Engineering Biomaterials for Regenerative Medicine

Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in basic research as well as in clinical transplantation of HSPCs. Currently, fluorescent-activated cell sorting (FACS) and immunomagnetic separation techniques are widely used for HSPC isolation. Although both methods offer relatively pure subpopulations, FACS can introduce potential sources of contamination and flow cytometers with high-speed sorting ability are prohibitively expensive for smaller laboratories. Immunomagnetic separation requires large amounts of starting material, involves several steps, and furthermore, cell-surface antibodies may prohibit cell proliferation and differentiation. Hence, development of simpler methods for the capture and purification of HSPCs are warranted. By exploiting the differential rolling behavior of CD34+ HSPCs from mature blood cells on selectins, we have developed a flow-based, adhesion molecule-mediated capture method, which may be a viable alternative approach to the isolation of HSPCs.

The extracellular environment is an essential mediator of cell health and provides both chemical and mechanical stimuli to influence single and collective cell behaviors. While historically there has been significant emphasis placed on chemical regulators within the extracellular matrix, the role of the mechanical environment is less well known. Here, we review the role of matrix mechanics on cell function and tissue integrity. Cellular responses to mechanical signals include differentiation, migration, proliferation, and alterations in cell–cell and cell–matrix adhesion. Interestingly, the mechanical properties of tissues are altered in many disease states, leading to cellular dysfunction and further disease progression. Successful regenerative medicine strategies must consider the native mechanical environment so that they are able to elicit a favorable cellular response and integrate into the native tissue structure.

Adequate oxygen transport is vital to the success of a tissue-engineered construct. Modulating oxygen tension within tissue-engineered constructs is necessary for the creation of devices with optimal functionality. Oxygen tension significantly influences cellular behavior through mechanisms which promote both cell proliferation and apoptosis. Given the negative consequences of low oxygen tension for most grafted tissues, many investigators have worked to improve oxygen tension within tissue engineered scaffolds through the use of synthetic oxygen carriers, natural or artificial heme, and polymeric oxygen generating thin films, or by inducing blood vessel growth into the matrix. Cellular oxygen consumption and transport within a scaffold can be calculated and predicted using diffusion models to improve device design. This section explores the interplay between fundamental engineering and biological processes used to modulate oxygen tension for the creation of functional tissue-engineered devices.

Adhesion is defined as the strength of an interface. The study of adhesion involves understanding the development of interfacial properties when two materials come into contact. Thus, it is a field that has relevance in nearly every aspect of our lives ranging from biological adhesion to microelectronics. In most instances, it is the control of adhesion that is important to the specific application. Additionally, adhesion is important to a diverse set of materials that ranges from soft tissues to hard inorganic materials. Therefore, it is important to discuss the general concepts and key materials properties that are involved in the adhesion and separation of an interface. As the scope of this book addresses properties of biomaterials, this chapter will focus on the adhesion of soft materials. To aid in this understanding, the theories of Hertz and JKR will be discussed as these theories address the coupling between contact geometry of the interfacial and materials properties. We will also discuss examples of the applications of these theories for characterization adhesion of different soft material applications. With the recent interests in biomimetic adhesion, i.e. gecko adhesion, we will also use these theories as foundation for understanding how patterned surfaces can tune interfacial properties and impact macroscopic performance.

Poly(lactide) – block – poly(ethylene oxide) – block – poly(lactide) [PLA–PEO–PLA] triblock copolymers are known to form physical hydrogels in water, due to the polymer’s amphiphilic architecture. Their biodegradability has made them attractive for use as soft tissue scaffolds and other biological applications such as drug delivery. In many cases, their mechanical properties have been poorly investigated and “rules” for tuning their stiffness are missing. Often the network junction points are only physical cross-links, not covalent, and in a highly aqueous environment, these hydrogels will absorb more water, transform from gel to sol, and lose the designed mechanical properties. New insights into the self-assembly of these materials have resulted in new PLA–PEO–PLA gels with novel structural and mechanical properties. Here, we summarize our recent efforts to understand these novel hydrogels and control their mechanical properties. This is highlighted by a new approach in which the system is allowed to self-assemble and then it is covalently cross-linked to capture this self-assembled architecture. This produces hydrogels with permanent cross-links and allows their mechanical properties to be studied in much more detail.

The implantation of a biomaterial-based device or drug carrier will elicit a localized or systemic inflammatory response. An immune response occurs regardless of the method of introduction of biomaterial into the body, as all methods of biomaterial insertion, including surgery and injection, result in the disruption of the host’s tissue. The extent of the inflammatory response, however, is dependent upon the location, implantation procedure, and compatibility of biomaterial. In general, inflammatory responses can be classified as acute or chronic, defined primarily by the duration of the response and the type of cells infiltrating a tissue in response to pro-inflammatory signals. This chapter discusses the general process of acute and chronic inflammatory response and gives details on the key immune cells and soluble blood plasma factors involved. Information is also provided on biomaterial design approaches employed to minimizing inflammatory response.

Oxidative stress originally gained attention as key pathological process in a variety of disease states and conditions (e.g., acute lung injury, sepsis, chronic degenerative neurological diseases). Furthermore, it oxidative stress has also been identified as one of the key mechanisms to tissue toxicity brought on by nanomaterials and implant biomaterials. Yet, despite these origins, newer research has started to view oxidative stress and not simply pathology, but as a physiologically relevant signaling system, working in concert with the more traditional cell signaling cascades (e.g., growth factor signaling, cytokine release). As a result, a reinvigoration of research in regenerative medicine has begun looking at oxidative stress as a potential tuning mechanism to enhance the natural wound healing process. In this chapter, a summary of the biological aspects of oxidative stress is presented as well as a current state of the art approaches used in designing biomaterials to actively participate in the oxidative stress signaling.

The next generation of surgical adhesives that are intended for internal applications will present considerable functional and regulatory challenges compared with topical adhesives that are currently available. These new adhesives must strongly adhere to wet surfaces and remain in place until the wound is leak-proof, while not being easily monitored. As adjuncts to current technologies such as sutures, staples, and tissue glue, there is a growing interest to reduce the incidence of trauma associated with surgical incisions and suture lines. Novel approaches are under development that can facilitate wound closure without causing excessive inflammation, ischemia, or necrosis to the wound. Degradable tissue adhesive tapes have recently been suggested as a potential method for wound closure/healing. One strategy to implement this technology utilizes biomimicry to achieve high levels of adhesion by replicating the nanotopography of the gecko’s footpad in biocompatible and biodegradable elastomers. Geckos can walk up vertical surfaces because the bottoms of their feet are covered with hierarchical fibrillar arrays which can maximize the interfacial adhesion to surfaces. Tissue adhesion of structures that mimic the gecko footpad can be further enhanced by applying a thin coating of a tissue-reactive biocompatible “glue” to the nanotopographically patterned surface. Using this two-component design, a tape-based adhesive can adapt to physiologic mechanical forces, while remaining strongly attached to the underlying tissue and eliciting minimal inflammatory response, eventually resorbing harmlessly without the damage caused by removal associated with other adhesives. These strongly adherent and minimally inflammatory biodegradable taped-based adhesives will provide a platform for many practical additions to the surgical armamentarium.

Over the past decade, the tissue engineering field has placed increasing emphasis on the development of materials that mimic the natural cellular microenvironment and the complexity inherent to biological systems. Understanding cell and molecular biology has become as important to the development of tissue engineering scaffolds as traditional material science. This emphasis on biological mimicry has led to interest in materials that incorporate the dynamic and complex interactions found in the native extracellular matrix (ECM); protein-saccharide interactions in the ECM are one such example. In particular, over the past decade the glycosaminoglycan (GAG) heparin has found a tremendous amount of use in the design of tissue engineering scaffolds and drug delivery devices, as well as in more traditional applications. Both heparin and its biological counterpart, heparan sulfate, interact – mainly electrostatically – with a number of proteins and play an important role in many biological processes. This has been put to use in tissue engineering approaches to sequester and deliver the factors that drive regenerative processes. Additionally, heparin has been utilized in the development of novel responsive materials that form hydrogels through physical assembly. The versatility provided by these approaches has afforded opportunities not only in the design of scaffolds for tissue engineering but also in the development of drug delivery devices.

Vocal fold is one of the most mechanically active tissues in the human body, producing a great variety of sounds through a regular, wave-like motion of the lamina propria (LP) at frequencies of 100-1,000 Hz and strains up to 30% [1]. Each vocal fold consists of a pliable vibratory layer of connective tissue, known as the lamina propria (LP), sandwiched between epithelium and muscle [2, 3]. The lamina propria plays a critical role in the production of voice as its shape and tension determine the vibratory characteristics of the vocal folds. Numerous environmental factors and pathological conditions can damage the delicate tissue; and patients suffering from vocal fold disorders are socially isolated due to their inability to phonate. This chapter summarizes recent endeavors in vocal fold regeneration using biomaterial-based, tissue engineering methodologies. Successful repair and regeneration of vocal fold lamina propria relies on the attainment of vocal fold-specific, biomimetic matrices that not only foster the attachment and proliferation of vocal fold fibroblast-like cells, but also induce the production of an extracellular matrix (ECM) that approximates the native tissue in terms of the biochemical composition, structural organization, and mechanical characteristics. The performance of the artificial matrices can be further enhanced by incorporating morphogenic factors and physiologically relevant biomechanical stimulations. Strategic combination of polymeric scaffolds, multipotent cells, defined biological factors, and biomechanical signals will lead to the successful reconstruction of functional vocal fold tissues that can be used as an alternative treatment modality for patients suffering from severe vocal fold disorders.

To promote tissue repair and regeneration, much research in the field of tissue engineering has been aimed at the development of synthetic three-dimensional scaffolds to maintain the space and provide the mechanical support necessary for tissue development. However, to regenerate functional tissue of the same quality as natural tissue, the release of biochemical cues from these synthetic matrices will be necessary. While both bolus injection as well as polymeric encapsulation of proteins has been shown to stimulate regenerative processes, proteins have a fragile three-dimensional structure, which can be costly and difficult to synthesize. Because of the increased stability of DNA in comparison with proteins, plasmids may be used to stimulate gene transfer and localized expression of plasmid-encoded proteins to promote tissue development. However, due to the multiple barriers to gene transfer, a gene delivery vehicle must be carefully designed to impart control over the spatial and temporal release of the DNA. Furthermore, as the cellular processes involved in directing tissue repair are complex, delivery must be well controlled and stimulate gene expression that mimics the natural release processes of target growth factors and other proteins. Thus, this chapter will discuss the delivery control imparted by various mechanisms of gene transfer including bolus, polymeric, and substrate-mediated delivery. Matrix-controlled delivery methods for regenerative medicine applications will be explored in depth. Ultimately, to reform tissue of the necessary quality and functionality, the appropriate spatial and temporal patterning of gene expression profiles within targeted cells will need to be attained by synthetic systems.

A variety of polymeric devices have been widely studied as a means to deliver drugs at an appropriate dosage, delivery sequence, and time period to improve and optimize the treatment. In the conventional therapy of drug administration, a major limitation is the initial burst release of drugs, inducing a rapid loss of therapeutic efficacy and increasing the risk of harmful side effects to patients. As an alternative approach, single or multiple therapeutic agents can be incorporated into an appropriate material to well regulate its residence time and dosages. In this regard, numerous synthetic and natural polymers have been employed as drug carriers to control drug release for a desired administration. Recently, chitosan, a cationic natural polymer, has gained considerably attention due to its potential broad application in tissue regeneration, chemotherapy, and wound healing. In this chapter, chitosan is introduced as a drug carrier in a variety of forms. This chapter discusses several properties of chitosan such as biocompatibility, biodegradability, and functionality. It will also discuss techniques for preparation of chitosan-based delivery systems, and strategies of controlled drug release for potential biomedical and pharmaceutical applications, including tissue regeneration, chemotherapy, and wound healing.

This volume has highlighted major advances in the design of biomaterials for regenerative medicine. Increasingly, tissue engineering is being recognized as a beneficial strategy for alleviating the global burden of disease. While significant progress has been made in the field of tissue regeneration, a number of scientific and engineering issues must be addressed before tissue-engineered scaffolds can be translated into clinical usage.