and lumpectomy. The progression of the breast cancer is the main determinant of procedure. If the cancer has spread beyond the tumor mass formation, then a mastectomy, or removal of the entire breast, is recommended. If the cancer has not progressed outside the initial tumor mass, then a lumpectomy, or removal of the cancerous mass, can be performed followed by the use of an adjuvant therapy, most often radiation [11]. A mastectomy involves the removal of the interior of the breast mound, the nipple, the areola, as well as a wide margin of tissue around the incision. Prior to surgery, the extent of the axilla tissue that must be removed in order to remove the cancerous tissue is determined. Cancer cells most easily spread through the lymphatic system, so the progression of the cancer can be determined by examining the lymph nodes around the cancerous breast [11]. Because of the ease of travel through the lymphatic system, it has become common practice to remove some of the surrounding lymphatic vessels and nodes.

BREAST RECONSTRUCTION There are no cosmetic surgical procedures available for lumpectomy patients. Following a mastectomy, patients are given the option of undergoing breast reconstruction surgery. The type and timing of reconstruction is determined first by the physical limitations of the patient and then by preference. An option that has grown in popularity is breast reconstruction immediately following mastectomy [12]. Previously, it was thought that reconstruction should be delayed to prevent any possible interference with an adjuvant therapy. However, benefits to immediate reconstruction include one surgery and hospital stay, better psychological outcomes, and improved aesthetic results. Risks involved with this combination surgery include extended surgical time and an increase in complexity of the procedure [12]. The risk and rewards of undergoing such a procedure are evaluated on a patient by patient basis. There are two primary types of tissue implants for breast reconstruction; synthetic and autologous.

Synthetic implants Generally, synthetic implants are simpler and require less surgical time, but the results are not as aesthetically satisfactory. The simplest reconstruction is a silicone breast implant, which is a silicone gel- or saline-filled silicone bag that is implanted in the submuscular position beneath the removed breast mound. In some instances, the void volume may be increased by the progressive inflation of a tissue expander [12] prior to placement of a silicone breast

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implant. Because an implant is a foreign body, it may trigger a substantial inflammatory response, resulting in fibrosis, thickening, capsular contraction and an unnatural shape and tactile quality. Also, implants may leak and require replacement. Silicone-filled implants are now rarely used due to fears of possible complications caused by the leakage of silicone.

Tissue flaps Autologous breast reconstruction relies on the use of the patient’s own tissue, is more complex, requiring more extensive surgery and a longer recovery time but the results are much more natural and aesthetically pleasing than those of prosthetic implants. There are several primary tissue retrieval sites, including the abdomen, the back, and the buttocks or thigh. The transverse rectus abdominis myocutaneous (TRAM) flap, located on the abdomen, is surgically excised, including fatty tissue, abdominal wall skin, often with the blood supply network intact, and is molded into the breast mound. A TRAM flap procedure requires that an additional surgery be performed to reconstruct the nipple-areola and to improve the shape of the reconstructed breast mound; a weakening of the abdomen as well as contour abnormalities of the abdomen can occur after this procedure [9]. The latissimus dorsi flap is removed from the back in a similar but less involved manner than removal of the TRAM flap. Disadvantages of this flap procedure include susceptibility to atrophy and lack of patient-to-patient tissue volume consistency. Muscle-free flaps from the buttock (gluteal) or thigh (tensor fascia lata) may also be used; in some instances a muscle sparing procedure may be used, wherein only fat/skin is transplanted. Adipose tissue matrices, devoid of lipids and cells, have been investigated for use in soft tissue defect repair [13].

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Some of the very first studies performed regarding adipose tissue replacement used a method called autologous fat transplantation. This methodology simply involved harvesting adipose tissue from a location on the patient and transplanting that tissue to the breast tissue void. This procedure was completed without a scaffold in place to guide the shape of the tissue replacement. The results of this type of procedure were very poor, with 50e70% reduction in volume due to the resorption of the grafted tissue. Resorption occurred because adipocytes are anchorage dependent and require a scaffold to survive. Additionally, the adipocytes found in the tissue graft were terminally differentiated and, therefore, could not proliferate [9]. After several failed attempts, it was determined that a scaffold was required for proper breast tissue replacement. The cellular material used for transplantation is obtained from lipoaspiration, a process that can damage and lyse the cells. Researchers are developing means with which to treat lipoaspirate, for example, the lipoaspirate may be washed in a polyoxamer, which is thought to stabilize the cellular membranes of damaged adipocytes, and lend greater stability for implantation and graft survival. Preliminary studies suggest that the treatment can increase implant viability post implantation [14].

Cellular scaffolds Breast tissue engineering is another reconstructive option, beyond that of synthetic or autologous implants. Breast tissue engineering may involve cellular or acellular scaffolds. Much of the current research is still focused on traditional approaches of ex vivo cell expansion; however, due to financial, regulatory and logistical obstacles, more focus must be placed on ‘just-in-time’ delivery options that do not incorporate cell expansion ex vivo. Currently, viable breast tissue-engineering technologies have not been translated from the laboratory to the clinic due to several existing challenges related to cell culture, scaffold type, and animal model selection that affect our ability to build a vascularized tissue that maintains volume in the long-term.

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

CELL TYPES AND RELATED CHALLENGES Many issues must be considered when choosing which cell type(s) should be used in human breast tissue engineering. The first consideration is that there is substantial variability in the size, shape and consistency of the breast. The breast changes over time, with a tendency for breast parenchyma (glands and ducts) to involute or regress as a woman ages, particularly after menopause, and be replaced by fat [15]. Also, comparing breast tissue among women of any given age, there is considerable variability in the size, shape, tactile, elastic and tensile characteristics of the tissue. The tensile and elastic characteristics of the breast are influenced by three major factors: 1) The amount and quality of fat within the breast; 2) The amount and quality of glandular and ductal tissue in the breast; 3) The mechanical characteristics of the fibrous support structures of the breast (Cooper’s ligaments). The creation of a functional breast with lactational ability is not needed and, in fact, may add to a woman’s breast cancer risk by introducing mammary epithelial cells that may be predisposed to cancer development. The major, immediate goal of breast reconstruction is to produce a breast mound with all of the aesthetic properties of a normal breast. Normal breast tissue comprises adipocytes; however, these lipid-laden fat cells (adipocytes) are terminally differentiated and will not divide further in vivo or in vitro. Indeed, studies have shown that if not maintained in a 3D culture environment, these differentiated cells will likely de-differentiate and become fibroblastic [5]. Hence, use of adipocytes requires harvesting fat in the exact volume required for the construct [16e18]. Additionally, the majority of mature fat cells in a lipoaspirate sample rupture [8,9,18]; accordingly, measures must be taken to compensate for or prevent this loss. Pre-adipocytes may be used for engineering soft tissue [19,20] as these cells are not as susceptible to retrieval damage and can potentially be expanded in culture. Pre-adipocytes are similar to fibroblasts in structure and possess the ability to expand in culture [21]. Several studies have shown ways of inducing differentiation into adipocytes. These cells can be successfully harvested and isolated from sites such as the subcutaneous tissues or the omentum. Investigations of autologous pre-adipocyte implantation with a sheep model have been promising [22]. Technologies are being developed to allow simple harvesting and fast isolation of pre-adipocyte cells for immediate re-implantation into the patient [23]. Studies in mice have shown that the incorporation of ’adipose-derived regenerative cells’ in fat grafts decreases cell apoptosis and increases expression of growth factors [24]. Possible other cell types include smooth muscle cells, fibroblasts, skeletal muscle and elastic cartilage. Fibroblasts contribute greatly to the support structure of the breast by laying down bands of collagen that connect the breast tissue to the skin and to the pectoral muscle, as well as helping maintain the overall shape. The density and firmness of the breast is determined primarily by the glandular epithelium and ductal structures, where tissues that have similar tactile and elastic properties are almost exclusively muscle. Smooth muscle cells can be readily isolated from a number of organs and expanded in culture; implantation of smooth musclecontaining polymers can lead to the reformation of significant tissue masses, with reorganization of the smooth muscle tissue into appropriate 3D structures [25]. Muscle myocytes can also be greatly expanded in vitro, and have been demonstrated to reform functional tissue masses under appropriate conditions [26,27]. However, it remains to be demonstrated that smooth or skeletal muscle myocytes will maintain a tissue mass over long periods of time without neural stimulation. An alternate approach might even be to incorporate chondrocytes in an engineered breast tissue. Elastic cartilage has many of the mechanical properties of glandular breast tissue that are potentially important for tissue engineering of breast (e.g., elasticity). Chondrocytes can be expanded in culture, and are able to survive in low

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oxygen tensions. Chondrocytes have been used extensively in tissue engineering to engineer a variety of tissue constructs both in vitro and in vivo [28e30]. However, the more commonly studied cell types for breast tissue engineering include human bone marrow-derived mesenchymal stem cells (hBMSCs) and human adipose-derived mesenchymal stem cells (hASCs) [31]. Both cell types have the ability to differentiate into adipocytes when introduced into an adipogenic differentiation medium. The hBMSCs are harvested from the bone marrow, through a relatively painful procedure, which lessens their clinical relevance, while hASCs can be harvested in large volume from adipose tissue obtained via biopsy or liposuction [31]. Recognizing that regeneration may most effectively occur in situ with cues from the native tissue, cell types such as mesenchymal stem cells and embryonic stem cells have rapidly become the most popular cells for breast tissue-engineering research. Human embryonic stem cells, hESCs, are readily able to differentiate into adipocytes [31]; however, a great deal of work will need to be done to ensure terminally differentiated cells, that once transplanted, do not have the potential to form a tumor mass. The implantation of embryonic stem cells has the potential to result in teratoma formation in vivo [32,33]. Studies in mice have shown that the formation may be site dependent and may be exacerbated in the presence of specific biomaterials [34].

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Standard cell isolation and expansion protocols must also be developed; critical steps influencing successful outcomes will include aseptic technique in the harvest, routine quality control testing of all cultures, long-term cell storage, and on-site operating room handling. It may also be possible to isolate all the cell types required for breast tissue engineering from a single tissue source; furthermore, it may be advantageous to retrieve tissue isolates rather than cellular isolates [35]. For example, fat, in addition to adipocytes, contains a large vascular network, composed primarily of capillary endothelial cells and some vascular smooth muscle cells as well as a collagen stromal structure produced by fibroblasts [36]. Hence, multiple cell types or cellular aggregates (tissue isolates) can potentially be obtained from this tissue. In attempting to produce fat tissue in vivo, it may be beneficial to expand the cellular components of fat without isolating each component, as the complexity of the mechanisms may be crucial and impossible to rebuild from cellular blocks. Given the complexity of tissue, it is most likely that a mix of cells, with respect to maturity and type, will result in the most promising tissue-engineering solution.

SCAFFOLDS An important consideration in engineering breast tissue is the scaffold selection. Selection of a material chemistry and form to use as a scaffold depends on the purpose and characteristics of the tissue that is being replaced as well as the physical characteristics and health of the patient. Scaffolds may be fabricated to induce tissue integration or they may be developed to house or attract cells which, in turn, assist in inducing tissue integration. Several key characteristics make a biomaterial suitable for tissue-engineering application. First, with in vivo endpoint, favorable biomaterials must be absorbable or degradable, and therefore facilitate new tissue integration with native tissue over time. The shape and texture of the material does not have to resemble that of natural tissue but it should induce growth of new breast tissue resembling native tissue, i.e., be soft and pliable. Cellular affinity is another important aspect of a biomaterial, whether considering in vivo or in vitro endpoint. The biomaterial must interact favorably with cellular components without negative impact (e.g., the material cannot be tumorigenic or toxic). Some element of porosity or surface texture is helpful and potentially allows cellular ingrowth into the material and/or transfer of nutrients and waste products, the establishment of a vascular network into the biomaterial scaffold [37], and/or differentiation of multipotent cells [38].

Synthetic materials Aliphatic polyesters of polyglycolide (PG) and polylactide (PL) are well characterized synthetic biodegradable polymers which are clinically familiar to biomedicine and are familiar also to

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tissue-engineering researchers [39]. PG is highly crystalline, has a high melting temperature, and has low solubility in organic solvents. PL is more hydrophobic than PG due to the presence of a methyl group. PL has a low water uptake and its ester bond is less susceptible to hydrolysis, due to steric hindrance by the methyl group. Therefore, PL degrades more slowly and has higher solubility in organic solvents than PG. Copolymers of PL and PG can be readily synthesized; their physical properties are modulated by the ratio of glycolic acid to lactic acid. Often incorrectly named in the literature, polylactide and polyglycolide are synthesized by ring opening polymerization, while the lower molecular weight polylactic acid and polyglycolic acid are synthesized by step growth. The latter are on the order of thousands of Daltons and are limited to use in structures with low mechanical demands, such as films and spheres [40]. Aliphatic polyesters can be readily processed into various physical forms appropriate for tissueengineering applications. A number of techniques have been proposed to generate highly porous scaffolds, including solvent casting/particulate leaching [41], phase separation [42], emulsion freeze-drying [43], fiber extrusion and fabric formation [44], and gas foaming [45,46]. Polyethylene glycol (PEG) scaffolds [47] are readily modified by degradation and adhesion peptides and thus have been the subject of pre-adipocyte studies. The combination of adhesion and degradation features appears to allow the highest adhesion and proliferation of pre-adipocytes. Non-degradable polymers have also been examined for tissue reconstruction applications. For example, in vitro studies have been conducted with fibronectin-coated polytetrafluoroethylene scaffolds (PTFE). Human pre-adipocytes have been shown to successfully attach, proliferate, and differentiate into adipocytes on the PTFE scaffolds [48]; however, these non-degradable scaffolds are likely better suited for 3D in vitro tissue test systems. A number of other synthetic polymers could be used to fabricate scaffolds for breast tissue reconstruction, including polycaprolactones, polyanhydrides, poly(amino acid)s, and poly (ortho ester)s [49]. Polycaprolactone (PCL) is an aliphatic polyester, a semi-crystalline polymer with high solubility in organic solvents, a low melting temperature, and a low glass transition temperature (Tg). The degradation rate of PCL is much slower than PG or PL; because of the low Tg, PCL has a flexible, sticky quality which can be advantageous in a scaffold. PCL has been tested in scaffold form in animals [50,51] and is used clinically in orthopedic applications. Polyanhydrides are usually copolymers of aromatic diacids and aliphatic diacids. These materials degrade by surface erosion, the rate of which can be controlled depending on the choice of diacids [52]. Poly(amino acid)s have been studied due to their similarity to proteins, and have been widely investigated for use in biomedical applications such as sutures and artificial skin [53]. Poly(amino acid)s are usually polymerized by ring opening of N-carboxyanhydrides; versatile copolymers can be prepared from various combinations of amino acids. However, due to the low solubility and limited processability of poly(amino acid)s, ’pseudo’-poly(amino acid)s were developed [54]. It has also been reported that poly(amino acid)s containing L-arginine, L-lysine or L-ornithine cause endothelium-dependent relaxation of bovine intrapulmonary artery and vein, and stimulate the formation and/or release of an endothelium-derived relaxing factor identified as nitric oxide [55]. Poly(ortho ester)s are biodegradable polymers, which degrade by gradual surface erosion and have been investigated for controlled drug delivery.

Naturally-derived materials Naturally-derived polymers have been used for adipose tissue engineering; scaffolds produced from these materials typically are hydrogels or structural forms like mesh, sponges [9], or beads. Investigations have been conducted, for example, using MatrigelÔ (reconstituted basement membrane of mouse tumor) and fibroblast growth factor 2 (FGF-2) to induce in situ adipogenesis. MatrigelÔ consists largely of type IV collagen, laminin, and perclan and, although a highly variable material with many ill-defined components, is considered the gold standard in cancer cell biology benchtop studies. Preliminary small animal studies have

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PART 8 Breast demonstrated the migration of native pre-adipocytes as well as endothelial cells into MatrigelÔ when this material is injected into subcutaneous tissue [9,37,56]. Hyaluronic acid (HA) is a natural component of the extracellular matrix of many tissues. HA comprises repeated sequences of glucuronic acid and acetylglucosamine; this material is susceptible to enzymatic degradation via hyaluronidase. Simple modifications have been made, such as cross linking the chains to form insoluble hydrogels [57]. In its natural form, HA plays a role in enriching wound healing by promoting early inflammation and stimulating angiogenesis [58]. Hyaluronan benzyl ester (HYAFFÒ 11) scaffolds are derived from hyaluronic acid that is esterified with benzyl groups at the glucuronic acid monomer. Researchers experimented with these sponges, seeding them with human pre-adipocytes and surgically implanting them into subcutaneous tissue of athymic nude mice. The sponges allowed good cellular penetration as well as the development of new vascular networks within the sponges. However, adipose tissue development remained sparse [59]. The researchers also compared collagen scaffolds to HYAFFÒ 11 scaffolds in vivo, and concluded there was increased implant weight, adipose tissue formation and distribution of cells in the HYAFFÒ 11 scaffolds [60]. Hydrogels of hyaluronic acid have been prepared by covalent cross linking with various kinds of hydrazides [61] and have been used in drug delivery [62].

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Collagen is the best-known tissue-derived natural polymer and is the main component of all mammalian tissues, including skin, bone, cartilage, tendon, and ligament. Collagen has been used as a tissue culture or artificial skin scaffold due to its high cell affinity. However, collagen offers a limited range of physical properties, can be expensive [63], is highly variable, and can elicit a strong immunologic response. Chemical modification and incorporation of fibronectin, chondroitin sulfate, or low levels of hyaluronic acid into the collagen matrix can change cell adhesion [64]. In vivo comparisons of freeze-dried collagen scaffolds with hyaluronic acid sponges and non-woven mesh, implanted in mice for 8 months, revealed a greater number of adipocytes in the hyaluronic sponges than in the non-woven mesh. This difference was mainly attributed to the porous nature of the sponge, which allowed greater surface area for adipocyte cell distribution and growth [60,65]. Recent studies also have assessed the idea of creating a fibrovascular tissue bed or natural scaffold, using a preexpansion vacuum into which fat cells are transplanted [66]. Alginate is a naturally occurring hydrogel that can be easily formed into an injectable gel or beads, but must be modified with a peptide sequence to allow cell attachment [22,67,68]. Interestingly, the human body does not contain alginase, the enzyme that breaks down the alginate chain, hence molecular weight is a crucial consideration for implantation as large molecular weight alginate chains will not be eliminated from the body. That is, the molecular weight of many alginates is typically above the renal clearance threshold of the kidney [69]. Alginate chains are bound together with divalent ions that migrate in areas of divalent ion deficiency, causing uncontrolled dissolution. To address this point, hydrolytically degradable, covalently cross linked hydrogels derived from alginate were developed [70]. Specifically, polyguluronate blocks with molecular weight of 6,000 Da were isolated from alginate, oxidized, and covalently cross linked with adipic dihydrazide. The gelling of these polymers could be readily controlled, and their mechanical properties depended on the cross linking density. It was also demonstrated that alginate gel degradation can be readily regulated by controlling the molecular weight distribution of the polymer chains in the gels, and their susceptibility to hydrolytic scission by partial oxidation [71]. Other materials have found limited, preliminary use in breast tissue engineering. Chitosan is relatively biocompatible and biodegradable [72,73], making it useful for breast tissue engineering [74] and wound healing [75]. Chitosan is abundant and easily derivatized by coupling molecules to the amino groups [75,76] and has shown early success in murine studies focused on injectable breast tissue-engineering systems [35]. Fibrin glue has been used as an adipocyte scaffold and, in small animal studies, has facilitated maintenance of adipose tissue up to

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one year after implantation [78]. Fibrin is a blood based product, a characteristic which has slowed its translational appeal in the United States.

Therapeutic scaffolds Some scaffolds provide more than a simple matrix on which cells will grow. Some scaffolds have an incorporated therapeutic agent such as a drug or growth factor, and are termed therapeutic scaffolds. The concept behind these scaffolds is that the therapeutic agent incorporated in the scaffold is released as the cells remodel the scaffold during cell growth and proliferation. The therapeutic agent therefore has a direct effect on the surrounding tissue where the scaffold is placed; accordingly, the use of growth factors and the stimulation of cell growth must be carefully evaluated in breast cancer related reconstruction, to avoid facilitating the cancer process. Indeed therapeutic agents can affect the tissue surrounding the implanted scaffold. An example can be seen in a study where the therapeutic agent, angiogenin, was incorporated into a scaffold [79]. Angiogenin is a drug that has been shown to promote neovascularization, so the intent of its incorporation was to help promote the growth of new vasculature throughout the scaffold, increase the chances for the success of an implanted tissue replacement, and improve the overall outcome of the procedure. The investigators subcutaneously implanted these scaffolds into rabbits and, after 28 days, the scaffolds were excised. The study showed that scaffolds with incorporated angiogenin had increased neovascularization. Other therapeutic scaffolds have been designed; for example, a collagen/chitosan/glycosaminoglycan scaffold was assessed [80]. The agent for this therapeutic scaffold, transforming growth factor-beta 1 (TGF-b1) targeted cells grown on the scaffolds rather than the surrounding tissue. Accordingly, TGF-b1 was incorporated into chitosan microspheres that were embedded into the scaffold. A final example of a therapeutic scaffold is one in which the agent targets and neutralizes a specific type of cell found in the surrounding tissue, such as a cancer cell. Researchers developed, for example, scaffolds with nanoparticles containing emodin, an anti-cancer drug [81]. These scaffolds were intended to fill a site where a cancerous tumor was removed from the breast. The concept of this scaffold is that as cells proliferate on the scaffold and remodel it, the emodin contained within the nanoparticles is released and neutralizes cancerous cells in the surrounding tissue. These scaffolds were implanted next to the mammary fat pads of nude mice in which cancerous cells had been injected. The results of this study indicated that the size and number of the tumors next to emodin-loaded scaffolds were reduced compared to those next to scaffolds without emodin.

Injectable scaffolds Early scaffolds took forms such as foams or mesh; they required implantation via an open surgical procedure. However, the reality of the transport and surgical limitations of large volume implants led to the development of alternate scaffolds [82]. Injectable scaffolds can be combined with cells and surgically implanted in a minimally invasive manner, including by syringe, catheter or endoscopic needles [83,84]. These injectable materials take forms such as gels, beads, or composite gels. Examples of injectable scaffold chemistries that have been assessed for breast tissue engineering include alginate, chitosan, hyaluronic acid, collagen, polyanhydride, degradable PEGs, decellularized adipose tissue, small intestinal submucosa, and blends thereof [13,35,82,85,86]. While gels support cell growth and readily conform to a defect, they typically do not support the necessary functions of anchorage dependent cells and are therefore useful as carriers but not as scaffolds in breast tissue engineering. Beads and composites (which contain beads or other filler) in contrast, are amenable to breast tissue engineering.

Combination scaffolds It is likely that a combination of biomaterials, with respect to chemistry and form, will result in the most promising tissue-engineering solution. Scaffold combinations that use two or

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more types of materials can be used to help combat and overcome the weaknesses and shortcomings of one material. For instance, a material that has excellent cell attachment characteristics but is not very durable can be combined with a more durable material, hopefully resulting in a scaffold that is both durable and able to support cell growth. One of the first combination breast tissue-engineering scaffolds was an injectable composite comprising beads in a delivery gel [82]. Indeed, cellular constructs that are approximately 500 mm in thickness or less, once implanted, may optimize diffusional transport of nutrients to the cells while each small cell-polymer unit becomes vascularized. Accordingly, these injectable composites were developed specifically to allow trafficking and infiltration of blood vessels, nutrients, waste products, other factors and cell types, within the discrete portions of the scaffold (i.e., between beads) [35,82]. The beads, or a fraction of the beads, may be selectively loaded with appropriate factors to induce tissue growth or prevent abnormal tissue growth. The gel is degradable and facilitates delivery of the composite through a needle, and also allows the composite to conform to and fill an irregular defect site. The gel may be loaded with factors for release on degradation. A variety of studies have been conducted to demonstrate the modularity of an injectable composite approach [35,68,82,87,88].

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It is unlikely that there is a one-size-fits-all biomaterial chemistry or form; accordingly, there is interest in a wide range of combination scaffolds. In one study, gelatin sponges, polyglycolide mesh, and monofilament polypropylene mesh were used to construct three-dimensional scaffolds of predefined shapes on which human adipose-derived mesenchymal stem cells (hAD-MSCs) were grown [89]. The scaffolds were made of an outer polypropylene mesh pocket that contained gelatin sponge cubes and polyglycolide mesh. Gelatin is an attractive scaffold for cell growth and attachment and can be molded into a desired shape but rapidly loses its dimensional stability over time. The polyglycolide mesh was used to increase the surface area available for cell attachment. Polypropylene mesh was used because of its ability to maintain dimensional stability after being implanted into the body. These scaffolds were seeded with a high density hAD-MSC suspension and cultured for 2 weeks. Subsequently, the scaffolds were implanted into the backs of nude mice for 2 months and then excised for analysis. Analysis showed that the scaffolds contained new adipose tissue as well as neovascular structures. The gelatin cubes, as well as the polyglycolide mesh, were completely absorbed by the body but the outer polypropylene mesh retained the predefined dimensions; the neovascular structures may simply be the transient part of the normal foreign body response.

STRATEGIES TO ENHANCE THE VASCULARIZATION OF ENGINEERED TISSUE A critical challenge to engineer breast tissue, or any tissue of significant thickness, remains the development of a vascular network to support the metabolic needs of the engineered tissue and integrate it with the rest of the body. Adipose tissue is highly vascularized, with a resting blood flow two to three times higher than that of skeletal muscle. The presence of vascularized networks in natural metabolic organs results in short diffusion distances between the nutrient source and the cells [90], and these vascular networks must be created in engineered tissues as well. Nutrient diffusion in vivo is constrained to a distance of approximately 150 mm. Most metabolically active cells that are located further than this distance from a nearby capillary are subject to hypoxia. Thus the success of any large engineered tissue hinges on its blood supply. The important interrelationship between pre-adipocytes and endothelial cells was demonstrated in hypoxic culture experiments. Frye and coworkers [91] exposed cell cultures of pure preadipocytes, as well as mixed cultures of pre-adipocytes and microvascular endothelial cells, to hypoxic conditions (5 to 2% O2) and found that pre-adipocytes co-cultured with microvascular endothelial cells had higher viability than cultures consisting of pre-adipocytes alone. Several general approaches have been taken to date to promote angiogenesis in engineered tissues; however, none provide a robust, consistent solution to this complex problem. Bland and colleagues [92] provide a detailed review of approaches to combat hypoxia in

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tissue-engineered systems. In short, this problem has been unsuccessfully addressed in the longterm by incorporating endothelial cells or angiogenic factors in tissue-engineered implants. Concerns about blood vessel promoting factors are high when targeting solutions for breast cancer patients. The composite injectable systems do provide the option of gradually building smaller volumes of tissue over time to collectively build a large volume [93]. Many studies highlight angiogenic response at short time points, suggesting that the foreign body response provides the necessary vascularity [94,95]. Indeed, while this is the case in the short term (and at later defined intervals as the material is further degraded or absorbed and elicits further response), the vascularity is temporary only. Blood vessel ingrowth occurs slowly with this approach, will likely not be sufficient to engineer large tissue volumes, and will likely subside with termination of the foreign body response. Other approaches attempt to actively modulate the vascularization process by either delivering angiogenic molecules or blood vessel forming cells (e.g., endothelial cells) to the site at which the tissue is being engineered. Prevascularization, either in vitro or in vivo, has also been proposed and investigated by numerous groups, but the problem of integrating newly developed vasculature with host tissue remains unsolved. Microstamping using nano-fluid chambers is one research approach of interest in liver tissue engineering [96], where the long-term goal is to develop a 3D vascular bed ex vivo that could be anastamosed to the host vasculature to support cellular engraftment. This approach could also be developed for breast tissue engineering. Another approach involves the direct association of an implant with a pre-existing blood supply. Experiments with nude mice were performed in which silicone molds packed with polyglycolide fibers were sewn to the inferior epigastric blood vessels. These silicone molds were injected with a combination of Matrigel and FGF-2. In situ adipogenesis was demonstrated over a 4 to 20 week time course. Direct application of a vascular pedicle to a construct is another promising approach. Prefabricated flaps have been created with vascular pedicles since the 1960s; vascular pedicles can be supplied in many different configurations. In general, however, conduits comprising an intact artery and vein fare better than those comprising a single vein alone [97,98].

Special considerations The need for nipple reconstruction occurs after mastectomy. Traditional implants include free composite grafts, local tissue transfer, and prosthetic devices. Free composite grafts were initially used and were created from autologous tissues such as the labia, inner thigh, cartilage (auricular or costal), the contralateral nipple, or the toe. Complications at the graft site made this technique less desirable. Local tissue flaps are the most popular option for nipple reconstruction. Commonly used techniques include the bell flap, the modified star flap, and skate flap [99e102]. Unfortunately these techniques can be hindered by flap necrosis and poor aesthetic results, including loss of nipple projection. Additionally, the presence of underlying subcutaneous fat is important for bulking; this layer is not always sufficient. Tissueengineering approaches include the use of tissue flaps coupled with acellular, naturally-derived (collagen, extracellular matrix, etc.) matrices [103] and/or fat grafts.

BREAST CANCER MODELING As Savage stated, ’Cancer is not a simple disease’ [104]. A significant challenge to both the development of breast cancer treatments and fundamental understanding of breast cancer processes is the development of a tool, a model, which allows isolation and control of specific parameters of interest. The study of basic disease processes and treatments in a patient is generally very inefficient because of the number of confounding biological (and ethical) issues. In response to this challenge, researchers have proposed animal models, mathematical models, and benchtop test systems as the means to more effectively study breast cancer.

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Animal models It is important to first consider the application for which an animal study is planned before the selection of the animal model itself. Either the nude (nu/nu) or severe combined immunodeficiency (SCID) mouse model is used for the transplantation of human-derived cells [105]. These animals have compromised immune systems to the extent that they will often accept xenogeneic transplanted tissues, and these mice have been particularly useful for transplantation and immunologic studies of human tumors, bone marrow, skin and other tissues [93,106,107]. SCID and nude mice are used to evaluate various polymer constructs with and without human cells in vivo, without the adverse effects of a major immunologic reaction. Furthermore, basic questions about human cells and polymers in vivo may be answered without launching human trials prematurely. Development of these models may not be straightforward since there are subtle differences between strains with respect to the acceptance of various tissues and growth of the implanted tissues in different sites (e.g., a tissue may grow in one mouse strain but not in another, or may grow in a subcutaneous site but not in an internal location). Even though mice are genetically altered for immunodeficiency, the particular genetic modifications can vary. These varying genetic modifications can cause varying levels of different hormones throughout the host body. Hormonal differences will result in adipose tissue development differences that will likely result in different biological reactions to the same implant. Several different genetic varieties of laboratory rats exist, which poses complications when attempting to compare studies [105]. Biologically, males and females of the same species have different hormone levels due to differences in development and maturation. Additionally, gender-induced differences in hormones cause a difference in adipose tissue development which may result in different biological reactions to similar scaffold constructs [105].

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Nude mice lack a T-lymphocyte response, while SCID mice lack both a T- and B-lymphocyte response. However, both types have natural killer cells that may interact with some transplanted materials. These mouse colonies must be monitored closely for changes in the immune status of the mice that, at times, occurs spontaneously. In addition, the human cells must be routinely screened for the human immunodeficiency virus, hepatitis and mycoplasma before being transplanted into immunosuppressed mice to assure the safety of the animals and workers, and the validity of the experiments. One other complicating factor is the size of the actual animal in question. Small size animals include animals such as mice and rats, while larger sized animals include sheep, domestic pigs, and cows [105]. When considering mammary tissue, the larger sized animals are more physiologically relevant to humans. This issue raises the question of applicability of relating animal model results to an expected human clinical trial outcome. Large animals can accommodate the same size implant as one that would be used in a human. Also, the internal anatomy of larger animals, such as the domestic pig, is much more similar to the human anatomy in regard to organ size and heart rate. It has also been shown that the histoarchitecture and hormonal control of the mammary glands of larger animal models is much more applicable to humans than those of rodents [108]. Studies have shown that bovine cells as well as human cells do not grow in the mammary fat pads of immune suppressed mice. This observation suggests that the fat pads of rodents do not provide an environment suitable for the proper growth of human or bovine mammary epithelium. It has also been shown that progesterone has a very different effect on the mammary tissue of mice when compared to that in larger animals, including humans. In mice, progesterone stimulates epithelial proliferation and ductal side branching, whereas progesterone has a limited effect on the mammary epithelium of larger animals and humans. There has been excellent success using inbred female Lewis rats as a small animal model for the development of transplantable tissues with absorbable polymers such as PG, PL, poly(lactideco-glycolide), and hydrogels such as alginate [109]. This model allows transplantation of cells

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

between individuals without concern for immunologic rejection, which parallels the likely autologous nature of cell transplantation for breast engineering. In addition, the Lewis rat is larger than many other strains, allowing the testing of larger (1e2 cm) or multiple constructs in the same animal. In evaluating larger animals as models for breast tissue engineering, animals with skin and subcutaneous tissues that are similar to humans are required in order to evaluate larger constructs subcutaneously. For this reason, the same animal must be used as a tissue donor and recipient. Porcine skin and subcutaneous tissues are very similar to that in humans, but most pigs continue to rapidly gain weight throughout their lives, which makes monitoring implants very difficult. Sheep have very little subcutaneous fat and, depending on the location, have welldefined subcutaneous space for cellular engraftment. Hence, if new adipose tissue is formed there is a high probability that this is developed from the implanted cells. One of the few locations in the sheep where there are significant fat deposits is the omentum. Researchers have shown that pre-adipocytes isolated from the omentum can be expanded in culture, seeded onto porous alginate-RGD fragments [22], where they attach, proliferate, and spread onto the biomaterial surface. Cellular alginate-RGD fragments were subsequently injected into the nape of the neck of sheep to determine if new adipose tissue would form. Although the cells were autologous and not labeled with a tracking marker, there appeared to be de novo adipose tissue formation in the cell implant sites compared to the acellular biomaterial control sites. Indeed, further investigation of breast tissue-engineering options within a large animal that has biological characteristics comparable to that of humans is required. The bovine mammary gland consists of the same anatomical structures and tissue types as that of the normal human breast [110]. Histological evaluation reveals that bovine mammary tissue is more similar to that of humans than is mammary tissue of traditional animal models such as mice and rats. The ductal structures in the human and cow are surrounded by relatively dense stromal tissue [111,112] unlike the ducts in the mouse which are almost completely enclosed by adipocytes [113]. Table 36.1 summarizes the similarities and differences seen in mammary gland development in mice, humans, pigs, and ruminant animals like sheep, goats, and cows. However, larger sized animals present logistical and financial issues. Large animals are much more expensive than smaller animals and fewer researchers are trained in, or have the facilities for, the proper care of these larger animals [108].

Breast tissue test systems To better mimic aspects of the in vivo setting, three-dimensional tissue test systems are used to model experimental clinical conditions. Events that are notable in a 2D setting may not be present in a 3D setting, or vice versa [5]. Breast tissue test systems can be used to inform tissueengineering reconstructive techniques, or to better understand and prevent the disease process. Many studies have been conducted to assess breast cell co-cultures in 3D. The 3D material, i.e., the scaffold, has enormous influence on cellular behavior, and can be selected according to the biological aspect of interest. Obviously, 2D cultures are not amenable to the study of ductal structures or questions regarding spatial co-location [114]. In order to distinguish tissue test systems from in vivo human or animal models and refine the expectations of an in vitro model, a tissue test system can be defined as a modular unit of ’useful’ biology. A tissue test system is depicted in Fig. 36.2 as a tissue model built using a LegoÔ -like approach with biological and non-biological components that can then be used to answer biological questions. The three primary questions that must be considered in designing a test system are: 1) What is the biological question of interest and how can this question be answered with an approximate biological model?

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TABLE 36.1 Comparative aspects of mammary gland development [108] Attribute

Mice

Humans

Pigs

Ruminaants

Morphology Stromal histology

Sparse ducts: alveolar Adipose » connective

No. of ductules/TDLU (Types 1, 2, and 3, respectively) No. of galactophores Epithelial proliferation

N/A

TDLU Intralobular, interlobular connective » adipose 11, 47, 81

TDLU Intralobular, interlobular connective » adipose 9, 24, 64

TDLU Intralobular, interlobular connective » adipose Not defined

~ 8e15 Concentrated in endbuds or TDLU Epithelial Negative, or No Effect Spontaneous

2 Concentrated in endbuds or TDLU Endbuds, TDLU No Effect Rare, few cases

1 Peripheral zones of TDLU Epithelial No Effect Rare, few cases

ADH, DCIS 50e60%

Unknown Unknown

Unknown Unknown

Response to estrogen Response to progesterone Mammary tumors Tumor precursor Tumor hormone dependence

1 Concentrated in endbuds and alveoli Endbud Alveoli Spontaneous, viral origin AH, ADH, DClS Rare

Abbreviations: TDLU: terminal ductal lobular unit, AH: alveolar hyperplasia, ADH: atypical ductal hyperplasia, DCIS: ductal carcinoma in situ, TDLU: terminal ductal lobular unit.

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

FIGURE 36.2

Top row shows the LegoÔ approach where plastic blocks are used to build complex models. The analogy to biofabrication of a tissue test system is shown in the bottom row where the elemental components, cellular units, biomaterials such as beads, fibers, or gels, and biochemical agents are assembled to produce a tissue model.

2) What are the elemental components (i.e., the Lego blocks) and how are the elemental components arranged to make the biological model? 3) How will the elemental components be assembled and the final tissue cultured? One cannot survive in the world without mentally forming cause and effect relationships e models e that provide useful advice about behaviors of the systems around us. Many natural systems are understood through models which provide useful information, such as predicting the future state of a system for a change in environmental conditions. As an example, the behavior of a resistance may be described by the simple Ohm’s law mathematical model of the voltage across the resistance equals the resistance multiplied by the current, v ¼ Ri. This model is ’useful’ to make calculations such as how many light bulbs could be connected to an electric circuit. The Ohm’s law model is easy to use, scales to more complex problems, is consistent and reliable since it works for anything that is a pure resistance, has been verified by experimentation, is cost effective to use, can be extended to account for additional physical phenomena, and increases understanding of the system. Thus, even though the Ohm’s law model is not a copy of the resistance it provides useful information. A tissue test system holds the same promise to provide a model that facilitates the creation and sharing of new knowledge of breast tissue processes. The Ohm’s law resistance model has obvious limitations. When the model is tested under unmodeled conditions like very high temperatures, the information from the resistor model becomes less accurate and hence less ’useful’. In this case the resistance model can be updated by changing the constant resistance to a function of temperature, i.e., R(T). Specifically, the resistor model must be designed to include the phenomena that affect the accuracy and resolution needed to provide useful information for a specific question of interest. Thus, a breast tissue test system must be defined to match the expectations of the user to provide useful information. Practically, a tissue test system must be defined to meet the user’s needs and likely should not be an exact duplication of the in vivo biology; rather, it should capture only the salient physiological, mechanical, biochemical, morphological, and biological elements needed to study a specific phenomenon. A general schematic of the process of designing, fabricating, and using a tissue test system is shown in Fig. 36.3. Tissue test system development begins with sampling the physical system

FIGURE 36.3 Process of developing biologically relevant tissue test systems.

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to identify the scope and features needed in the test system model. The first challenge in using a tissue test system is defining what behaviors in the native system must be included to address a specific question of interest; that is, what does one intend to learn from the tissue test system and what are the first-order factors that affect the behavior under study. Model accuracy is used here to describe the translation of a biological question into a test pattern or structure for the tissue test system. Note that high model accuracy does not mean that the test pattern must be an exact reproduction of the biological structure, in this case the breast; rather, it must be a representation of the underlying chemical, physical, electrical, etc. phenomena that are useful for answering a specific biological question regarding breast tissue. In fact, a well-designed test system will exclude factors that confound answering the biological question of interest. Hence, different biological questions will require different test system models. As an example, there are currently test systems, such as those addressing adhesion assays, that comprise cells seeded onto a plate. These are relatively straightforward test systems to implement and hence are often the first assays performed. Such a flat, two-dimensional (2D) system can answer important questions; for example, adhesion assays were used to study the expression and function of Laminin-511 during metastatic breast cancer progression [115]. These results can inform decisions regarding incorporation of Laminin-511 in a breast tissue implant, or regarding selection of reconstructive biomaterials that may promote or inhibit production of this extracellular protein. Note, however, that 2D cultures are not amenable to the study of breast ductal structures or questions regarding spatial co-location [114]. Following Fig. 36.3, the test pattern must be fabricated, for example by cell printing, and then cultured. The fabrication system to produce a desired test system and then the culture system, e.g., a bioreactor, to produce the environmental conditions, such as temperature or pH, present significant instrumentation challenges. Test system accuracy describes the overall degree to which the biological question of interest can be studied in the in vitro culture. An established tool in breast cancer studies that is also applicable to breast reconstruction research is the breast tissue organoid, which is formed by digesting harvested tissues into duct-like structures that resemble the original organ in appearance or function. While it is obvious that an organoid model formed by removing tissue and allowing the remaining tissue to grow in a new manner is not a physical copy of the breast tissue, it has been shown that this simplified model can produce useful results. For example, in the work by Cellurale and coworkers [116] the authors were able to isolate the role of cJun NH2-terminal kinase signaling in mammary gland development and tumorigenesis using mammary organoid cultures. In other studies [117], organoids were used as the basic building block of human mammary epithelial cell cultures. The organoid model uses an elemental component that already has significant structure and is directly derived from the biological source. There are more general approaches to building test systems that use biofabrication techniques to assemble smaller elemental pieces. Fig. 36.4 shows additional considerations in biofabricating a test system

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FIGURE 36.4 Sources of error in biofabrication of tissue test systems include the quantization error that arises from resolution of the fabrication instrumentation, the fabrication errors that arise from the precision and repeatability of the fabrication instrumentation, and the culture errors that arise from the ability of the culture controller to reproduce environmental conditions.

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

model. The first source of potential error is the discretization of a proposed test pattern based on the resolution of the fabrication system. The two-dimensional pixel error is illustrated in the second column of Fig. 36.4 where it can be seen that the biofabrication system with the coarser resolution produces a lower-fidelity replica of the test pattern. The 3D equivalent of pixel error is the voxel error which includes the pixel error as well as a depth component. For example, if a test pattern is defined at the cellular level using micron resolution, such as a scan of a histology slide, then a biofabrication technique that has a smallest fabrication component on the order of a millimeter can only produce an approximate copy of the desired pattern. Thus the approximation of continuous biology with discrete fabrication components leads to quantization error. The second fabrication error type is in the ability of the biofabrication technique to result in placement of elemental components at a desired location. As illustrated in the third column of Fig. 36.4, high precision means that the system can closely replicate the discretized test pattern. The ability of a process to produce identical replicates of a test pattern is then defined as the pattern repeatability. This is defined as different from the machine precision because occurrences such as cell settling in storage reservoirs can cause evolution of the biofabrication instrumentation precision over time. Note that there is much debate about how much quantization error and fabrication error can be tolerated in building a useful tissue structure. One consideration is that tissue structures will self-assemble from an appropriate starting condition [118]. Specifically, placing cells ’close enough’ will allow them to move and assemble based on their natural behaviors. Any approach to tissue fabrication will require that nature take over for the final stage of tissue assembly, the idea of ’close enough’ is actively debated. There are two main technologies used to place the cellular components to fabricate a tissue system (see Fig. 36.5a). In the first approach, known as drop-on-demand deposition, a fixed quantity of cells and medium are deposited as a single drop. A representative method of forming these droplets is inkjet printing [119]. Inkjet printing uses either a thermal or mechanical process to eject a fixed droplet size (on the order of 100pL). This same process is used in many home color printers. A biofabrication system based on thermal inkjet printing is shown in Fig. 36.5b. A second technology is the deposition of a small block or pellet of cells through extrusion or separation from a feedstock. Speed, cost, damage to cells, and resolution are the differences that distinguish these technologies. The ability of the tissue culture system to reproduce environmental conditions can greatly affect overall results, the culture error shown in the last stage of Fig. 36.4. An important part of specifying the tissue test system is to define the environmental conditions, e.g., dissolved oxygen level, that will be used to incubate the tissue. The capability of the culture system to produce these conditions must be considered; limitations occur in the ability of the instrumentation to measure a quantity of interest and the ability of the control system to modulate that quantity. As with all aspects of the tissue test system, the environment is a simplified model of the actual environment and as such must be designed at the start of a project to address a specific question of interest.

FIGURE 36.5 (a) Two general approaches to cell deposition, printing of cells in medium and extrusion of small cell pellets. (b) Photo of a biofabrication system with multiple deposition stations for cells and biomaterials.

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As a final consideration, the state or feature of interest must be measured in the tissue or medium during and/or after culture. A tissue test system can present the same imaging and measurement challenges as in vivo experiments e real-time measurements like oxygen levels, discrete measurements like magnetic resonance imaging, and endpoint analysis like classical histology may all be used but have the same fundamental instrumentation limitations. However, there are several important advantages of a tissue test system over an in vivo model. First, there is the possibility that sensors can be embedded directly into the tissue design. For example, it is possible to embed a temperature sensor in the center of a tumor mass as it is fabricated. Second, the tissue is potentially more amenable to direct observation than in vivo, e.g., a tumor model could be grown on a microscope stage and images automatically taken at regular intervals to observe spreading or growth. There is generally improved access to both inputs and outputs of the culture in a test system, agents can be applied without metabolism or complex pharmokinetics distorting the input. As an example, it is possible to apply an anti-angiogenic agent to a cell culture and know the local concentration without measurement because the pharmokinetics can be easily modeled as a mixing problem in a fixed volume. Similar arguments suggest that observation of outputs, such as metabolic byproducts, are more accessible in a test system. Perhaps the most important advantage that the tissue test system can have over an in vivo test is that, given low fabrication errors and culture errors, each copy of the test tissue should be nearly identical. This means that replicates can increase the power of observations and increase the ability of outside groups to repeat experiments and results. In general, a tissue test system should make the biology of interest more accessible for observation and manipulation.

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In summary, tissue test systems are an evolving approach to modeling complex biological systems in vitro. Ethical, economic and scientific drivers will ensure that this technology continues to evolve. However, the use and expectations of the test system must recognize that the tissue and culture environment is an approximate model that must be designed to address a specific question of interest. As researchers and clinicians begin to appreciate all of the subtleties of designing, fabricating, culturing, and assessing tissue test systems, these approximate models will help reveal the complexities of breast biology and disease processes and therefore will provide the insight to engineering breast tissue. As tissue test systems, the basic units of useful biology, become standardized they can be integrated to produce systems capable of answering ever more complex questions.

In silico breast cancer models Mathematical modeling of breast cancer continues to advance rapidly, so much so that any summary is nearly obsolete at the time of writing. However, a brief overview of some approaches helps demonstrate the potential and challenges of mathematical and computer models. Generally speaking, there are two starting points for deriving a mathematical model, the first is the application of basic chemical and physical equations (first principles) to create a model, and the second is the observation of physical behavior and use of mathematical tools to capture this behavior. A model may be proposed to work at a specific scale, such as at the molecular level, the cellular level, the organ level, the organism-wide level, or the population level. Initially, models were developed to capture the behavior of an isolated system at a fixed scale. As modeling sophistication has increased, multi-scale models [120] of connected subsystems have evolved. Often the models are referred to as ’in silico’ to indicate that the simulation is performed on a computer and to highlight their similarity in role to in vivo or in vitro models. As with other modeling approaches, the best model is the one that allows the user to address the question at hand. As an example of a simple model, tumor angiogenesis is modeled as a set of two interconnected differential equations, one subsystem models tumor growth and one subsystem models the carrying capacity of the vascular network [121]. This model appears to be a gross

CHAPTER 36 Reconstruction Implants and Three-Dimensional Tissue Test Systems

summary (as an ordinary differential equation) of the physical diffusion processes and the effects of the biological signals that stimulate or inhibit angiogenesis. However, analysis of the model has provided new insight into the scheduling of anti-angiogenic treatments. The traditional dosing of such a therapy centers on applying a constant dose at regular time intervals; however, the model provides the structure to apply optimization and control techniques that suggest a more efficient use of the treatment agent [121,122]. There will always be questions about the resolution and accuracy of any mathematical model. For example, the angiogenesis model may not be sufficient to model clinical cancer treatment. By connecting a second subsystem model, the movement of bone-marrow-derived endothelial progenitor cells to the tumor site and their effect on tumor growth can be used as a starting point to model vasculogenesis [123]. Such a model can clarify further the possible treatment strategies, including chemotherapy and anti-angiogenic therapies aimed at suppressing vascularization, which may be incorporated in a tissue-engineered implant.

CONCLUDING REMARKS Tissue engineering may provide a means of both assessing and building reconstructive breast implants for a woman undergoing lumpectomy or mastectomy. The development of vascularization and the long-term retention of tissue volume are keys to viable breast reconstruction options. Benchtop tissue test systems will be critically important in addressing these two major issues, by facilitating markedly improved understanding of the normal and diseased states and, accordingly, assessing and developing advanced reconstructive options.

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