In This Issue
Fall Issue of The Bridge on the Convergence of Engineering and the Life Sciences
October 1, 2013 Volume 43 Issue 3

Regenerative Engineering Materials, Mimicry, and Manipulations to Promote Cell and Tissue Growth

Wednesday, September 25, 2013

Author: Cato T. Laurencin, George Q. Daley, and Roshan James

Since time immemorial literature and art have depicted the desire to recreate or regenerate human life or to transplant parts from one individual to another. An early example is the Fra Angelico painting Healing of Justinian, which showed the transplantation in the third century AD of an allograft limb onto an injured soldier.

More recently, elements of this vision have been captured and defined as tissue engineering, the use of isolated cells or cell substitutes, tissue-inducing substances, and cells placed on or in matrices (Langer and Vacanti 1993). Tissue engineering has rapidly progressed in the past 25–30 years to revolutionize treatment strategies and define new approaches in tissue repair. This new frontier, enabling the reconstruction of complex tissues and whole organs, requires the combination of top-down engineering approaches with bottom-up strategies that integrate materials science and tissue engineering with stem cell and developmental biology (Laurencin and Khan 2012).

Regenerative engineering takes tissue engineering a step further, integrating it with advanced materials science, stem cell science, and areas of developmental biology. Whereas tissue engineering involves interdisciplinary teams from the fields of engineering, science, and medicine, we see regenerative engineering as an expansion of this approach—a “convergence” (Sharp and Langer 2011) of tissue engineering with nanotechnology, stem cells, and developmental biology (Reichert et al. 2011). Our hope is that this new interdisciplinary synthesis will usher in transformative opportunities for advancing human health.

Materials of Tissue Engineering
The Scaffold

Virtually all cells in the human body (apart from circulating blood cells) are anchorage-dependent and reside in a scaffold-like structure called the extracellular matrix (ECM). There exist numerous types of ECM in human tissues, each with several unique components, but they all provide (1) structural support and a physical environment for cells to reside (attach, migrate, and grow) and (2) structural integrity to the tissue and mechanical properties associated with tissue function (e.g., elasticity in tendon tissues, high rigidity for bone). The ECM sequesters bioactive molecules and sends cues to the cells to modulate cellular functions. It is a highly dynamic microenvironment that undergoes active remodeling by cells in response to developmental, physiological, and pathological challenges during morphogenesis, homeostasis, and wound healing (James et al. 2011b).

Figure 1

The best architecture for guided tissue regeneration is a structure or scaffold that mimics the ECM of the target tissue in its native state (Figure 1). The complexity and dynamic nature of the native ECM are difficult to duplicate, requiring the development of new materials and structures that copy the ECM in bioactivity and mechanical properties.

To be effective, an engineered scaffold for tissue regeneration should encompass features such as biocompatibility, porosity, mechanical functionality, bioactivity, and degradability. Biocompatibility—the ability of a biomaterial to perform its desired function without eliciting undesirable local or systemic effects in the patient—is critical to the success of every implantable medical device. Porosity is the percentage of void space in a solid object; macroporosity (pore size >50 µm) in bone grafts supports the growth of new bone and vasculature, enabling the transport of oxygen and nutrients throughout the depth of the scaffold. For load-bearing applications, an engineered scaffold must be mechanically competent to tolerate and transfer local forces; this is most critical for applications involving tissues such as tendon, bone, and ligament. Bioactivity refers to an inherent property of a material or molecule to direct desired biological activities; for example, hydroxyapatite, a material of chemical composition similar to native bone, provides a skeletal framework that promotes the formation of new bone tissue and is thus identified as being osteoinductive. Degradation of biomaterials is modulated strongly by the local environment, and it is important to ensure that degradation byproducts will not elicit local and systemic immune response, which would significantly compromise the success of implant-host integration.

Stem Cells

A readily accessible and robust source of cells is essential for tissue engineering, and increasingly stem cells represent just such a source.

Based on their differentiation potential, stem cells are categorized as pluripotent or multipotent. Pluripotent stem cells—embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)—are characterized by continuous self-renewal in culture and the capacity to differentiate into any of the three germ layers: endoderm, ectoderm, and mesoderm. Thus they can theoretically give rise to any and all cells of the developing embryo and are a plentiful source of tissues that might not actively regenerate from stem cells in the adult.

Multipotent stem cells, isolated from adult tissues, are comparatively rare and restricted in their differentiation potential to the tissues in which they reside. They may be hematopoietic stem cells (HSCs), limbal/corneal stem cells, bone marrow–derived mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs), skin or epidermal stem cells, or neural stem cells (NSCs). HSCs have been used in bone marrow transplantation for decades and represent the most therapeutically advanced of the multipotent stem cells. Limbal stem cells have proven effective at engendering corneal grafts to cure ocular injuries and blindness (Rama et al. 2010). However, virtually all other uses of adult multipotent stem cells should be considered experimental. Bone marrow–derived MSCs, for example, can be coaxed to become bone, cartilage, and fat in vitro and have undergone extensive clinical trials for orthopedic applications, but have yet to win regulatory approval for such indications. They are in clinical trials for cardiac applications and autoimmune disease, but it is not clear that these clinical settings reflect the native functions of MSCs.

Among the most compelling recent innovations is the synthesis of tissue engineering with new techniques for culturing stem cells and adhering them to an engineered organ, as when autologous MSC-derived chondrocytes and epidermal cells were seeded onto a decellularized donor trachea to provide an airway for a patient with severe bronchomalacia (Jungebluth et al. 2011; Macchiarini et al. 2008). These surgical solutions to challenging clinical problems require a marriage of anatomic structures, cells, and tissues as well as advanced understanding of the means by which cells reproduce, interact, and differentiate in order to guide them.

Mimicry

A major goal of new therapeutic strategies is to develop advanced biomaterials that closely mimic what the body already does perfectly.

Cells in tissues are situated in microenvironments composed of insoluble macromolecular proteins, soluble stimulatory factors, and neighboring cells. This landscape is highly specialized, dynamic, unique to every tissue, and subject to change with age, disease, and injury. The behavior and function of cells are determined by mechanical, structural, and chemical signals encoded in the chemical composition, localization, duration, and context of the specific microenvironment. Spatial cues range from nanometers to centimeters and are largely depicted by ECM fibers and tissue structures.

Nanofiber Scaffolds

Integration of nanotopographical cues is important in engineering complex tissues that have multiple cell types and require precisely defined cell-cell and cell-matrix interactions in a 3D environment. Nanoscale materials and structures therefore play a paramount role in controlling cell fate and regenerative capacity, and nanofibers have been one of the most widely investigated nanostructures for tissue regeneration (Li et al. 2002).

Nanofiber scaffolds are characterized by ultrathin continuous fibers, high surface-to-volume ratio and porosity, and variable pore size distribution (Figure 2). Their interconnecting porous structure provides a large surface area for cell attachment and sufficient space for nutrient exchange. Cell performance has been found to be significantly enhanced on nanofiber matrices.

Figure 2

Polymeric nanofibers can be fabricated using a simple, reproducible, and scalable technique called the electrospinning process. Scaffolds composed of electrospun nanodiameter fibers recapitulate ECM structural features and, via modifications to the fiber material or surface, biochemical cues as well. This type of artificial scaffold with enhanced biofunctionality constitutes a more biomimetic microenvironment for ex vivo stem cell culture and has been shown to support multiphasic MSC differentiation into chondrogenic, osteogenic, tendinogenic, and adipogenic phenotypes (Deng et al. 2011; James et al. 2011a; Soliman et al. 2010; Xin et al. 2007). The electrospinning process thus enables the combined benefits of micro and nano features for desirable cellular responses and mechanical characteristics.

Biodegradable Polymer Scaffolds

The micro- and nanohierarchical structure of bone tissue—comprising woven and lamellar bone, interstitial networks, and gap junctions—is very difficult to mimic in bone substitutes for modulating repair/regeneration. Porous bone substitutes can partially mimic the canal systems and interconnected networks of native bone, and 3D macroporous scaffolds have been fabricated using a salt leaching technique that achieves porosity similar to trabecular bone. These heat-sintered scaffolds are made of a degradable polyester that represents a negative template of trabecular bone, so that newly forming bone tissue will occupy the pore structure while the microsphere matrix slowly degrades, leaving voids that will form the pores of new trabecular bone. MSCs seeded on such scaffolds manifest bone phenotypes, indicating their osteoinductive potency.

Problems with cell migration into the scaffold, and the lack of diffusion of nutrients into and wastes out of the scaffold system, limit the dimensions and functionality of regenerative implants. Exploiting the chemistry of two biodegradable polymers, a 3D polylactic acid (PLA) nanofiber mesh was successfully incorporated in the void spaces between sintered microspheres and thereby achieved cell migration and ECM production throughout the scaffold (Brown et al. 2010).

Previous scaffold designs incorporated signals from the cellular microenvironment but were restricted to one or two stationary cues. The focus now is on dynamically encoding multiple cues to integrate signaling and direct cell fate in the host microenvironment. This ambitious goal requires control over architecture at multiple size scales, spatiotemporally regulated release of signaling molecules, and dynamic polymer design.

Dynamic Polymer Scaffolds

Developments of new biomaterials and composite scaffold systems have led to dynamic scaffolds in which the substrate recapitulates the stem cell microenvironment by dynamically encoding the localization, duration, and context of numerous cues to integrate the cellular response in the host. The cellular responses in turn remodel the scaffold; for example, stem cell–induced enzymatic changes in the local microenvironment will change the substrate stiffness or induce the release of factors that will accelerate differentiation.

Figure 3

A unique polymer erosion system was designed with a polyphosphazene-polyester blend polymer platform, a simple mixture of two polymers (Deng et al. 2010). Blend polymer matrices evolve from a solid coherent film to an assemblage of microspheres with an interconnected 3D porous structure (Figure 3). Rapid hydrolysis of the polyester component permits the formation of 3D void spaces filled with self-assembled polyphosphazene spheres. Characterization of the spheres reveals macropores between them as well as micro- and nanopores on their surface. The hydrolysis-driven remodeling of the scaffold substrate into self-assembled spherical structures gives an insight into dynamic matrices and how they may be controlled to achieve structures with gradient porosity.

Manipulation

Biomaterials may improve the impact of transplanted stem cell populations through the delivery of (1) cells in a protective gel to enhance viability; (2) diffusible factors to mobilize the endogenous cells involved in repair (e.g., angiogenic factors to form blood vessels); and (3) regulatory proteins to enhance survival, self-renewal, and stimulation of tissue-specific phenotype maturation.

Regulation of Cell Fate: Directed Differentiation

As a fledgling field, stem cell biology has come to embody the ambition to understand both the intrinsic internal clues and microenvironmental influences that specify, maintain, or destabilize a cell’s identity (Cherry and Daley 2012). Among the most vexing challenges in cell biology is how to control cellular behavior for medical applications, in particular how to promote the differentiation of pluripotent cells or specialized adult stem cells without loss of their “stemness” attributes. The challenge is common to pluripotent and multipotent stem cell types: directed differentiation—coaxing cells toward a specific desired terminal cell type—is difficult, inefficient, and fraught with technical and theoretical limitations.

Stem cell self-renewal is a major asset because it enables the large-scale cell production required for clinical applications, but only certain multipotent stem cells can be cultured in vitro for periods adequate to generate a large enough dose of cells. Here, rigorous bioprocess and manufacture optimization are proving crucial (Kirouac and Zandstra 2008). For pluripotent stem cells, which are immortal, the propagation and expansion to clinical scale is less daunting than the challenge of directed differentiation (Ungrin et al. 2012). In most circumstances, pluripotent stem cells must be bathed in media that replicates the cues provided in the embryo by precise temporal and positional stimulatory gradients, and by the ECM and native tissue environment.

Directed differentiation of stem cells requires a deep mechanistic knowledge of development, whereas the routines of cell biology (static culture in two-dimensional petri dishes at ambient oxygen tension) generally do not mimic the biochemical or biomechanical milieu of the developing embryo. Here again, the interface of cellular and developmental biology with biomaterials science and nanotechnology provides novel opportunities to engineer the cell-scaffold interaction and thus a solution to an otherwise intractable problem of cellular alchemy.

A kind of cellular alchemy in fact rules in stem cell biology today. Because in many instances the stepwise application of developmental principles to direct cell fates in vitro has proven inadequate, alternative and creative strategies have emerged. This new wave has been inspired largely by the work of Yamanaka and colleagues, who demonstrated that a small set of transcription factors could reset the epigenome of a differentiated cell back to a pluripotent state (Takahashi and Yamanaka 2006). Within a year of the production of iPSCs in mice, several groups succeeded in reprogramming human somatic cells back to a state equivalent to embryonic stem cells (Park et al. 2008b; Takahashi et al. 2007; Yu et al. 2007), and quickly turned to modeling human disease (Park et al. 2008a). iPS cells represent an accessible supply of immortal embryonic tissue for any given patient and, when differentiated into somatic cells and tissues, can become a source of autologous tissues for regenerative engineering.

Rather than await the painstaking elucidation of methods for directed differentiation in a dish, some intrepid investigators have attempted to harness transcriptional regulators to convert one differentiated adult somatic cell type into another with therapeutic relevance. Melton and colleagues succeeded in introducing transcriptional regulators via adenoviral vectors into exocrine pancreas cells to convert them into insulin-producing beta cells in situ (Zhou et al. 2008), a dramatic demonstration of cellular alchemy for therapeutic regenerative engineering. Wernig and colleagues then applied this alchemy in a dish to convert dermal fibroblast into neurons (Vierbuchen et al. 2010), setting off a modern gold rush of sorts among scientists who have scurried to convert fibroblasts into any number of adult somatic tissues such as cardiomyocytes and hepatocytes (reviewed in Morris and Daley 2013).

It is now up to bioengineers and stem cell scientists to collaborate to configure appropriate biomaterials and microenvironments that can promote cell fate transitions in a controlled manner for regenerative purposes.

Controlling Cell Fate

Two approaches have been proposed using biomaterials to control the fate of stem cells in vivo. In one approach, biomaterials function as carriers to introduce stem cells into damaged, diseased, or aged tissue, and in the other biomaterials are used to augment the endogenous stem cell function. The survival of the transplanted stem cells is extremely poor and their behavior is poorly controlled in the absence of instructive signals at the injury site due to tissue necrosis.

Biomaterial scaffolds decorated with stimulatory molecules, particularly peptide sequences such as fibronectin, bone sialoprotein, and osteopontin, accelerate bone formation and improve osteointegration of implants. In addition, large molecules delivered from polymeric biomaterials could directly or indirectly signal endogenous cells to undergo osteogenic differentiation. Ceramic surfaces and composite scaffold mediate cellular responses including cell differentiation.

To accelerate the evaluation of novel materials, an array-based technology has been developed to study simultaneously several hundred polymer formulations to determine optimal biomaterial-cell interactions for specific applications. Such array-based technologies enable rapid and efficient screening to identify biomaterials and other analogues that can be used as scaffold components to direct stem cell fate and differentiation.

Opportunities and Challenges

In the future it is expected that several cell-matrix interactions will be layered together in a single continuous material to create a dynamic, bidirectional feedback system. For example, as stem cells proliferate in response to a scaffold loaded with stimulatory factors, cellular signaling events may trigger a reorganization of scaffold components or matrix softening. In response to this new microenvironment, the stem cells may differentiate along a specific pathway.

In the context of rapidly developing materials, hierarchical designs, and material morphogenesis, an emerging understanding of the plasticity and pliability of stem and tissue cells presents remarkable new opportunities for regenerative engineering. However, it will be challenging to define the mechanogradients, hierarchical levels, optimal differentiation factors, and gradients that will effectively direct cell fates, ensure tissue integration, and restore tissue functionality. Moving beyond individual tissue repair to complex tissues, organs, and whole biological systems is a long-term ambition.

These challenges can be addressed through the convergence of advanced materials science, stem cell science, and developmental biology. Teams of scientists, engineers, physicists, and clinicians, all with integrated training in the foregoing disciplines, will be necessary in the biomedical laboratory of the future.

Summary

Cells can be a fundamental building block of regenerative strategies, and novel means of manipulating stem cells and their progeny will prove essential to realizing the bold long-term goals of limb and organ regeneration. Strong understanding of cell-matrix interactions and cell-cell signaling events will guide the development of novel biomaterials and scaffold structures. Furthermore, an understanding of the parameters that regulate cell fate will influence the fabrication of scaffold systems that recapitulate the features of stem cell microenvironments.

Breakthroughs will emerge from the convergence of varied entities, such as materials science, biomimetics, and developmental biology. No single entity is enough by itself. The combinatorial approach will enable scaffold composition and hierarchical organization to provide physical support, nurture and sustain pluripotent or multipotent cells, and direct their fates while providing a native microenvironment in which they can regenerate and remodel to restore tissue and organ function. Through interdisciplinary approaches the ultimate ambitions of regenerative engineering will become a dominant paradigm for 21st century medicine.

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About the Author:Cato T. Laurencin (NAE/IOM) is the Albert and Wilda Van Dusen Chair in Academic Medicine and Distinguished Professor of Orthopaedic Surgery and Chemical, Materials and Biomolecular Engineering at the University of Connecticut. George Q. Daley (IOM) is the Samuel E. Lux IV Professor of Hematology/Oncology and director of the Stem Cell Transplantation Program at Children’s Hospital Boston. Roshan James is a postdoctoral fellow at the Institute for Regenerative Engineering and Department of Orthopaedic Surgery at the University of Connecticut Health Center.