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

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MDSCs expressing BMP-4. In addition to improved vasculature, we observed increased mesenchymal cell

inﬁltration and cartilage formation, decreased cell apoptosis at the injured site, earlier cartilage resorp-

tion, and a subsequent increase in mineralized bone formation within the regenerated bone that had been

treated with the combination of BMP-4 and VEGF compared to that treated with BMP-4 alone [58]. Thus

VEGF appears to enhance BMP-4-induced endochondral bone formation by inﬂuencing steps before and

after cartilage formation. Intriguingly, we have also demonstrated that the beneﬁcial effect of VEGF on the

bone healing elicited by BMP-4 requires proper dosing, with high doses of VEGF leading to detrimental

effects on bone healing [58]. These ﬁndings open a new avenue by which to improve bone repair based

on the synergistic effects of osteogenic and angiogenic factors delivered via MDSCs.

20.3.3 Can We Isolate Human Muscle-Derived Stem Cells That Display

Similar Osteogenic Behavior?

To translate this research to the clinical setting, our research group is pursuing the isolation of a similar

population of MDSCs from human skeletal muscle. We have recently evaluated whether muscle-derived

cells isolated from the skeletal muscle of humans (∼50 years of age) via the preplate technique can be

used in gene therapy and tissue engineering applications to support bone regeneration and repair. We

have explored the use of primary human muscle-derived cells (isolated via the preplate technique) for

ex vivo gene therapy to deliver BMP-2 and produce bone in vivo. Our ﬁndings indicate that certain

cells isolated from a primary cell culture taken from human skeletal muscle responded to BMP-2 by

differentiating into osteogenic cells, which suggests that these cells possessed osteocompetence [60]. Using

ex vivo gene transfer, we were able to transduce these cells to secrete BMP-2 at levels sufﬁcient to induce

radiographically detectable ectopic bone formation within skeletal muscle [60]. Histological assessment

of the injected, transduced, human muscle-derived cells suggests that the cells may have responded to the

secreted BMP-2 in an autocrine fashion by becoming osteoblasts, thereby contributing to the induction

of bone formation [60].

We also have used adenoviral and retroviral constructs to genetically engineer these freshly isolated

human skeletal muscle cells to express human BMP-2. We implanted these cells into nonhealing bone

defects (skull defects) in severe combined immunodeﬁcient (SCID) mice and monitored the closure of

the defect grossly and histologically. Mice implanted with BMP-2–producing human muscle-derived cells

displayed full closure of the defect by 4 to 8 weeks after transplantation [61]. Remodeling of the newly

formed bone was histologically evident during the 4 to 8 week period [61]. Analysis by ﬂuorescent in situ

hybridization (FISH) revealed a small fraction of the injected human muscle-derived cells within the

newly formed bone in the location where osteocytes normally reside [61]. These results indicate that

genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2,

although a small fraction of the cells seems to differentiate into osteogenic cells. We are investigating

whether this small fraction of human muscle cells is the human counterpart of the mouse MDSCs that

display osteogenic behavior.

20.3.4 Can We Regulate the Expression of Osteogenic Proteins and the

Resultant Bone Formation?

Regulated therapeutic gene expression has become increasingly important to the success of various gene

therapy applications — particularly stem cell-based ones — due to the potential long-term persistence

of genetically engineered cells within the body. To develop a system by which to regulate the expres-

sion of osteogenic proteins, we ﬁrst investigated the use of antagonist proteins to limit bone formation.

Heterotopic ossiﬁcation (HO) of muscles, tendons, and ligaments is a common problem encountered

by orthopaedic surgeons. We evaluated the ability of noggin, a BMP antagonist, to inhibit HO in vari-

ous mouse models. First, we developed a retroviral vector carrying the gene encoding human noggin

and used this vector to transduce MDSCs. MDSCs transduced with BMP-4 were implanted into both

hind limbs of mice along with 100,000, 500,000, or 1,000,000 noggin-expressing MDSCs (treated limb)

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Gene Therapy and Tissue Engineering 20-7

or with an equivalent number of nontransduced MDSCs (control limb). The mice were sacriﬁced and

radiographed 4 weeks after implantation to look for evidence of HO. In a second set of experiments,

80 mg of human demineralized bone matrix (DBM) was implanted into the hind limbs of SCID mice

along with 100,000, 500,000 or 1,000,000 noggin-expressing MDSCs (treated limb) or nontransduced

MDSCs (control limb). The mice were sacriﬁced and radiographed 8 weeks after cell implantation. In a

ﬁnal set of experiments, immunocompetent mice underwent bilateral Achilles tenotomy along with the

implantation of 1,000,000 noggin-expressing MDSCs (treated limb) or nontransduced MDSCs (control

limb). The mice were sacriﬁced and radiographed 10 weeks after surgery.

Our in vitro results showed that the MDSCs expressed noggin at a high level (280 ng/million cells/24 h)

[62]. Four weeks after implantation, we found that the BMP-4-expressing MDSCs combined with the three

varying doses of noggin-expressing MDSCs had formed 53, 74, and 99% less HO in a dose-dependent

manner (p < .05) [62]. Similarly, DBM formed 91, 99, and 99% less HO when combined with the three

varying doses of noggin-expressing MDSCs (p < .05) [62]. Additionally, all 11 animals that underwent

Achilles tenotomy developed HO at the site of injury in the control limbs, whereas the limbs treated

with the noggin-expressing MDSCs exhibited 84% less HO formation; 8 of the 11 animals exhibited no

radiographic evidence of HO formation (p < .05) [62]. These ﬁndings suggest that the delivery of noggin

mediated by MDSCs can inhibit HO in three different animal models. Gene therapy to deliver noggin

may consequently serve as a powerful method to inhibit HO and perhaps regulate the amount of bone

formation mediated by osteogenic proteins delivered by MDSCs [62].

We have also developed a retroviral vector that appears to be suitable for regulated gene therapy to

improve bone healing. To date, one of the most intensively studied inducible gene expression systems

comprises the tetracycline (tet)-controlled gene and its corresponding synthetic promoter [63]; research-

ers have used this system for either tet-on (gene expression activated by the presence of tet) or tet-off

(gene expression activated in the absence of tet) applications. Scientists have developed retroviral vectors

containing tet-on- or tet-off-controlled therapeutic genes with varying degrees of success [64]. We tried

to optimize the most promising design to date — the self-inactivating (SI) tet-on retroviral vector — to

develop a retroviral vector suitable for regulated gene therapy to improve bone healing. After extensive

investigation, we identiﬁed and optimized a retroviral vector that confers a high level of inducible trans-

gene expression (i.e., BMP-4 expression) to transduced, muscle-derived stem cells (MDSCs) [65]. This

novel retroviral vector also enables us to regulate the amount of bone formation elicited by the transduced

cells after transplantation [65]. We believe that the ﬁndings generated by this study could be translated to

a wide variety of other gene-therapy applications that require stringent regulation of a transgene’s thera-

peutic effect, such as the codelivery of inducible VEGF and its speciﬁc antagonist to achieve regulated

angiogenesis.

20.3.5 Is the Bone Regenerated by BMP-Producing Muscle-Derived Cells

Biomechanically Relevant?

We have evaluated the ability of muscle-derived cells transduced with retroBMP4 to heal a long bone defect

both structurally and functionally. Primary muscle-derived rat cells were genetically engineered to express

BMP-4 and implanted into 7-mm rat femoral defects; muscle-derived cells transduced with retroLacZ were

used as the control. Bone healing was monitored via radiography, histology, and biomechanical testing.

Our results indicate that 78.6% of the defects treated with muscle-derived cells expressing BMP-4 formed

bridging callous by 6 weeks after surgery, and 12 of the 13 femora exhibited radiographically evident union

12 weeks after implantation. Histological analysis at the 12-week time point revealed that the medullary

canal of the femur was restored and the cortex was remodeled between the proximal and distal ends of

each BMP-4-treated defect. In contrast, nonunions were observed at all tested time points in the defects

treated with muscle-derived cells expressing β-galactosidase (a marker gene). The maximum torque-to-

failure in the treatment group (BMP-4–producing cells) indicated up to 73% strength of the contralateral

intact femur, while the torsional stiffness and energy-to-failure were not signiﬁcantly different between

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the treated and the intact limbs. This study demonstrates that retroBMP4-transduced muscle-derived cells

can elicit both structural and functional healing of critical sized segmental defects in rat femora [66].

20.4 Articular Disorders

Various articular disorders, including arthritis, chondral and osteochondral defects, meniscal tears, and

damaged ligaments, are potential candidates for novel treatment using gene therapy and tissue engineering

applications.

20.4.1 Articular Cartilage

Articular cartilage defects and progressive osteoarthritis are among the most frequent and challenging

conditions encountered in orthopaedics. In contrast to bone, articular cartilage heals poorly. Current

repair techniques include cartilage debridement and resurfacing [67,68], subchondral drilling [69], arth-

roscopic abrasion, and microfracture. Common to these techniques is the disruption of subchondral

bone to allow osteochondral progenitors from the marrow to gain access to and participate in repair of the

cartilage defect. The repaired cartilage, however, is structurally inferior to native cartilage and contains

signiﬁcantly less proteoglycan [70]. Newer strategies for cartilage repair seek to provide an alternate source

of cells to stimulate healing. Strategies have included the use of autologous chondrocyte transplantation

[71] and transplantation of cartilage plugs [72]. Although adult or embryonic chondrocytes may be used

for transplantation, the limiting factor is chondrocyte supply. Other researchers have tried to overcome

this limitation by generating chondrocytes from mesenchymal stem cells [73]. These cells then are induced

to become osteochondral progenitor cells in culture before reimplantation.

Partly because muscle-derived cells are easily accessible, we have explored their capacity for repair-

ing articular defects. The ability of muscle-derived cells to promote cartilage formation/restoration and

bone formation through an endochondral pathway (see earlier sections) further justiﬁes the evaluation

of their potential use for articular cartilage repair. For these reasons, we have investigated the utilization

of transplanted allogeneic muscle-derived cells embedded in collagen gels for the repair of full thickness

articular cartilage defects [74]. The results were compared to chondrocyte transplantation and control

(nontreated) groups. Although grafted cells were found in the defects only up to 4 weeks after transplanta-

tion, histological assessment at 12 and 24 weeks after transplantation revealed that the tissue repaired with

muscle-derived cells or chondrocytes displayed comparably higher levels of healing than observed in the

control groups [74]. Notably, at 24 weeks the repaired tissues in the muscle-derived cell and chondrocyte

groups were composed primarily of type II collagen, indicating the presence of hyaline articular cartilage

[74]. These results demonstrated that allogeneic muscle-derived cells could be used as both gene-delivery

vehicles and a cell source to promote full-thickness cartilage repair. The implantation of muscle-derived

cells appeared to improve the healing of the defects with an efﬁciency equivalent to that of chondrocyte

cartilage transplantation. Muscle-derived cell transplantation is a particularly attractive option given the

fact that the muscle biopsy required to obtain muscle cells is less invasive than the arthroscopy required

to obtain chondrocytes [74].

20.4.2 Meniscus

Allograft meniscal transplantation is one of the few available treatment options after menisectomy. Despite

acceptable early results, considerable controversy exists due to the subsequent poor graft regeneration,

shrinkage, and biomechanical failure of transplanted menisci. Meniscal injuries are promising candidates

for treatment via gene therapy and tissue engineering applications. The possible in vitro creation of a cus-

tom, replacement meniscus by using scaffolds, cells, and gene therapy for subsequent in vivo implantation

is intriguing. Alternatively, gene therapy could be used to genetically engineer meniscal cells in order to

promote the healing of certain injuries. Meniscal cells are amenable to gene transfer of both marker genes

and various growth factor genes via either direct or ex vivo gene therapy, with gene expression persisting

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Gene Therapy and Tissue Engineering 20-9

for up to 6 weeks [75]. Successful ex vivo gene transfer to the meniscus has been accomplished using either

muscle-derived cells [20] or meniscal cells [75]. Identiﬁcation of the growth factors that best promote

meniscal healing, techniques to improve long-term gene expression, and the optimal scaffold required to

create de novo menisci is the focus of ongoing research.

We have recently investigated the feasibility of using gene transfer in meniscal allografts in rabbits to

eventually deliver speciﬁc growth factor genes that may improve the regeneration of meniscal allograft

[76]. Four different viral vectors encoding marker genes, including LacZ, luciferase, and green ﬂuor-

escence protein, were used to investigate viral transduction in 50 lapine menisci for 4 weeks in vitro.

Subsequently, 16 unilateral meniscus replacements were performed with ex vivo retrovirally transduced

meniscal allografts; the expression of the LacZ gene was examined histologically at 2, 4, 6, and 8 weeks after

transplantation. Gene expression in the superﬁcial cell layers of the menisci was detected for up to 4 weeks

in vitro, but the level of gene transfer declined over time [76]. The retrovirally transduced cells displayed

more persistent transgene expression and penetrated the menisci more deeply after implantation [76].

In vivo, declining numbers of β-galactosidase-positive cells were detected in the retrovirally transduced

allografts for up to 8 weeks. Transduced cells consistently were found at the meniscosynovial junction of

the transplants and in deeper layers of the menisci. There was no evidence of cellular immune response

in the transduced transplants. This investigation demonstrates the promise of growth factor delivery in

auto- and allografts. We will conduct additional experiments to assess the potential of vectors expressing

therapeutic proteins (e.g., growth factors) to improve remodeling and healing of meniscal allografts [76].

20.4.3 Ligaments

Researchers also are investigating the use of gene-therapy techniques for treatment of injured ligaments.

LacZ gene transfer to ligaments via either direct or ex vivo adenoviral or retroviral approaches has proven

feasible in animal models. Ex vivo gene transfer to ligaments has been achieved using either ligament

ﬁbroblasts or skeletal muscle-derived cells [21]. Many growth factors, such as basic ﬁbroblast growth factor

(bFGF), platelet-derived growth factor (PDGF), VEGF, insulin-like growth factor (IGF)-1 and -2, TGF-β,

and BMP-12, are believed to possibly play roles in ligament healing. Data suggest that PDGF stimulates

cell division and migration, whereas TGF-β and the IGFs promote extracellular matrix synthesis [77]. Use

of a viral-liposome conjugate vector for the direct gene transfer of PDGF-β into rat patellar ligaments was

found to initially improve angiogenesis and subsequently enhance extracellular matrix synthesis [78].

The integration of tendon grafts typically used for anterior cruciate ligament replacement continues

to be unsatisfactory and may be associated with postoperative anterior–posterior laxity. We have evalu-

ated whether BMP-2 gene transfer can improve the integration of semitendinosus tendon grafts at the

tendon–bone interface after anterior cruciate ligament reconstruction in rabbits [79]. Anterior cruciate

ligament reconstruction using autologous double-bundle semitendinosus tendon grafts was performed in

46 adult rabbits. The semitendinosus tendon grafts were genetically engineered in vitro with adenovirus–

luciferase, adenovirus–LacZ (AdLacZ), or adenovirus–BMP-2 (AdBMP-2); untreated grafts served as

controls. The anterior cruciate ligament grafts were examined histologically 2, 4, 6, and 8 weeks after

surgery. In an additional series of experiments, the structural properties of the femur–anterior cruci-

ate ligament graft–tibia complexes were investigated in 10 untreated controls and 10 specimens with

AdBMP-2-transduced semitendinosus tendon grafts. The animals were sacriﬁced 8 weeks after anterior

cruciate ligament surgery, and the femur–anterior cruciate ligament graft–tibia complexes were tested

under uniaxial tension. The stiffness (N/mm) and ultimate load at failure (N) were determined from

load-elongation curves.

Our results indicate that genetically engineered semitendinosus tendon grafts expressed reporter genes

and BMP-2 in vitro [79]. The AdLacZ-infected anterior cruciate ligament grafts showed two different

histological patterns of transduction. Intra-articular, infected cells were mostly aligned along the surface

and decreased in number during the 6 weeks after surgery. In the intra-tunnel portions of the anterior

cruciate ligament grafts, the number of infected cells did not decrease during the observation period.

Moreover, we observed a high number of transduced cells in the deeper layers of the tendons. In the

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control group, granulation-type tissue at the tendon–bone interface showed progressive reorganization

into a dense connective tissue, and subsequent establishment of ﬁbers resembling Sharpey’s ﬁbers.

In the specimens receiving AdBMP-2-infected anterior cruciate ligament grafts, a broad zone of newly

formed matrix resembling chondro-osteoid was observed at the tendon–bone interface 4 weeks after

surgery. This area had increased by 6 weeks after surgery, showing a transition from bone to mineralized

and nonmineralized cartilage. In addition, in the BMP-2-treated specimens, the tendon–bone interface

in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness

(29.0 ± 7.1 vs. 16.7 ± 8.3 N/mm) and the ultimate load at failure (108.8 ± 50.8 vs. 45.0 ± 18.0 N) were

signiﬁcantly enhanced in the specimens with BMP-2-transduced anterior cruciate ligament grafts when

compared with controls. This study demonstrated that BMP-2 gene transfer signiﬁcantly improves the

integration of semitendinosus tendon grafts in the tunnels after anterior cruciate ligament reconstruction

in rabbits [79]. Before introducing gene transfer as a therapeutic method in orthopaedics, however,

researchers must answer questions regarding safety and regulatory issues.

20.5 Summary

This paper summarizes the current knowledge and most recent achievements in gene therapy and tis-

sue engineering for the musculoskeletal system; many of these advances are attributable to or strongly

inﬂuenced by the research ﬁndings of our laboratory. Although this review focuses on a population of

muscle-derived stem cells isolated by our research team, we are not excluding the possible use of altern-

ative cell populations, including stem cells from various sources (e.g., hematopoietic cells, mesenchymal

stem cells, or fat-derived cells) for future applications. Bringing tissue engineering technology to clinical

fruition, however, will require multidisciplinary collaboration to identify and optimize the appropriate

cell type, growth factor, and scaffold construct for each application. Future investigations should elaborate

upon the utility of stem cells derived from various adult tissues in autologous, allogeneic, or perhaps xeno-

geneic settings. Details regarding these cells’ proliferative potential, susceptibility to genetic engineering,

and immunogenic potential remain to be characterized. Furthermore, researchers must learn to combine

stem cells with growth factors in a viable spatiotemporal relationship in order to re-create the in situ

microenvironment. Much work remains before the regeneration of tissue for healing the musculoskeletal

system—acomplicated endeavor with vast potential applicability — becomes a clinical reality.

Acknowledgments

The authors wish to thank Marcelle Pellerin, James Cummins, and Brian Gearhart (Children’s Hospital of

Pittsburgh, Pittsburgh, PA) for technical support and Ryan Sauder (University of Pittsburgh, Pittsburgh,

PA) for editorial assistance with the manuscript. This work was supported by grants to Dr. Johnny Huard

from the Muscular Dystrophy Association (USA), the National Institutes of Health (NIH P01 AR 45925-

01, R01 AR 49684-01, R01 AR 47973-01, R01 DE 13420-01A2), the Orris C. Hirtzel and Beatrice Dewey

Hirtzel Memorial Foundation and the William F. and Jean W. Donaldson Chair at Children’s Hospital of

Pittsburgh, and the Henry J. Mankin Endowed Chair at the University of Pittsburgh.

The authors also want to thank the clinical fellows Chan Y.S., Fukushima K., Shen H.C., Adachi N., and

Martinek V. for their contribution to this work.

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Tissue Engineering

Applications — Bone

Ayse B. Celil

Scott Guelcher

Jeffrey O. Hollinger

Carnegie Mellon University

Michael Miller

University of Texas

21.1 Biology of the Bone ...................................... 21-1

21.2 Signaling Molecules for Bone ........................... 21-4

Growth Factors • Stem Cells and Gene Therapy

21.3 The Ideal Scaffold ........................................ 21-8

21.4 Clinical Reconstruction of Bone Defects ............... 21-9

21.5 Conclusion ............................................... 21-12

References ....................................................... 21-14

21.1 Biology of the Bone

Bone is a complex living tissue that provides internal support for all higher vertebrates. It develops by

osteogenesis, the process of ossiﬁcation, starting out as a highly specialized form of connective tissue.

Two major players in the formation of bone are the bone cells called osteoblasts (bone-forming) and

the osteoclasts (bone-resorbing). During the process of ossiﬁcation, osteoblasts secrete type I collagen,

in addition to many noncollagenous proteins such as osteocalcin, bone sialoprotein, and osteopontin.

Osteoblast-secreted extracellular matrix may initially be amorphous and noncrystalline, but it gradually

transforms into more crystalline forms [1]. Mineralization is a process of bone formation promoted by

osteoblasts and is thought to be initiated by the matrix vesicles that bud from the plasma membrane [2]

of osteoblasts to create an environment for the concentration of calcium and phosphate, allowing crys-

tallization. Collagen serves as a template and may also initiate and propagate mineralization independent

of the matrix vesicles [3,4]. Eventually, some osteoblasts are surrounded by the bone matrix that they

help to form and are called osteocytes. Despite their location, osteocytes are not metabolically inactive;

they dissolve and resorb some bone mineral though osteolysis [5]. Bone resorption is in fact the primary

function of another bone cell, the osteoclast, which can also digest calciﬁed cartilage and is then called the

chondroclast. Formation by the osteoblasts and resorption by the osteoclasts maintains bone in constant

renewal as a dynamic tissue.

Bone formation in the developing embryo occurs by two developmental processes: intramembranous

and endochondral ossiﬁcation. Intramembranous ossiﬁcation is the direct differentiation of osteoblasts

from mesenchymal cells. Several craniofacial bones and parts of the mandible and clavicle develop by

intramembranous ossiﬁcation [6]. Most other bones are formed by endochondral ossiﬁcation. In this

process of bone formation, mesenchymal cells condense and differentiate into cartilage. The cartilage

anlage matures, undergoes hypertrophy, mineralizes, and subsequently becomes invaded by blood vessels

as well as osteoprogenitor cells [7,8].

21-1