Biofabrication

Micro- and Nano-fabrication, Printing, Patterning and Assemblies

Gabor Forgacs, Wei Sun, Gabor Forgacs, Wei Sun

• Publisher: William Andrew
• Year: 2013
• ISBN:
  9781455730049
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

Biofabrication is a practical guide to the novel, inherently cross-disciplinary scientific field that focuses on biomanufacturing processes and a related range of emerging technologies. These processes and technologies ultimately further the development of products that may involve living (cells and/or tissues) and nonliving (bio-supportive proteins, scaffolds) components. The book introduces readers to cell printing, patterning, assembling, 3D scaffold fabrication, cell/tissue-on-chips as a coherent micro-/nano-fabrication toolkit. Real-world examples illustrate how to apply biofabrication techniques in areas such as regenerative medicine, pharmaceuticals and tissue engineering.

In addition to being a vital reference for scientists, engineers and technicians seeking to apply biofabrication techniques, this book also provides an insight into future developments in the field, and potential new applications.

• Discover the multi-disciplinary toolkit provided by biofabrication and apply it to develop new products, techniques and therapies
• Covers a range of important emerging technologies in a coherent manner: cell printing, patterning, assembling, 3D scaffold fabrication, cell/tissue-on-chips...
• Readers develop the ability to apply biofabrication technologies through practical examples

Chapter 1

In Vitro Biofabrication of Tissues and Organs

Koichi Nakayama, Graduate School of Science and Engineering, Saga University, Japan

Contents

Introduction

1.1 Problems with scaffold-based tissue engineering

1.1.1 Immune reactions

1.1.2 Degradation of scaffolds in vivo

1.1.3 Risk of infection

1.1.3.1 Potential risk of disease transmission by scaffolds

1.1.3.2 Biofilms

1.2 “Scaffold-free” tissue engineering

1.2.1 Classification of present scaffold-free systems

1.2.1.1 Cell sheets

1.2.2 In vitro self-produced ECM-rich scaffold-free constructs

1.2.3 The rotating wall vessel bioreactor system

1.3 Aggregation/spheroid-based approaches

1.3.1 Preparation of multicellular spheroids

1.3.2 Molding MCSs

1.3.3 Bio-printing

1.3.4 Alternative approach for MCS assembly technique for biofabrication

Conclusion

References

Introduction

After the sensational images of the mouse growing a human ear were broadcast around the world in the late 1990s, the in vitro fabrication of tissues and the regeneration of internal organs were no longer regarded as science fiction but as possible remedies for the millions suffering from chronic degenerative diseases. Although some mistook it as a genetically engineered mouse expressing a human ear [1], these striking images nonetheless highlighted the medical promise of “tissue engineering” and ignited widespread interest from researchers in many fields, including cell and molecular biology, biomedical engineering, transplant medicine, and organic chemistry.

While there have already been successful clinical reports documenting the treatment of severe burn patients with culture-expanded skin cell sheets since the introduction of this tissue engineering technology in 1981 [2], fabrication of three-dimensional (3D) tissue constructs in vitro remains a challenge.

In the above-mentioned study, Cao et al. prepared a biodegradable polymer scaffold in the shape of a human ear and seeded its surface with bovine chondrocytes. This “tissue engineered ear” was then implanted under the skin of a nude mouse. As nutrients were provided by the in vivo environment, the implanted chondrocytes gradually started producing extracellular matrix (ECM) components such as collagen and glycoproteins. While a cell-free ear-shaped polymer could not have maintained its original shape in vivo due to the hydrolytic degradation of the polymer, the chondrocytes seeded onto the polymer maintained the original scaffold shape for 12 weeks after implantation. Indeed, the geometry was similar to and as complex as the original human ear.

After the study of the mouse with the human ear, many researchers attempted to create tissues or organs in vitro by constructing scaffolds composed of various biocompatible materials, such as animal-derived collagen [3], synthetic polymers [4], artificially synthesized bone substitutes (calcium-phosphate cement) [5], and autologous fibrin glue [6]. These scaffolds were seeded with a large array of somatic cells or stem cells to reconstruct target tissues such as skin [7], bladder [8], articular cartilage [9], liver [10], bone [11], vascular vessels [12], and even a finger [13].

The combination of a scaffold with cells and/or growth factors became the gold standard of tissue engineering [14]. Successful application of scaffold-based tissue engineering depends on three steps: (1) finding a source of precursor or stem cells from the patient, usually through biopsy or isolated from accessible stem cell-rich tissues, (2) seeding these cells in vitro onto scaffold material of the desired shape (with or without growth factors) that promotes cell proliferation, and (3) surgically implanting the scaffold into the target (injured) tissue of the patient.

This tissue engineering method overcomes a number of problems associated with allogeneic organ transplantation: the perpetual shortage of donors, the possibility of rejection, ethical issues such as organ trafficking [15], and the need for prolonged immunosuppression, which may lead to opportunistic infections and increased risk of cancer [16].

Many researchers tried to fabricate organs by combining cells, proteins/genes, and scaffolds. The various biomaterials used to fabricate scaffolds are classified into three types: (1) porous materials composed of biodegradable polymers, such as polylactic acid, polyglycolic acid, hyaluronic acid, and various co-polymers; (2) hydroxyapatite or calcium phosphate–based materials; and (3) soft materials like collagens, fibrin, and various hydrogels and their combinations.

In addition to providing a 3D structure for transplanted cells, scaffolds also dramatically enhance cell viability (e.g., a few exogenous cells were detected after the transplantation of single isolated cells into infarcted myocardium [17,18]). Anchorage-dependent cells cannot survive for long when detached from the surrounding ECM or culture surface. When there is loss of normal cell–cell and cell–ECM interactions, unanchored cells may undergo a specific form of programmed cell death called “anoikis” [19,20]. Thus, seeding anchorage-dependent cells onto scaffolds allows for efficient transplantation, especially if scaffolds are pretreated with growth factors. Indeed, some scaffold-based tissue engineered systems, such as bladder [21], articular cartilage [22], epidermis [23], and peripheral pulmonary arteries [24], have already been translated into the clinical stage.

1.1 Problems with scaffold-based tissue engineering

The ideal biodegradable scaffold polymer should be (1) nontoxic; (2) capable of maintaining mechanical integrity to allow tissue growth, differentiation, and integration; (3) capable of controlled degradation; and (4) nonimmunogenic; also, it should not cause infection or a prion-like disease. Although there are many clinical reports on the successful use of various biomaterials, there is still no “ideal” biomaterial for scaffold construction. Furthermore, concerns such as immunogenicity, long-term safety of scaffold degradation products, and the risk of infection or transmission of disease, either directly or concomitant with biofilm formation, remain to be resolved.

1.1.1 Immune reactions

A serious concern is that scaffolds may induce undesirable immune reactions [25], including inflammation, acute allergic responses, or late-phase responses. Scaffolds might even stimulate an autoimmune response, such as that produced by type II collagen in mice [26–28] used as models for rheumatoid arthritis. Immune responses may also be triggered by scaffold degradation byproducts. Metallosis is a specific form of inflammation induced by tiny metal particles that are shed from the metallic components of medical implants, such as debris from artificial joint prostheses [29]. Accumulation of scaffold degradation byproducts may elicit chronic diseases associated with inflammatory responses.

1.1.2 Degradation of scaffolds in vivo

Classic biodegradable polymers are defined as materials that are gradually digested by environmental bacteria through a process that is distinct from physiological degradation processes like digestion. Biodegradation can lead to toxicity in two ways: either a degradation product is directly toxic or it is metabolized to a toxic product (i.e., by liver enzymes). “Biodegradable” is distinct from “biocompatible.” In most industrialized countries, only certified biomaterials that have passed multiple tests for severe toxicity and safety are permitted for use as medical implants.

Most synthesized biodegradable polymers are broken down by hydrolysis, resulting in the accumulation of acids that may alter the pH of the microenvironment or exert more direct toxicity. Some scaffolds are destroyed by macrophages, inducing an inflammatory reaction.

While bone substitute scaffolds may be replaced gradually by true bone through the activity of osteoclasts and osteoblasts, degradation of most other biomaterial scaffolds will leave a potential space that can impede repair. Biodegradable biomaterials are used extensively for cartilage repair, since articular cartilage (hyaline cartilage) has a low regenerative capacity and is usually replaced by weaker, rougher fibrous cartilage after injury [30]. When the scaffold is degraded and disappears, the space that once occupied it may no longer be filled with chondrocytes due to the cells’ low proliferative capacity. These spaces might eventually form tiny cracks that trigger further deterioration of the smooth cartilage surface.

1.1.3 Risk of infection

There are two potential sources of infection from implanted scaffolds: pathogens transmitted directly from the scaffold or cells and infections emerging from the bacterial biofilm formed around the scaffolds after implantation.

1.1.3.1 Potential risk of disease transmission by scaffolds

Some scaffolds, such as collagen gels and amniotic membranes, are animal-derived. Recent outbreaks of severe infectious diseases like bovine spongiform encephalopathy and severe acute respiratory syndrome highlight the fact that animals harbor pathogens that may be lethal or cause severe infections in humans. Moreover, it is safe to assume that there are many undiscovered animal pathogens with the potential to cause human disease or death. Preclinical studies may minimize this risk, but there is no guarantee that these materials do not harbor unknown human pathogens.

1.1.3.2 Biofilms

Another source of infection from implanted scaffolds is the biofilm that forms on the scaffold surface [31]...