Why regenerative medicine needs an extracellular matrix

Abstract

Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term ‘stem cell therapy’ is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call “design for optimal functionality.” Third, we describe an alternative approach to sECM design and implementation, called “design for simplicity,” in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

Keywords:

• cell delivery
• cell differentiation
• cell retention
• cell therapy
• clinical biomaterial
• extracellular matrix
• regulatory barriers
• stem-cell transplantation
• tissue engineering
• translational medicine

1. Cells need context and support

Natural extracellular matrices (ECMs) perform the tasks necessary for tissue formation, maintenance, regulation and function, providing a powerful means of controlling the biological performance of regenerative materials. For transplantation of stem or progenitor cells to succeed in the clinic, we opine that scalable production of clinically viable synthetic ECMs (sECMs), rather than natural ECMs, are critical to deliver and retain the therapeutic cells at the site in need of repair [1,2]. Moreover, the sECMs should allow nutrient input and waste outflow, vascularization and controlled differentiation in the context of the biological tissue. In the context of translating biomaterials to the clinic, it is our opinion that biomaterials scientists who are concerned with producing a clinical grade product not only recapitulate the biological, mechanical and chemical properties of the native ECM environment (Table 1), but create a material that meets practical design criteria for a clinical product (Table 2) [1,3]. These innocent-looking tables represent a thoughtful and concise statement of the difference in ‘research’ design thinking and ‘product’ design thinking that we maintain is extremely important as a reminder and a ‘reality check’ between the academia and industry. That is, we believe that researchers and corporate scientists alike must incorporate the importance of the financial, production, regulatory, intellectual property, marketing and reimbursement constraints at the beginning of the technology development. Moreover, we maintain that all scientists should regularly re-evaluate the clinical potential for the technology product development in light of the conundrum implicit in developing cell-device combination products [4,5]. Importantly, we must all recognize that as products approach the clinic, decisions based on the needs of the end-users become increasingly important [3]. In this editorial, we identify two complementary approaches for creating sECMs for clinical stem-cell transplantation: design for optimal functionality and design for simplicity. Whether minimal or instructive, we maintain that an sECM is the most essential contributor for improving the outcomes of cell therapy.

Table 1. Technical design criteria for biomaterials.

Create temporary sECM with correct mechanics and chemistry

Easy delivery of cells and molecules to target site

Retain cells at site of repair

Stimulate cells to control and direct differentiation

Enable cells to recreate tissue-appropriate ECM

Facilitate tissue regeneration

Achieve clinically meaningful outcome

ECM: Extracellular matrix; sECM: Synthetic extracellular matrix.

Table 2. Real-world design criteria for biomaterials.

Meet needs of end users: patients and physicians

Simple, safe, effective composition

Easy to use by physicians

Affordable, scalable cGMP manufacturing process

Robust IP portfolio

Viable business model

Clear regulatory path

Reimbursable by health-care payers

2. Design for optimal functionality

Precise spatial and temporal coordination of physical and chemical cues is required throughout biology. The microenvironments surrounding cells provide the signals needed to coordinate cellular activities in tissue development, wound healing and remodeling. Bioinspired materials for stem-cell transplantation attempt to recapitulate the natural processes of tissue dynamics and morphogenesis by providing guidance through cell-instructive and physically responsive matrices. Designs can be inspired by the natural extrinsic cell response to the surrounding niche, including interactions with other cells, the surrounding matrix and receptor–ligand interactions (Figure 1A). Highly regulated signals in the stem-cell microenvironment, such as adhesion ligand density, matrix mechanics and growth factor presentation and concentration have been implicated in modulating stem-cell proliferation and maturation [6-8]. Maintaining independent control over presentation of biochemical and mechanical cues presented to cells allows assessment of relative and combined effects on stem-cell function and survival. Accordingly, sECMs can be created in a modular fashion with solid phase adhesion ligand and growth factor presentation [9], protease labile crosslinkers (e.g., MMP) [10,11] and material moduli spanning physiologically relevant ranges (100 – 10,000 Pa) that can be tuned for either a specific stem-cell type or application [12-14]. Synthetic ECMs with these design parameters present ligands that direct intracellular signaling and gene expression, create an architecture that resembles their native environment, and have controlled mechanical properties that enable adhesion and the development of contractility in the cellular cytoskeleton [15].

Figure 1. A. In the natural ECM (left image), cells engage in a niche full of bidirectional feedback between the cell and its matrix that results in reciprocal dynamics. Binding events include integrin receptors, growth factor receptors and cell–cell adhesions that induce intracellular signaling that crosstalk through common transduction molecules. These signals are also integrated through transcription factor and gene expression networks to alter protein translation. Modern instructive approaches to sECM design (right image) focus on recapitulation of the native ECM attributes such as cell adhesion binding domains, growth factor presentation and material physical properties such as elasticity and viscoelasticity. B. In designing for simplicity, the ECM is deconstructed as a crosslinked hydrogel containing the minimal components required for cell attachment and survival. Key: HA, hyaluronan, blue circles; Gtn, gelatin, green squares; black lines, chemical crosslinks. Cells, growth factors and the deconstructed sECM are gelled together in situ and the cells and surrounding tissues remodel the sECM into a tissue-appropriate ECM [1,23].

Synthetic ECMs can be in the form of porous solids, hydrogels or nanofibrous materials (e.g., electrospinning). While the interconnected nature of solid porous scaffolds promotes mass transport, facilitates cell seeding and migration, and improves integration with existing tissue, they appear as two-dimensional (2D) surfaces at the scale of cells. Hydrogels and nanofibers best recapitulate the structure of the natural ECM, and provide cells with a true 3D environment with which to interact. An advantage of hydrogels is that they can be designed for in situ crosslinking so that they allow immediate cell infiltration to create a true 3D cellular structure. In contrast, electrospun matrices with nanoscale pores cannot be formed in situ and preclude immediate cell movement into the spun matrix [16]. Indeed, hydrogels form a highly flexible and diverse class of materials, and many hydrogels meet the requirements to become optimized sECMs.

Accordingly, synthetic or semisynthetic hydrogels for in situ formation provide the opportunity for enormous chemical diversity and range of biophysical properties due to the extensive libraries of monomers, macromers and crosslinkers available to construct sECMs [15,17]. This diversity has allowed strategies for cell encapsulation and transplantation and subsequent in situ formation via redox, photo initiation and Michael-type addition reactions. Synthetic polymers for sECM types include polyacrylamides, polyacrylates, polyethers, polyesters, polyhydroxy acids, polyfumarates, polyphosphazenes and others. Modified natural polymers that use elements of the native ECM (e.g., hyaluronic acid [16], collagen, fibrin) or structural components of other organisms such as cellulose from plants, agarose and alginate from algae, and chitosan derived from crustacean shells can also be used to form the basis of an sECM. Depending on the polymer, degradation and subsequent remodeling of the sECM can occur by nonenzymatic or enzymatic hydrolysis.

Ligands modulate stem-cell phenotype based on identity, density and presentation to a stem cell. To improve cell adhesion and interaction with sECMs, peptides that engage with the cell-surface integrins are identified from the literature [18] or by screening with either phage or bacterial libraries [19]. ‘Presentation’ includes effects from ligand mobility, conformation and orientation; spacer arm length and chemistry can be adjusted to optimize ligand availability and activity. Additionally, robust influences on stem-cell fate and survival can be achieved by sequestering exogenous growth factors within the matrix at the time of synthesis [20]. Novel and improved methods of growth factor presentation have been recently developed [21].

We believe that the level of complexity in the design of the sECM should reflect the desired goals and application. Although stem cells respond with exquisite sensitivity to cell-extrinsic biological and physical stimuli, it is critical not to overdesign the system. The main design parameters discussed (ligand and growth factor presentation, enzymatic degradation, material mechanics and matrix architecture) coordinate the interplay between intrinsic and extrinsic determinants of stem-cell fate that promote successful tissue regeneration.

3. Design for simplicity

In response to the potential for overdesign, some teams have adopted a minimalist approach, in which the complexity of the ECM is deconstructed into the minimum essential components. As shown in Figure 1B, an sECM can have as few as one or two biomolecules, crosslinked into a hydrogel with a bifunctional crosslinker [1,22,23]. The simplest version is based on a chemically modified hyaluronan (HA), available with a wide variety of ‘living’ chemistries that permit crosslinking in vivo in the presence of cells, growth factors or other small molecules, and tissues [2,16]. The HA-only hydrogels have been most successful for self-renewal of stem cells [16], for scar-free wound repair and for prevention of post-surgical adhesions [24]. As one example, the thiol-modified HA can be combined with thiol-modified gelatin and crosslinked with polyethylene glycol diacrylate to give a hydrogel. This research-grade composition, known as HyStem, has been extensively used for cell and growth factor delivery [2]. The clinical version, Renevia, is in human clinical trials for delivery of autologous lipoaspirate-derived cells [3]. The deconstructed minimal sECMs are remodeled enzymatically in situ, and cells secrete tissue-appropriate ECMs during tissue regeneration and vascularization.

During these natural developmental and healing processes, the loose macromolecular networks of both the simple sECMs and the optimally functionalized sECMs serve as provisional matrices that are replaced by functional tissue. Matrix components are produced and deposited by resident cells, depending on their phenotype and surroundings. Thus, biomimetic materials only need to set in motion the natural process of tissue regeneration, as the ECM, signals and cellular connections produced by cells within the construct will stimulate and guide later stages of tissue growth. The sECM is the essential cornerstone of effective cell therapy and regenerative medicine, providing a simple and effective means to deliver, retain and support cells, and concomitantly to support cell growth, proliferation and tissue remodeling.

4. The imperative for translational medicine

In our opinion, the imperative for translational medicine can be summarized in just six words [1]. As biomaterial scientists and clinicians, we should embrace complexity – biology is complex and offers chemical and engineering challenges, and many different patient needs. However, no one material composition can fulfill every need and we cannot allow complexity to dominate the design of the clinical material. To move toward the clinic, we must engineer flexibility into products, so that approvable devices can be developed that serve most clinical needs. Finally, the business marketing of a clinical product must deliver simplicity. These six words move us as scientists from technology push to market pull, by recognizing that beyond safety and efficacy, the true factors limiting translation to the clinic are cost, familiarity and ease of use. In our opinion, successful translation from bench to business to bedside cannot occur without incorporating design thinking from the very beginning into any research program that aspires to reach human patients. Moreover, successful translation in regenerative medicine requires continuous interactions between the academic, government and industry teams, as well as within the multidisciplinary academic team of that includes (but is not limited to) engineers, physicians and cell biologists. Taken together, in the context of this editorial, we believe that a scalable and FDA-approved sECM, whether minimal or instructive, will improve the outcomes of cell therapy and regenerative medicine.

Declaration of interest

GD Prestwich is a shareholder of BioTime, Inc., which is developing sECMs for clinical use. KE Healy is a founder of Valitor Inc. and CardioRegenX Inc. developing molecular therapuetics and sECMs for clinical use respectively. The authors have no additional relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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