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Journal of Investigative Medicine:
doi: 10.231/JIM.0b013e3181efbc61
Symposium and Meeting Reports

Regenerative Medicine: Basic Concepts, Current Status, and Future Applications

Corona, Benjamin T. PhD; Ward, Catherine L. BS; Harrison, Benjamin S. PhD; Christ, George J. PhD

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From the Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston Salem, NC.

Received May 14, 2010, and in revised form June 29, 2010.

Accepted for publication June 30, 2010.

Reprints: George J. Christ, PhD, Wake Forest Institute for Regenerative Medicine, Wake Forest University Baptist Medical Center, Richard H. Dean Biomedical Research Building, Room 442, Medical Center Blvd, Winston-Salem, NC 27157. E-mail: gchrist@wfubmc.edu.

This symposium was supported in part by a grant from the National Center for Research Resources (R13 RR023236).

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Abstract

A recent report demonstrated that a laboratory-grown neobladder tissue could be successfully used for cystoplasty in young patients with myelomeningocele who were otherwise healthy. This remarkable achievement portends well for the application of tissue engineering/regenerative medicine technologies to the treatment of end-organ failure due to a variety of causes (ie, congenital, acquired, age or disease related). Nonetheless, the broader clinical use of these groundbreaking technologies awaits improved understanding of endogenous regenerative mechanisms, more detailed knowledge of the boundary conditions that define the current limits for tissue repair and replacement in vivo, and the parallel development of critical enabling technologies (ie, improved cell source, biomaterials, bioreactors). This brief report will review a number of the most salient features and recent developments in this rapidly advancing area of medical research and detail some of our own experience with bladder and skeletal muscle regeneration and replacement as examples that highlight both the promise and challenges facing regenerative medicine/tissue engineering.

Regenerative medicine may be simply defined as the repair and replacement of damaged cells, organs, and tissues. Stocum codified it quite clearly as follows:

"Regenerative biology seeks to understand the cellular and molecular differences between regenerating and non-regenerating tissues. Regenerative medicine seeks to apply this understanding to restore tissue structure and function in damaged, non-regenerating tissues."1

Without question, regenerative medicine is an international, interdisciplinary, and pioneering translational research effort with enormous potential to mitigate the inevitable adverse health consequences and health care costs of an aging US and world population. Regenerative medicine encompasses a spectrum of technologies and approaches ranging from cell, biomaterial, and drug therapy for promotion of endogenous regenerative capacity to whole organ/tissue replacement with laboratory-grown organs or biomimetics (ie, tissue engineering). As such, tissue engineering/regenerative medicine spans the fields of cell and molecular biology, chemical and material sciences (ie, nanotechnology), engineering, molecular genetics, physiology and pharmacology, surgery, and cell transplantation toward the creation of technologies that would restore and maintain normal tissue/organ function and/or promote endogenous regenerative capacity.

The size of the unmet medical need and the potential importance of regenerative medicine have been noted in recent government publications (National Institute of Health Fact Sheet (http://www.nih.gov/about/researchresultsforthepublic/Regen.pdf)). The current world market for replacement organ therapies is estimated to be in excess of $350 billion, whereas the worldwide market for regenerative medicine will approach $500 billion by 2010. In light of the growing shortage of donor organs, regenerative medicine and tissue engineering technologies are poised to have a tremendous impact on extending both the quality and duration of patient's lives. Certainly then, there is a tremendous need for the continued development of these technologies; yet, despite the decades of research conducted thus far, there has been relatively limited clinical success.2-5 Nevertheless, the field has made great strides in recent years, and the requisite formation of extensive interdisciplinary collaborations is now well underway in a global fashion. In particular, one recent example of a large US government-sponsored effort in regenerative medicine is the creation of the Armed Forces Institute of Regenerative Medicine (AFIRM). The press release for the AFIRM can be viewed at http://www.defense.gov/releases/release.aspx?releaseid=11842. Briefly, AFIRM has pulled together more than 30 institutions across 2 consortia (Wake Forest-Pittsburgh and Rutgers-Cleveland Clinic) to tackle the challenges associated with restoring function to soldiers with traumatic war injuries (www.afirmwakepitt.org). This translational research effort is organized around 5 core therapeutic areas pertinent to military injuries: burn repair, compartment syndrome, craniofacial injuries, healing without scarring, and limb and digit repair. The implications to the civilian population are clear, and the existence of such coordinated research enterprises is critical to advancing the field and bodes well for the clinical application of tissue engineering and regenerative medicine technologies to meet patient needs. Nonetheless, numerous challenges remain, and these have been well described in several recent reviews devoted to this subject.2-7 Not surprisingly, a variety of substantive strategies are currently being contemplated to overcome the existing barriers to organ and tissue repair and replacement. The focused aim of this report is to highlight a few critical technologies and their importance to the tissue engineering/regenerative medicine process and to provide some examples from our own group's experience with bladder and skeletal muscle regeneration and repair to emphasize the point.

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THE REGENERATIVE MEDICINE/TISSUE ENGINEERING PROCESS AND BASIC COMPONENTS

Figure 1 highlights the spectrum of potential therapeutic applications of regenerative medicine/tissue engineering technologies. The approach taken will clearly depend on the nature and extent of tissue/organ dysfunction. As such, the development of tissue-specific applications seems inevitable. Moreover, it corresponds to intuition that the more complex or complete the degree of end organ/tissue dysfunction, the more comprehensive the corresponding tissue engineering/regenerative medicine strategy that will be required.

Figure 1
Figure 1
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Although numerous possibilities exist, and moreover, the details may vary, the overall conceptual framework for tissue/organ creation will be similar (Fig. 1). That is, regardless of the precise strategy used for reconstruction, restoration, or repair of the tissue/organ of interest, cells and biomaterials (ie, scaffolds) still provide the basic components required for building new tissue. If sufficient tissue/organ viability remains in vivo, then either cells (ie, cell therapy) or scaffolds (biomaterial therapy) alone may be used to assist with the regenerative process. At this point, it is still possible to leverage existing endogenous mechanisms for tissue repair and/or restoration of organ function. However, for many congenital or acquired conditions, or after traumatic injury, the degree of end organ/tissue damage may exceed the intrinsic mechanisms for endogenous repair because of the paucity of the remaining viable tissue. In this instance, endogenous repair mechanisms may require augmentation via the use of more mature, native-like tissue/organ biomimetics (ie, biological substitutes). The creation of more native-like biomimetic tissues, in turn, will require more in vitro exposure. For example, one such approach to creation of more native-like biomimetics uses laboratory devices, referred to as bioreactors, to recapitulate relevant aspects of the in vivo physiological environment of the tissue or organ of interest. For this method, cells may be seeded on a biomaterial, placed in a bioreactor, and subjected to environmental cues that are critical to tissue formation, such as, stress, strain, stretch, flow, or pulsation. As such, bioreactors may be used to create more advanced 3-dimensional tissue formation in vitro before implantation in vivo.

Having provided a general overview of the process, we now proceed to a description of some of the key components of this process as well as their contributions to the further development of tissue engineering/regenerative medicine technologies.

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CELL SOURCE: STEM CELLS FOR REGENERATIVE MEDICINE/TISSUE ENGINEERING

Because cells represent one of the primary "raw materials" required for building tissues and organs, a renewable and expandable source of cells is clearly of paramount importance to the tissue engineering process. Autologous cells, because they are immune compatible, provide the current gold standard for tissue engineering/regenerative medicine applications. However, not all tissues provide a readily available source for autologous primary cell expansion (eg, brain, heart, pancreas), and moreover, as previously noted, depending on the extent of end-organ/tissue damage, there may not always be a sufficient pool of viable tissue or cells available for biopsy and subsequent expansion. Thus, development of alternative human stem cell sources for tissue engineering/regenerative medicine is required to meet the demands of these circumstances.

In this regard, stem cells are capable of self-renewal and may differentiate into multiple cell lines, making them advantageous for regenerative medicine applications.8,9 Depending on the type and maturity of the tissue, stem cell potency and capacity for self-renewal can vary. Embryonic stem (ES) cells derived from the blastocyst inner cell mass of developing embryos are pluripotent, giving rise to progeny of ectoderm, endoderm, and mesoderm germ layers.10,11 Although stem cell pluripotency diminishes with development, adult tissues do possess stem cells that exhibit multipotency.9,12,13 Examples of such adult stem cells include mesenchymal stem cells,13,14 which may differentiate in culture into a variety of cell types including chondrocytes, osteocytes, adipocytes,15-17 and hematopoietic stem cells, which may replenish both myeloid and lymphoid cells in lethally irradiated mice.18 Additionally, adult tissues, such as skeletal muscle, liver, and kidney tissue, among others, possess progenitor cells capable of proliferating and differentiating into specific end organ cell types.19-22 For example, in response to skeletal muscle fiber injury, satellite cell progeny may fuse with damaged fibers during the regeneration process aiding in the recovery of skeletal muscle function.23-25 Thus, the purpose of adult stem cells is to assist in repair and regeneration of postnatal tissues.

Pluripotent stem cells provide great flexibility for cell-based therapies and tissue engineering because of their capacity to generate the multiple types of cells required for organogenesis (eg, the kidney is composed of at least 26 different cell types) and the large number of each cell type required for such therapies. However, an optimal pluripotent stem cell source for regenerative medicine and tissue engineering therapies has been elusive. In this regard, ES cells may someday be used to fulfill this purpose. Alternatively, stem cells collected from amniotic fluid (ie, amniotic fluid stem cells) also have pluripotent stem cell characteristics.26,27 However, one potential drawback of either ES or amniotic fluid stem cells is that they would represent an allogeneic cell source that may require administration of immune suppressant drugs. In this regard, induced pluripotent stem (iPS) cell technology is under development, whereby nonpluripotent somatic cells are reprogrammed via transduction with subsets of genes (eg, Oct3-4, Sox2, c-Myc, Klf4, Nanog) that are thought to convey pluripotency in ES cells.28-31 In this scenario, autologous somatic cells could be isolated and transformed to iPS cells, which could then be used for in vitro organogenesis or cell injection therapy without immune rejection. Since the recent advent of this technology,29 induction methodologies have been developed to remove some translational concerns of retroviral mutagenesis-mediated tumor formation,32 thus making these stem cells a potentially promising pluripotent cell source for the development of a variety of clinical therapies, including pancreas and kidney regeneration as well as a treatment for Parkinson disease.33-35

Multipotent adult stem cells also hold great promise as tools for further development of regenerative medicine technologies. For example, the use of mesenchymal stem cells derived from primarily bone marrow, but also adipose and placental tissues in animal studies has prompted the use of mesenchymal stem cells in more than 100 clinical trials (http://clinicaltrials.gov/). Mesenchymal stem cells have been shown to ameliorate disease states and degenerative conditions in animal models of myocardial infarction, type I diabetes mellitus, and graft-versus-host disease, among other conditions.13,36-40 The mechanistic basis for the beneficial effects of MSCs are not entirely understood. One possibility is that injected MSCs may directly augment host tissue function by differentiating and integrating with the relevant cell type of the injured tissue. For example, MSCs when injected into sarcoglycan deficient hamsters were reported to integrate with host skeletal muscle fibers, presumably, improving the functional phenotype of the muscle.41 However, given the low efficiency of injected MSC engraftment with injured host tissue,13 a more feasible mechanism is that MSCs release soluble factors that support and enhance the endogenous regenerative capacity of the host. For example, bone marrow- and adipose tissue-derived MSCs may primarily work via immune modulation and angiogenic stimulation to improve recovery of injured or diseased tissues.13,42-45 The former characteristic provides the rationale for the use of MSCs as a potential allogeneic cell source for development of therapies in humans.

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BIOMATERIALS FOR REGENERATIVE MEDICINE/TISSUE ENGINEERING

In a recent article, Williams46 outlined the nature and evolving role of biomaterials in the tissue engineering process. In short, biomaterials can be used to provide both the mechanical and molecular signals required for development of an artificial tissue.46 Although a material is not always necessary for regeneration per se, the support provided can greatly advance and accelerate the growth and maturation of a regenerating or tissue-engineered construct. Generally, 3 main types (or combinations) of biomaterials have been used for tissue engineering: (1) naturally derived materials (eg, fibrin, collagen, chitosan, and alginate); (2) synthetic polymers (eg, polyglycolic acid [PGA], polylactic acid [PLA], polylactic-coglycolic acid [PLGA]), polyhydroxyalkanoate (PHA), and polydioxanone (PDS); and (3) acellular tissue matrices (eg, bladder acellular matrix and small intestinal submucosa). Although several methods have been used to fabricate biomaterials for numerous tissue engineering applications, recent advances have focused on accelerating the field of "active" biomaterials, that is, biomaterials that (actively) facilitate 3-dimensional tissue engineering and maturation of regenerated tissue. Numerous approaches are currently being evaluated, including manipulating the structure of biomaterials for better oxygen and nutrient diffusion, developing "intelligent" biomaterials with the ability to release growth factors, and advancing the use of gels and injectables for less invasive in vivo applications, just to name a few.47-49 Because a detailed review of all of these approaches is well beyond the scope of this report, we provide a few relevant examples from our own experience.

In this regard, 3 techniques routinely used in our laboratory provide control over the formation, viability, and construction of engineered tissue, respectively. Specifically, we use electrospinning to provide control over the nanomorphology of the scaffold biomaterial, oxygen-generating biomaterials to extend the viability of the tissue until a vascular system can be established, and inkjet printing to assemble cells and biomaterials/scaffolds simultaneously. All of these are accomplished with relatively precise spatial control, and a short description of each of these 3 technologies is provided below. However, it is important to keep in mind that, although each of these technologies can be used independently, they also can be combined, as required, to yield further enhancements to the regenerative process.

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ELECTROSPINNING

Electrospinning provides a simple technique to generate a nanofiberous structure while simultaneously providing macroscopic control over scaffold formation and architecture. Briefly, electrospinning uses a high electric field to overcome the surface tension of a viscous polymer or aqueous solution. Once the surface tension is overcome, a polymeric jet is generated, known as a taylor cone, and deposited on a collecting grounded electrode (schematic seen in Fig. 2). The fibers are approximately 100 nm in diameter, although they can be manipulated by adjusting parameters, such as polymer concentration, solvent, and electrode distance. Deposition on a stationary electrode usually forms a random orientation, nonwoven mat. However, by rapidly rotating the collecting electrode, aligned fibers can be formed. Several recent studies have used electrospinning as a controllable method of creating a scaffold using both synthetic and natural biomaterials for regenerative medicine applications.50-53 For example, in a study by Choi et al.,54 aligned electrospun polycaprolactone/collagen nanofibers were prepared to evaluate the impact of fiber alignment on the development of tissue-engineered skeletal muscle in vitro. The goal was to create appropriately aligned nanofibers that more directly mimic the underlying architecture of uniformly aligned skeletal muscle in vivo. This study demonstrated that the aligned electrospun architecture guided and enhanced myotube formation to favor the presence of longer myotubes. In fact, an approximate doubling was observed in myotube length.54

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OXYGEN-BASED BIOMATERIALS

In addition to the control of scaffold configuration, there are many other aspects of biomaterial development that are relevant to tissue engineering. When an engineered tissue is implanted, it usually does not have a vascular system established to easily integrate into the host. This situation typically results in a large loss of cell numbers within the 3-dimensional scaffold because of the ensuing hypoxia/ischemia at the center of the construct. Methods have been used to accelerate the formation of a vascular supply, such as adding growth factors or endothelial cells to facilitate blood vessel formation.55,56 Alternatively, temporarily providing oxygen and nutrients while a permanent vascular system is being established is a potential approach to mitigate the deleterious consequences associated with immediate lack of blood supply after implantation.57-59

To this end, we have been working with particles that generate oxygen (ie, particulate oxygen generators [POGs]). Particulate oxygen generators represent a novel class of biomaterials for providing oxygen in situ for a temporary period (currently, up to 2 weeks).58,59 These materials begin to generate oxygen upon contact with aqueous solutions. Studies by our group have used these materials for in vivo tissue salvage with an implantable film form in an ischemic skin flap model in mice,58 which delayed the onset of necrosis via early supplementation of oxygen. The model showed that many of the normal degradation processes typically associated with hypoxia, such as apoptosis, discoloration, and lactate increase, seemed to slow down with oxygen supplementation. A second form of oxygen-generating biomaterial incorporated POGs in a 3-dimensional porous polymer construct, formed using poly(lactic-co-glycolic acid) through a porogen leeching process, for cell viability in vitro. Constructs with incorporated oxygen-generating biomaterials were seeded with cells and cultured in a hypoxic environment, where the only oxygen source was from the POG supplement. This study showed an increase in cell viability in both a hypoxic and 3-dimensional environment,59 which has significant implications for the tissue engineering field. Taken together, our results to date illustrate the potential use of these novel oxygen-incorporating biomaterials for extending cell viability and tissue salvage/preservation both in vitro and in vivo until vascularization can be established.58

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CELL AND BIOMATERIAL PRINTING

Cell and biomaterial printing technologies can combine rapid prototyping procedures with microencapsulation techniques to print free-form structures of virtually any shape and size.60-64 For example, one can use inkjet printing technologies where the ink cartridge is filled with cells and culture medium, termed bioink, or with biomaterials such as alginate. The materials and/or cells are printed layer by layer, deep to superficial, onto an existing scaffold, or to create a new scaffold.65 Several cell types and materials have been used for printing, allowing for formation of viable 3-dimensional structures as well as the simultaneous creation of microstructures within the constructs.66,67 This method provides control over the geometry and architecture of a construct in a 3-dimensional environment, which has obvious implications for further development of tissue engineering technologies.

As previously noted, each of these 3 technologies also can be combined to yield further advantages for the tissue engineering process. For example, oxygen-generating materials can be incorporated into electrospun meshes to demonstrate the capability of using a structured scaffold with the added benefit of oxygen production (Fig. 2). In addition, electrospun meshes also have been used as scaffolding materials for 3-dimensional cell printing, aiding in the layer-by-layer tissue engineering technique.68 Finally, oxygen-generating biomaterials can provide a supplement to cell printing techniques with the idea of simultaneous dispersion of particles throughout the 3-dimensional scaffold. In short, the versatility of these technologies and their various combinations can provide tissue-specific customization for acceleration of the tissue engineering process.

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CURRENT STATUS

Without question, it is clear that tissue engineering/regenerative medicine technologies are poised to revolutionize treatment of end-organ/tissue disease and dysfunction. However, despite the armamentarium of existing regenerative medicine/tissue engineering technologies and the wealth of preclinical achievements, to date, there have been few demonstrable clinical or commercial successes. In fact, Carticel (which uses autologous chondrocytes to repair articular cartilage damage; see www.carticel.com), Apligraf (wound care and healing of foot or leg ulcers; see www.apligraf.com), and Integra (dermal regeneration for burn and reconstructive surgery; see www.integra-ls.com/home/catalogs.aspx) are among the small number of success stories. Below, we briefly review the first successful implantation of a laboratory-grown neo-organ. The creation of the neobladder represents an important translational technology that captures the essence of the current state of the field.

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IMPLANTATION OF A LABORATORY-GROWN NEOBLADDER: "STATE OF THE ART"

In 2006, Atala and colleagues69 were the first to report on the successful implantation of a laboratory-grown, tissue-engineered neobladder. In this initial pilot study of 7 young patients with myelomeningocele (aged 4-19 years), the investigators created a collagen scaffold seeded with cells either in the presence or absence of omental coverage or used a combined PGA-collagen scaffold seeded with cells and omental coverage, according to the process outlined in Figure 3.69 The use of the reconstructed neobladders created with PGA-collagen cell-seeded scaffolds was reflected by an increased compliance, decreased end-filling pressures, increased capacities, and longer dry periods.70 Despite the unequivocal importance of this seminal clinical study, the implanted neobladders lacked the normal innervation required for micturition and thus provided a neoreservoir, rather than a functional bladder. Nonetheless, this study represented a critical advance for the field and laid the groundwork required for additional clinical studies (see the Tengion Web site, www.tengion.com, for more details on the latest clinical trials).

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Importantly, the clinical use of the neobladder also provides an important reference point for future translational research investigations. It seems logical then to look to animal models of functional endogenous regeneration for guidance regarding further improvements in the development of regenerative medicine technologies for humans. In fact, there is quite a bit of evidence indicating that the urinary bladder possesses a significant capacity for regeneration. Some salient details of regeneration in animal models, in particular as they apply to bladder regeneration, are discussed below in the context of providing a potential bridge to new clinical applications in the future.

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BLADDER REGENERATION

Species-dependent variations in organ and tissue regeneration are well documented in the animal kingdom.71 For example, a tremendous capacity for regeneration can be found in lower vertebrates, such as the axolotl and the newt, where regeneration of body parts, including limbs, jaws, tail, skin, spinal cord, brain, and apex of the heart, has been demonstrated.72,73 By comparison, adult mammals have a much more restricted capacity for repair and replacement of damaged tissues/organ systems, and furthermore, regenerative potential is thought to be limited to epidermis, muscle, bone, and liver.72 Excellent models for studying regenerative biology do already exist in nature, and one less well-known example of this is the urinary bladder-a fact established more than 100 years ago. More specifically, in 1891, Schwarz74 reported the growth of a normal-sized bladder after a subtotal cystectomy (ie, removal of a majority of the bladder; STC). More recent studies also have confirmed this supposition in dogs,75,76 and similar observations have been made in rats.77,78 A significant capacity for bladder regeneration was clearly demonstrated in humans as early as 1939.79 Other investigators have since confirmed these initial observations in humans.77,78,80 In this scenario, perhaps it is not surprising that after many years of preclinical work, Atala and colleagues69 were able to leverage the regenerative capacity of the bladder and extend it to the clinic.

In conclusion, the literature supports the concept of functional bladder regeneration after subtotal cystectomy in both humans and rats. This indicates that the latter is a reasonable model of the former. Given the suboptimal therapeutic alternatives for bladder repair currently available in humans (eg, detubularized bowel segments), improved understanding of regenerative biology in the rodent urinary bladder would seem an important prerequisite to the development of novel and more efficacious therapeutic treatment options for bladder disease/dysfunction. As such, we have begun multidisciplinary studies to elucidate the time-dependent characteristics of native bladder regeneration after STC in rats. Our initial observations in this animal model have recently been published, and the salient features of our findings are described below.

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A RODENT MODEL OF BLADDER REGENERATION IN VIVO

For these studies, surgical removal of approximately 70% of the bladder (STC) of female rats was used as a model system to gain insight into the normal organ regeneration process in adult mammals in vivo. At 2, 4, and 8 weeks after STC, the extent of bladder regeneration was monitored via a host of methods, including micro computed tomography (CT) scans, urodynamic studies (bladder function studies conducted in conscious rats via an indwelling catheter in the bladder; see references81-84), pharmacological and physiological studies conducted on isolated bladder strips, and immunohistochemistry. It is important to note that power of this model is enhanced by the fact that comparisons were made between the native and regenerated bladders from the same animal. That is, all of the aforementioned observations were obtained on both the native bladder and retrieved strips from the excised portion of that bladder (ie, after STC) as well as on the regenerated bladder from the same rat (Fig. 4). After STC, CT imaging revealed a time-dependent increase in bladder size at 2, 4, and 8 weeks after STC (Fig. 4) that positively correlated with restoration of bladder function. Moreover, the maximal contractile responses observed after pharmacological activation and electrical field stimulation increased over time in isolated tissue strips from regenerating bladders. Nonetheless, the observed responses were still lower at all time points after STC compared with the responses observed on bladder strips from age-matched control animals. Consistent with these observations, urodynamic studies (ie, bladder function studies) revealed that the pressures observed in the bladder during emptying (ie, micturition) also were significantly lower in the regenerated than the native bladder. However, despite these physiological alterations, at all time points studied, the bladders emptied completely-testament to the fact that the regenerated bladders functioned essentially normally. In addition, immunostaining of the bladder wall of STC rats suggested a role for progenitor cells and cellular proliferation in the regenerative response. Finally, immunostaining and the presence of electrical field stimulation-induced contractile responses verified the presence of functional innervation of the regenerated bladder. In short, these initial studies illustrate the potential of the rodent model for studying natural tissue regeneration in vivo. Studies are still ongoing and are expected to provide important experimental details that could lead to the development of novel technologies to optimize and enhance the intrinsic regenerative capacity of the bladder, and perhaps other tissues/organ, for clinical applications.

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SKELETAL MUSCLE TISSUE ENGINEERING AND REPAIR

Virtually every aspect of daily living is dependent on the proper function of skeletal muscle. As such, the loss of skeletal muscle function can lead to a drastic reduction in the quality of life, disability, and death. Functional deterioration may occur with traumatic injury (eg, volumetric muscle loss [VML]), disease (eg, Duchenne muscular dystrophy), and aging. Although a smaller degree of dysfunction/disease may be treated with technologies, such as cell therapy and regenerative pharmacology, traumatic injuries resulting in VML, for example, currently have no satisfactory treatment options available. Thus, the need for the development of more effective technologies for functional skeletal muscle reconstruction and/or replacement is critical. We discuss herein the merits of using tissue engineering to this end.

In this regard, as with the neobladder previously described, skeletal muscle tissue engineering incorporates basic cell culture techniques with other enabling technologies, such as biocompatible scaffolds and in vitro bioreactors, to generate skeletal muscle tissue ex vivo. In fact, we have developed a method for skeletal muscle tissue engineering in which cultured myoblasts derived from rat primary cells are seeded onto an acellular collagen matrix derived from the bladder (ie, bladder acellular matrix). After expansion and differentiation of myoblasts on the scaffold, these tissue-engineered constructs are then placed in a bioreactor, in which uni-axial stretch is applied for 1 week, to provide mechanical cues for further tissue maturation/development.85 Other groups have used qualitatively similar approaches to skeletal muscle tissue engineering by using different scaffold materials (eg, fibrin gels86-89) and/or distinct forms of bioreactor preconditioning (eg, electrical stimulation88,90). However, the goals for doing so can be very different. For example, one goal is to create tissue-engineered skeletal muscle for in vitro study only, such as that required for high throughput drug screening or improved understanding of certain aspects of developmental biology.91-94 However, another goal is to create tissue-engineered skeletal muscle in vitro that can subsequently be implanted for restoration of muscle function in vivo.95 Examples of each approach are briefly reviewed below.

During the past several years, tissue-engineered constructs that recapitulate some relevant functional characteristics of mature skeletal muscle have been successfully generated in vitro. For example, constructs have been shown to exhibit active isometric contractile responses to membrane depolarizing stimuli (ie, electrical stimulation or potassium chloride exposure).85,86,88,89,96 That is, tissue-engineered skeletal muscle created in fibrin-based gels have been shown to produce a voltage-induce maximal isometric tetanic forces of approximately 36 kN/m2,89 exhibit a graded force response to increasing frequency of electrical stimulation, and exhibit dependency of length on maximal isometric contraction force (ie, length-tension relationship).86,89

As previously noted, one application for tissue-engineered skeletal muscle in vitro is to leverage this technology for high-content drug screening. Because tissue-engineered skeletal muscle constructs exhibit functional characteristics similar in nature to skeletal muscle generated de novo, high-content drug screening may be used to identify, on a physiological basis, drugs effective in the treatment of various diseases and disorders.92,93 In this light, muscle precursor cells could be harvested from human patients with neuromuscular disease, for example, for the purpose of a patient-specific tissue-engineered drug screening. As a proof of concept for potential human applications, myoblasts from mdx mice, a putative Duchenne muscular dystrophy model, were recently engineered to form constructs, the contractile function of which was assayed for drug screening.94 Although drug screening using skeletal muscle tissue-engineered constructs by no means encapsulates the intricacies and complexities of neuromuscular disease in humans, this approach could be extremely useful in focusing drug development studies before animal experimentation.

After creation of tissue-engineered skeletal muscle in vitro, some groups have implanted these constructs subcutaneously in vivo. For example, 3 weeks after a fibrin-based construct was implanted near femoral vessels in a rodent model, retrieved implants produced a voltage-induced isometric contractile response of approximately 0.04 kN/m2.87 Additionally, using a bladder acellular matrix-based skeletal muscle construct, Moon et al.,85 demonstrated that after bioreactor preconditioning, constructs implanted subcutaneously in nude mice produced approximately 0.9 kN/m2 of isometric force in response to maximal electrical stimulation. Although the development of these tissue-engineered constructs is an important step in the right direction, it should be recognized that the forces currently generated by such constructs are only a fraction of maximal forces (∼200 kN/m2) generated by native skeletal muscle tissue.

As part of the logical progression to clinical translation, one of the ultimate goals of development of tissue-engineered skeletal muscle is functional reconstruction and/or replacement. Three general approaches are currently being pursued to restore function after VML, and they are as follows: (1) implantation of scaffold only,97 (2) implantation of scaffold followed by injection of stem cells,98 or (3) implantation of scaffold seeded with cells followed by bioreactor preconditioning.95 With respect to the scaffold only approach, Merritt et al.,97 created a 1 × 1 cm2 full-thickness surgical defect in the rat gastrocnemius muscle to model VML. Creation of this surgical defect resulted in an initial 25% reduction in force, and implantation of a decellularized gastrocnemius muscle matrix at the site of injury did not facilitate functional recovery 42 days after injury.97 However, in the same injury model, when bone marrow-derived mesenchymal stem cells were injected into the scaffold 7 days after creation of the surgical defect and scaffold implantation, the injured muscle produced approximately 85% of contralateral control muscle force 42 days later, which was statistically significant and indicative of the importance of incorporating a cellular component for treatment of VML.98 Our group has leveraged the initial findings of Moon et al.,85 and further optimized the bioreactor-preconditioned, cell-seeded, collagen-based scaffold approach. In short, we have created a tissue-engineered skeletal muscle construct that can be implanted in a 50% surgical defect of the rodent lattisimus dorsi muscle. Using this approach, we have observed robust functional recovery within 2 months of implantation.95 Taken together, the initial work in the field bodes well for the use of using a tissue engineering platform for the treatment of VML and related skeletal muscle disease/dysfunction.

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SUMMARY AND CONCLUSIONS

The current climate bodes well for the future development of regenerative medicine technologies. Key advances in stem cell biology and the development of novel biomaterials are driving the field forward. Continued improvements in these basic "raw materials" for tissue and organ building and regeneration are essential for more widespread clinical applications. Moreover, the parallel development of critical enabling technologies (eg, bioreactor, bioprinters, materials fabrication [ie, electrospinning], encapsulation/drug delivery) will further enhance and accelerate the translational potential of regenerative medicine by creating new opportunities and targets for tissue engineering and regeneration. Finally, the formation of multidisciplinary teams across institutional and international boundaries (eg, AFIRM) will create a global community of scientists with expertise in this cutting-edge field of medical research, providing the synergies and cross-fertilization required for both increased clinical success and sustainable commercialization of regenerative medicine technologies.

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Keywords:

tissue engineering; regenerative medicine; biomaterials; scaffolds; bladder; skeletal muscle

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