Fisher John P. e.a. (ed.) Tissue Engineering

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19-18 Tissue Engineering

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Gene Therapy and

Tissue Engineering

Based on

Muscle-Derived Stem

Cells: Potential for

Musculoskeletal

Tissue Regeneration

and Repair

Johnny Huard

Baohong Cao

Yong Li

Hairong Peng

University of Pittsburgh

20.1 Introduction.............................................. 20-2

Tissue Engineering • Growth Factors and Gene Therapy •

Isolation of Muscle-Derived Stem Cells

20.2 Skeletal Muscle ........................................... 20-4

Muscle Disease: Duchenne Muscular Dystrophy •

Sports-Related Muscle Injuries

20.3 Bone Healing ............................................. 20-5

The Problem of Bone Healing • The Use of Muscle-Derived

Cells to Improve Bone Healing • Can We Isolate Human

Muscle-Derived Stem Cells That Display Similar Osteogenic

Behavior? • Can We Regulate the Expression of Osteogenic

Proteins and the Resultant Bone Formation? • Is the Bone

Regenerated by BMP-Producing Muscle-Derived Cells

Biomechanically Relevant?

20.4 Articular Disorders ...................................... 20-8

Articular Cartilage • Meniscus • Ligaments

20.5 Summary ................................................. 20-10

Acknowledgments............................................... 20-10

References ....................................................... 20-10

20-1

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20-2 Tissue Engineering

20.1 Introduction

Recent discoveries in cell biology have signiﬁcantly advanced our knowledge of disease mechanisms.

Newly developed techniques, coupled with advances in cell biology and polymer chemistry, are enabling

the evolvement of novel tissue engineering approaches for the treatment of various disorders. Surgeons,

who have traditionally used the tools of excision and reconstruction to treat patients, may now act as

surgical gardeners who create microenvironments conducive to tissue regeneration. Our research team

has isolated a novel population of muscle-derived stem cells (MDSCs) that display enhanced regener-

ative capability due to their multipotency, self-renewal ability, and immune-privileged behavior. This

paper presents evidence supporting the existence of MDSCs and reviews some current MDSC-based gene

therapy and tissue engineering applications designed to improve the healing of various musculoskeletal

tissues, including skeletal muscle, bone, and intra-articular tissues. This review updates readers on the

foundational principles and current advances in muscle-based gene therapy and tissue engineering for the

musculoskeletal system.

20.1.1 Tissue Engineering

“Tissue engineering” refers to the science of creating living tissue to replace, repair, or augment diseased

tissue. The engineered tissue may be created in vitro and subsequently implanted into the patient or

the tissue may be created entirely in vivo. Regardless of the technique, tissue engineering requires at

least three components: a growth-inducing stimulus (induction), a scaffold to support tissue formation

(conduction), and responsive cells (production). The mechanical stimulation that occurs after the appro-

priate tissue engineering constructs have been introduced also strongly inﬂuences the remodeling of the

newly regenerated tissue.

20.1.2 Growth Factors and Gene Therapy

“Growth factors” are soluble proteins that promote cell division, maturation, and differentiation. Recom-

binant DNA techniques have increased our understanding of the function of these proteins, which

inﬂuence various tissue regeneration and repair processes. These techniques also have enabled the pro-

duction of large quantities of growth factors for use in experimental investigations. Currently, a great

deal of research is focused on the identiﬁcation of optimal growth factors for particular applications.

After identifying the ideal growth factor, scientists must determine the optimal method for delivering

the growth factor within the surgical microenvironment. Direct application of a particular recombinant

protein is the most straightforward means of delivering growth factors in situ. Research conducted during

the 36 years since the discovery of bone morphogenetic proteins (BMPs) has demonstrated that one of the

most important factors for in vivo healing is the presence of high, sustained doses of growth factors. Most

growth factor proteins have half-lives of minutes and are cleared rapidly by the bloodstream. Even when

the protein is bound to a collagen scaffold, its half-life is limited to several days. Although microgram

doses are sufﬁcient for in vitro manipulation of cells, milligram doses typically are required for in vivo

regional regeneration. Researchers thus have turned to regional gene therapy for the sustained delivery of

agrowthfactortoalocalsitein vivo.

One common gene therapy approach involves the transfer of a gene encoding a desired protein into

cells via viral or nonviral vectors. The transduced cells subsequently secrete the desired protein into the

microenvironment. Gene therapy generally assumes one of two forms: in vivo or ex vivo (indirect). The

in vivo approach, which involves the direct injection or implantation of a gene-carrying vector into a

recipient or patient, is attractive due to its technical simplicity. However, when using in vivo gene-therapy

techniques it is impossible to perform in vitro safety testing on the transduced cells. In the ex vivo approach,

cells are isolated from a tissue biopsy, expanded, and then transfected or transduced in vitro. Before being

introduced into the recipient or patient, the genetically altered cells can be tested in vitro to assess the

efﬁciency of gene transfer or to identify any abnormal behavior.

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Gene Therapy and Tissue Engineering 20-3

Clinical application of a particular gene therapy or tissue engineering technique requires identiﬁcation

of the protein required for proper tissue function, the gene that expresses this protein, and a reproducible

method by which to deliver the protein to the diseased or injured tissue in sufﬁcient quantities with

adequate persistence. During the last few years, muscle cells have emerged as promising vehicles for gene

therapy and tissue engineering in the musculoskeletalsystem. Several attributes make muscle an ideal tissue

for tissue engineering such as, (1) Obtaining a muscle biopsy or tissue harvest involves a simple surgical

procedure that can be repeated without compromising patients’ health; (2) Muscle-derived cells tolerate

ex vivo manipulation well and it is possible to obtain a large number of muscle-derived cells rather

quickly (within 1 to 2 weeks) via expansion [1,2]; (3) After reinjection in vivo, these cells naturally fuse

to form permanent postmitotic myotubes that allow for long-term expression of the desired protein;

(4) Muscle cells can be transduced easily by a variety of viral vectors [3–6]; and (5) Muscle tissue contains

a population of muscle stem cells that display an enhanced regenerative capacity when used in tissue

engineering applications [1,2,7,8].

Because of the aforementioned characteristics, muscle cells have been used extensively as vehicles

in gene therapy protocols for various muscle-related diseases, including Duchenne muscular dystrophy

(DMD) [9–12]. Researchers also have used these cells as vehicles in many nonmuscle-related gene-therapy

applications, such as the transfer and expression of factor IX for hemophilia B [13]; systemic delivery of

human growth hormone for growth retardation [14]; gene delivery of human adenosine deaminase

for the adenosine deaminase deﬁciency syndrome [15]; gene transfer of human proinsulin for diabetes

mellitus [16]; expression of tyrosine hydroxylase for Parkinson’s disease [17]; expression of FasL to

prevent immunorejection of pancreatic islet cell transplants [18]; and injection of muscle cells into the

joint (i.e., the meniscus, synovium, and ligament) for the treatment for arthritis and improvement of

intra-articular tissue healing (see the sections that follow in this chapter) [19–21].

20.1.3 Isolation of Muscle-Derived Stem Cells

Muscle is composed of a heterogeneous population of cell types. Skeletal muscle contains satellite cells,

which are resting, mononucleated, myogenic precursor cells capable of fusing to form postmitotic, mul-

tinucleated myotubes and myoﬁbers. Skeletal muscle also contains stem cells. These unique cells are

identiﬁable based on their capacity for protracted renewal; production of daughter cells, which may pro-

ceed toward lineage commitment and terminal differentiation; multipotency; and the ability to exhibit

such behaviors throughout life [7]. Accordingly, muscle-derived stem cells produce populations of daugh-

ter cells that can undergo terminal differentiation into muscle cells, some of which subsequently can

differentiate into myoﬁbers while others remain undifferentiated [22]. These stem cells do not express

markers of mature muscle tissue, but are capable of prolonged production of progeny and are multipotent

[7,8,23–25]. Of the various cells that constitute any given postmitotic muscle sample, approximately 1%

are committed satellite cells; the percentage of muscle-derived stem cells is substantially lower — roughly

1% of the number of satellite cells [7,8].

Researchers have used a variety of techniques to isolate stem cells from skeletal muscle digest. These

cells can differentiate into myogenic, hematopoietic, osteogenic, adipogenic, chondrogenic, neural, and

endothelial lineages [7,26–33]. Importantly, these stem cells also appear to display distinct marker pro-

ﬁles that distinguish them from satellite cells. Expression proﬁles for satellite cells vary depending on the

activated state and the degree of differentiation. In our laboratory, we have used the preplate tech-

nique to isolate a novel population of highly puriﬁed MDSCs [7,8]. These cells express both early

myogenic markers (e.g., desmin, c-met, MNF, and Bcl-2) and stem cell markers (e.g., Sca-1, Flk-1,

and CD34) [7,8]. Because these cells can differentiate into various lineages (mesodermal, endodermal,

and ectodermal) in vitro and in vivo and display an enhanced ability to regenerate various tissues, they

constitute ideal cellular vehicles for gene therapy and tissue engineering applications. During the last

few years, we have investigated the use of this technology to improve the healing of different com-

ponents of the musculoskeletal system, including skeletal muscle, bone, and various intra-articular

structures.

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20-4 Tissue Engineering

20.2 Skeletal Muscle

20.2.1 Muscle Disease: Duchenne Muscular Dystrophy

Patients with DMD lack the dystrophin protein in the sarcolemma of the muscle ﬁber; its absence dis-

rupts the integration of the cytoskeleton with the extracellular matrix, a process normally controlled

by the dystrophin associated proteins (DAPs), and consequently renders muscle ﬁbers more susceptible

to damage after contraction. Intensive efforts have been made to develop an effective cell therapy for

DMD [9–12]. Although myoblast transplantation can transiently deliver dystrophin and improve the

strength of injected dystrophic muscle, this approach is hindered by immune rejection, poor cellular

survival rates, and limited dissemination of the injected cells. Animal and human clinical trial results have

demonstrated that myoblast transplantation, although feasible, is rather inefﬁcient [10–12]. In animal

experiments, immunodeﬁcient animals and immunosuppressive regimens, preirradiation of the injected

muscle, or myonecrotic agents have been used extensively to improve the efﬁciency of this technique

[34–43]. Although these approaches have ameliorated the restoration of dystrophin in mdx mice, success

remains quite limited. More importantly, these attempts to improve myoblast transplantation in mdx

mice are not applicable to human DMD patients and are therefore clinically irrelevant.

Our research team has used a modiﬁed preplate technique to isolate a novel population of MDSCs that

display an improved transplantation capacity when injected into skeletal muscle [8]. These cells can be

expanded in vitro for more than 30 passages while preserving their phenotype (Sca-1[+], CD34[low/−],

c-kit[−], CD45[−]) and their ability to differentiate into various lineages both in vitro and in vivo.The

transplantation of these MDSCs, in contrast to transplantation of other myogenic cells such as satellite

cells, improved the efﬁciency of muscle regeneration and dystrophin delivery to dystrophic muscle in mice.

The number of dystrophin-positive myoﬁbers observed 90 days after injection of these MDSCs was not

signiﬁcantly different than the high number observed 10 days after transplantation, despite the fact that

the mdx mice used as recipients in this experiment were not immunosuppressed and the injected muscles

were not preirradiated or injured with a myonecrotic agent. Examination at 30 and 90 days after cell

transplantation revealed that the injection of these MDSCs resulted in ten times more dystrophin-positive

myoﬁbers in the injected muscle than did the transplantation of satellite cells, although the same number

of cells was injected in both groups. The ability of these MDSCs to proliferate in vivo for an extended

period of time — combined with their strong capacity for self-renewal, their multipotency, and their

immune-privileged behavior — reveals, at least in part, a basis for the beneﬁts associated with their use

in cell transplantation [8].

Although the above results show that allogeneic transplantation of normal MDSCs can restore dys-

trophin within dystrophic animals, autologous transplantation of MDSCs genetically engineered to

express dystrophin may be more suitable for DMD patients. Our research group currently is investig-

ating such autologous transplantation of genetically engineered MDSCs to determine the feasibility of this

technology.

20.2.2 Sports-Related Muscle Injuries

Muscle injuries, particularly pulls and strains, pose a challenging problem in traumatology and are among

the most common and most frequently disabling injuries to afﬂict athletes [44,45]. Although injured

muscles can heal, such healing occurs very slowly and often results in incomplete functional recovery

[44,45]. The regeneration initiated in injured muscle shortly after injury is inefﬁcient and hindered by

ﬁbrosis, that is, scar tissue formation [44,45]. The scar tissue that often replaces the damaged myoﬁbers is

a potential contributing factor in the tendency of strains to recur.

We have identiﬁed various growth factors with the ability to enhance muscle regeneration by increasing

myoblast proliferation and differentiation. The delivery of these growth factors via genetically engineered

muscle cells improves the regeneration of injured muscle, but ﬁbrosis still impedes full recovery [46–48].

The overexpression of transforming growth factor (TGF)-β1 in various injured tissues has been identiﬁed

as the major cause of ﬁbrosis in animals and humans [49]. We have observed that TGF-β1 plays a central

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Gene Therapy and Tissue Engineering 20-5

role in skeletal muscle ﬁbrosis [50] and, more importantly, that the use of antiﬁbrotic agents that inactivate

this molecule (e.g., decorin, suramin, γIFN, and relaxin) can reduce muscle ﬁbrosis and consequently

improve muscle healing to near-complete recovery levels [51–53].

The overall goal of our research on sports-related muscle injuries is the development of biological

approaches to improve muscle healing after common muscle injuries. Because the development of muscle

ﬁbrosis has greatly hindered our attempts to improve muscle healing by enhancing muscle regeneration,

the prevention of such ﬁbrosis has become the major emphasis of our ongoing research. Although numer-

ous studies have demonstrated the involvement of TGF-β1 in ﬁbrosis in various tissues, very few have

examined the signiﬁcance of ﬁbrosis or the role played by TGF-β1 in the scarring process in injured skeletal

muscle. As described above, our recent data show that TGF-β1 plays a central role in skeletal muscle ﬁbrosis

and that the use of antiﬁbrotic agents (e.g., decorin, suramin, γIFN, and relaxin) to inactivate this molecule

can reduce muscle ﬁbrosis and consequently improve muscle healing to near-complete recovery levels. We

believe that the ultimate approach to improving muscle healing after sports-related muscle injuries may

involve the transplantation of muscle cells genetically engineered to express antiﬁbrotic agents in order to

block scar tissue formation while promoting muscle regeneration.

20.3 Bone Healing

20.3.1 The Problem of Bone Healing

Fracture healing constitutes a fundamental problem in orthopaedic surgery. Although the majority of

fractures heal well, difﬁculty in the form of delayed healing or nonunions can be devastating. Fractures

in anatomically compromised locations (e.g., the talar neck or scaphoid), with inadequate ﬁxation or

infection, resulting from high-energy-type injuries with soft tissue stripping or segmental bone loss,

or occurring in poor bone stock (osteoporosis) often result in delayed healing. According to a recent

review, 5 to 10% of the 5.6 million fractures that occur annually in the United States exhibit delayed

or impaired healing [54]. Treatment options for the orthopaedic surgeon confronted with these com-

plex fractures, particularly fractures involving segmental bone loss, include bone autograft, vascularized

bone grafting, allograft supplemented with osteogenic proteins, bone transport, or amputation [55,56].

Unfortunately, patients generally must endure a lengthy recovery period, numerous procedures, potential

donor site morbidity, and, often, a less-than-satisfactory end result [57]. Similar difﬁculties are associated

with closure of the residual bony defects in craniofacial reconstruction and oncology or trauma surgery.

Consequently, research directed toward improving fracture treatment and the development of optimal

bone substitutes continues to garner intense interest.

20.3.2 The Use of Muscle-Derived Cells to Improve Bone Healing

We have demonstrated that a population of MDSCs isolated from mouse skeletal muscle produced alkaline

phosphatase in a dose-dependent manner after stimulation with recombinant human BMP-2 and BMP-4

[1,2,7,58,59]. After being genetically engineered to express the osteogenic proteins BMP-2 and BMP-4

and injected into skeletal muscle, these mouse muscle-derived cells induced and participated in ectopic

bone formation [1,2,7,58,59]. These MDSCs were also able to differentiate toward the osteogenic lin-

eage and consequently improve bone healing in calvarial defects in both immunodeﬁcient mice and

immunocompetent mice [1,2,7,58,59].

Although angiogenesis is integral to normal bone development, it remains largely unknown whether

bone regeneration can be further improved by the use of angiogenic factors, either alone or in combination

with BMPs — the most promising growth factorsfor inducing bone formation. To address this question, we

transduced MDSCs to express human BMP-4, vascular endothelial growth factor (VEGF), or both growth

factors. VEGF signiﬁcantly improved the efﬁcacy of BMP-4-elicited bone formation and regeneration by

enhancing angiogenesis, but VEGF alone did not improve bone regeneration. We have also character-

ized the mechanism by which VEGF accelerates the bone formation mediated by genetically engineered