The appropriate viral vector for a given gene therapy application depends on the duration of gene expression desired, the cell type to be transduced, the immune pro-tected or unpropro-te
Trang 1Advances in molecular biology, cell
biology, and polymer chemistry are
providing novel approaches for
treating musculoskeletal disorders
These advances are facilitating
bio-logic approaches that complement
and could even replace traditional
biomechanical techniques in
ortho-paedic surgery The explosion of
research has resulted in a myriad
of potential applications for these
innovative biologic approaches
Many probably will never reach
clinical fruition; however, effective
implementation of only a few may
revolutionize the way we manage
certain musculoskeletal disorders
Clearly, orthopaedic surgeons must
have a fundamental understanding
of the terminology (Table 1) and
techniques of these biologic
ad-vances, and of current research, to
be able to interpret the literature,
direct future investigations, and
best serve their patients
Tissue Engineering
Tissue engineering is the science of creating living tissue to replace, repair, or augment diseased tissue.1 Tissue engineering thus refers to a broad variety of techniques The engineered tissue is created either entirely in vivo or, in an ex vivo technique, is created in vitro and subsequently implanted into the patient Regardless of the tech-nique, however, tissue engineering requires three components: a growth-inducing stimulus (growth factor), responsive cells, and a scaffold to support tissue formation
Growth Factors and Gene Therapy
Cytokines are small, soluble pro-teins that influence cell behavior
A subset of cytokines, known as growth factors, promotes cell mito-sis The term growth factor,
how-ever, often is used generically in reference to any number of cyto-kines that promote cell division, maturation, and/or differentiation Recent advances in molecular biology have facilitated the genetic sequencing of various cytokines Consequently, the genetic sequences can be inserted into cells of labora-tory animals, producing large quan-tities of “recombinant” human cyto-kines A particular cytokine often has multiple, diverse effects, and these can vary depending on the different cell types
Determining the influence and pharmacokinetics of various cyto-kines is an area of active, ongoing investigation In vitro research typ-ically is performed to identify the optimal cytokine for a particular
Dr Musgrave is Chief Resident, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa Dr Fu is David Silver Professor and Chairman, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh Dr Huard is Associate Professor, Departments of Orthopaedic Surgery, Molecular Genetics and Biochemistry, and Bioengineering, University of Pittsburgh, Pittsburgh.
Reprint requests: Dr Huard, Room 4151, Rangos Research Center, 3705 Fifth Avenue, Pittsburgh, PA 15213-2583.
Copyright 2002 by the American Academy of Orthopaedic Surgeons.
Abstract
A new biologic era of orthopaedic surgery has been initiated by basic scientific
advances that have resulted in the development of gene therapy and tissue
engi-neering approaches for treating musculoskeletal disorders The terminology,
fundamental concepts, and current research in this burgeoning field must be
understood by practicing orthopaedic surgeons Different gene therapy
approaches, multiple gene vectors, a multitude of cytokines, a growing list of
potential scaffolds, and putative stem cells are being studied Gene therapy and
tissue engineering applications for bone healing, articular disorders,
interverte-bral disk pathology, and skeletal muscle injuries are being explored Innovative
methodologies that ensure patient safety can potentially lead to many new
treat-ment strategies for musculoskeletal conditions.
J Am Acad Orthop Surg 2002;10:6-15
Gene Therapy and Tissue Engineering
in Orthopaedic Surgery
Douglas S Musgrave, MD, Freddie H Fu, MD, and Johnny Huard, PhD
Trang 2application The optimal method
by which to deliver the cytokine in
vivo also must be determined The
simplest method is to inject or
im-plant the recombinant protein
di-rectly into the anatomic region of
interest However, the inherently
short half-lives of recombinant
pro-teins and the dilutive effects of
body fluids limit this technique
Consequently, the injected
cyto-kines may not be present in
ade-quate concentrations and at the
critical time periods to effect a
maximal response Therefore,
in-vestigators have devised two basic approaches to deliver temporary, sustained quantities of a desired protein
The first method is to impreg-nate the cytokine onto a polymer scaffold Such a method requires a thorough understanding of the chemical interactions between the protein and the scaffold, the bind-ing characteristics of the protein to the scaffold, and the pharmacoki-netics of both the protein and the scaffold The second method is to deliver the gene encoding for the
protein into the patient, resulting in
in vivo cytokine production When the gene is delivered only to a spe-cific region of the body and not systemically, the method is termed regional gene therapy
Gene therapy is simply the transfer of a particular gene into a cell so that the cell transcribes the gene into messenger ribonucleic acid (mRNA); the cell’s ribosomes then translate the mRNA into a protein (cytokine) To accomplish this, the particular gene must be packaged and inserted into the
Table 1
Common Terms Used in Gene Therapy
Term Definition
cDNA Complementary DNA DNA created from a protein’s mRNA by enzyme reverse transcriptase
Contains only exons
mRNA RNA that is produced by transcription of DNA This genetic material leaves the nucleus, moves to the
cytoplasm, and is translated by ribosomes into a protein
RNA A string of ribonucleotides that is similar in structure to DNA There are several classes of RNA,
including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)
Nucleotide Building block of DNA and RNA comprising a base, a deoxyribose or ribose sugar, and a phosphate Exon A piece of DNA encoding a gene that is transcribed into mRNA and then translated into a protein
(that is, a nucleotide sequence of a gene that encodes for a specific protein)
Intron A piece of DNA of unknown function that is interspersed between exons (that is, a noncoding
nucleotide sequence of a gene)
Transcription The process of conversion of DNA into RNA (that is, creation of an mRNA from DNA)
Transduction Gene transfer using viral vectors
Transfection Gene transfer using nonviral vectors
Translation The process of conversion of mRNA into an amino acid sequence (that is, creation of a protein from
mRNA by ribosomes)
Plasmid A self-replicating piece of DNA naturally found in bacteria and yeast Plasmids are commonly used to
carry foreign genes into cells for the production of recombinant DNA pharmaceuticals and in gene expression studies
Liposome A gene-delivery system based on the production of lipid bodies that contain pieces of DNA These
lipid-DNA complexes fuse with the cell surface and are internalized by phagocytosis, depositing the encapsulated DNA into the target cells
Promoter A regulatory element in DNA that functions to induce transcription of a gene
Cytokines Small proteins that influence cell behavior
Ex vivo/In vitro
gene transfer Genetic manipulation of cells outside the host (body)
In vivo gene
transfer Genetic manipulation of cells within the host (body)
Vector A disabled virus or DNA structure used as a vehicle to transfer genes into cells
Trang 3is reverse-transcribed in the
labora-tory by the enzyme reverse
trans-criptase to create a complementary
deoxyribonucleic acid (cDNA)
Because the cDNA is created
di-rectly from mRNA, the noncoding
nucleotide sequences (introns) of
the gene are not present, leaving
only the protein-coding nucleotide
sequences (exons) This fact
distin-guishes a protein’s cDNA from its
DNA sequence found naturally,
which includes both introns and
exons
The cDNA is inserted into a
cir-cular plasmid with various non–
amino acid-coding sequences at its
leading and trailing ends (Fig 1, A)
A plasmid is a self-replicating,
cir-cular piece of DNA that is used to
transfer specific genes into cells
The cDNA plasmid is then digested
with restriction enzymes and
in-serted into a larger plasmid
contain-ing a promoter sequence (among
other sequences), thereby creating
an expression plasmid (Fig 1, B)
The promoter sequence induces
transcription of the cDNA once
gene transfer into a cell has
oc-curred Frequently, the promoter is
constitutive, meaning it is always
“on” and the gene will be
tran-scribed continuously until the gene
is lost The cytomegalovirus
pro-moter is a commonly used
consti-tutive promoter Investigators are
currently developing regulated
pro-moters, which can be turned “on”
or “off” with an exogenous stimulus
(i.e., a drug) Regulated promoters
are obviously desirable so that gene
expression may occur only at the
desired, critical time periods Once
a functional expression plasmid
has been created, one must
deter-mine the optimal vector with which
to transport the expression plasmid
safely and efficiently into the
de-sired cells
Vectors are vehicles that
facili-tate transfer of a particular gene
into cells Vectors are subdivided
into viral and nonviral vectors The most commonly used nonviral vec-tors are liposomes Liposomes are phospholipid vesicles capable of fusing with a cell membrane,
there-by delivering their contents (ex-pression plasmid) into the cell
Liposomes are relatively cheap and nonpathogenic, but their use results
in inferior gene-transfer efficiency compared with viral vectors Other nonviral methods include the gene gun (DNA loaded onto gold beads injected into the cell using a helium
“gun”), DNA conjugates (DNA conjugated to certain polycations that improve DNA adherence to cell membranes, thereby improving gene transfer), gene-activated matrices (matrices, or scaffolds, im-pregnated with expression plas-mids), and nonviral-viral hybrids (viral DNA sequences that facilitate longer gene expression or integra-tion into host cell DNA spliced into
an expression plasmid)
Successful gene transfer using nonviral vectors is termed trans-fection Alternatively, successful
gene transfer using viral vectors is termed transduction Using current methods, the efficiency of nonviral transfection is generally inferior to that of viral-mediated transduction Therefore, despite the nonpatho-genic nature and economic advan-tages of nonviral vectors, most cur-rent gene therapy applications use viral vectors
Viruses are highly efficient at infecting cells to deliver genetic material Various viruses have dif-ferent advantages and disadvan-tages when applied to gene therapy (Table 2) The most commonly used viruses in current gene therapy ap-plications include adenoviruses, retroviruses, herpes simplex viruses (HSVs), and adeno-associated viruses (AAVs) Certain viral DNA se-quences are often removed to ren-der the viral replication defective before gene therapy applications Adenoviruses have the advan-tage of infecting both dividing and some nondividing cells, thereby achieving a high level of transient gene expression The adenovirus
G E
N E
restriction sites
polyadenylation
restriction sites
restriction sites
cDNA
restriction sites
A A
G E NE
P R O M
OT ER
A M
P
N E O
EXPRESSION PLASMID
Figure 1 A, Complementary DNA (cDNA) consisting of desired gene and restriction enzyme sites in circular form B, An expression plasmid is created by inserting the desired
gene into plasmid containing other sequences A promoter initiates transcription of the gene, and the polyadenylation sequence helps stabilize the mRNA Ampicillin (AMP) and neomycin (NEO) resistance genes also may be present to allow in vitro selection of trans-duced or transfected cells.
Trang 4genome exists as an episome within
the cell’s nucleus, not being
inte-grated into the cell’s genome
Low-level production of adenoviral
anti-gens often results in an immune
response to infected cells, resulting
in loss of gene expression after
sev-eral weeks Research is ongoing to
develop “gutted” adenoviral
vec-tors, whereby the majority of viral
protein expression is eliminated to
minimize the immune response to
adenoviral antigens
Retroviruses are small RNA
viruses that, once inside the cell,
have their RNA transcribed by the
enzyme reverse transcriptase into
stranded DNA The
double-stranded DNA enters the cell
nu-cleus and is integrated, although at a
random site, into the cell’s genome
Consequently, the therapeutic gene
is expressed for the life of the cell
The disadvantages of retroviral
vec-tors are that they infect and
trans-duce only actively dividing cells
and that their DNA is randomly
integrated into the host cell genome
This random integration
theoreti-cally can alter oncogene expression
and adversely affect cell behavior
HSV is capable of infecting both
dividing and nondividing cells,
car-rying large amounts of DNA,
infect-ing many different cell types, and establishing latency in neuronal cells First-generation HSV vectors demonstrated toxicity in certain cell types and expressed significant anti-genicity, resulting in only transient gene transfer in certain cell types
Next-generation HSV vectors thus are being engineered to minimize these drawbacks
AAV has the advantages of infecting nondividing cells and of stable integration of its DNA into a specific site on chromosome 19, which appears to be nonpatho-genic.2 Furthermore, AAV is not known to cause any disease.3 The main disadvantage of AAV is its capacity to accommodate only small amounts of exogenous DNA, on the order of 5.2 kilobase (kb) pairs
The appropriate viral vector for
a given gene therapy application depends on the duration of gene expression desired, the cell type to
be transduced, the immune pro-tected or unpropro-tected environment
in which the cells reside, and the method chosen to ensure patient safety (i.e., in vitro and/or in vivo safety testing)
As mentioned, two fundamental approaches to gene therapy exist: in vivo (direct) and ex vivo (outside
the host) (Fig 2) The in vivo ap-proach consists of directly injecting
or implanting the gene-carrying vector into the patient This ap-proach is attractive for its technical simplicity although it is limited by the inability to perform in vitro safety testing on the transduced cells The alternative ex vivo approach consists of isolating cells in vitro from a tissue biopsy taken from the patient The cells are expanded and then transfected or transduced in the laboratory The genetically altered cells can be tested in vitro for both successful gene transfer and abnormal behavior before they are reimplanted in the patient After in vitro testing, the cells are introduced into the patient so that they may produce the genetically specified protein The ability to test for successful gene transfer and abnormal cellular behavior before cell implantation is an advantage of the ex vivo approach However, the
ex vivo method is technically more complex than the in vivo approach
Mesenchymal Stem Cells
Growth factors are capable of acting on many different cell types However, mesenchymal stem cells (MSCs) are the ideal cell type for
Table 2
Common Viral Vectors
Adenovirus Infects dividing and nondividing cells Viral antigens can induce immune response
Infects many different cell types DNA remains episomal; does not integrate Straightforward vector production
Retrovirus DNA integrates into cell’s genome Random DNA integration
Accomodates 8 kb Infects only dividing cells Straightforward vector production
Herpes simplex virus Infects dividing and nondividing cells Toxicity
Accomodates >35 kb Transient expression in certain cell types Adeno-associated virus Infects nondividing cells Accomodates only 5.2 kb
Nonpathogenic Difficult vector production Infects many different cell types
Trang 5therapy approaches MSCs are
rest-ing stem cells capable of
differenti-ation into multiple connective
tis-sue lineages (such as muscle, bone,
ligament, tendon, and cartilage).4,5
MSCs are not to be confused with
hematopoietic stem cells, which are
obtained from fetal umbilical cord
blood and are capable of
differenti-ation into multiple hematopoietic
lineages (such as megakaryocytes,
erythrocytes, lymphocytes).6
The existence of MSCs facilitates
seemingly endless tissue engineering
possibilities Theoretically, an MSC
could be stimulated to undergo
dif-ferentiation down a desired lineage,
thereby recreating a certain tissue for
therapeutic use Isolation of fully
differentiated cells (i.e.,
chondro-cytes) for a certain tissue engineering
application can be hindered by
donor site morbidity and limited cell
availability The use of MSCs would
obviate the need to obtain fully
dif-ferentiated cells for a specific tissue
engineering application
MSCs are thought to reside in the
bone marrow and thus are obtained
by bone marrow biopsy Resting
osteoprogenitor stem cells reside in
the bone marrow stroma, and bone
marrow–derived MSCs have been
used in animal studies to heal
carti-lage defects7as well as produce
cel-lular elements that could modify the
clinical course of muscle diseases.8
Additionally, the feasibility of using
MSCs in gene therapy has been
de-monstrated in animal models.9
Furthermore, the effective human
clinical use of MSCs to treat
neo-nates with osteogenesis imperfecta
has been reported.10,11 Clinical
im-provement was demonstrated in
neonates with osteogenesis
imper-fecta who received allogeneic bone
marrow transplantation aimed at
replacing their abnormal MSCs
MSCs also can reside outside the
bone marrow: evidence supports
the existence of resting stem cells in
skeletal muscle that are capable of
either myogenic or osteogenic dif-ferentiation.12-14 The availability of skeletal muscle and the relative ease
of cell isolation make skeletal mus-cle an attractive source of potential stem cells The relationship of these muscle-derived stem cells and MSCs still is unclear and remains to be in-vestigated fully
In addition, stem cells possibly may reside in other tissues, such as skin, brain, kidney, or perivascular tissue Further research must be conducted to elucidate fully the existence and residence of stem cells and to refine efficient isolation tech-niques
Scaffolds
A scaffold to support tissue growth is the final component of any tissue engineering approach
This scaffold can take the form of
in situ host tissue (e.g., meniscus), transplanted host tissue (e.g., skele-tal muscle flap), naturally derived polymers (such as collagen and hyaluronic acid), synthetic
poly-mers (such as poly[L-lactic acid] [PLLA], polyglycolic acid [PGA], and poly[DL-lactic-co-glycolic acid] [PLGA]), or injectable polymers that cross-link in situ (such as algi-nate and polyethylene oxide [PEO]) (Table 3) Scaffolds influence cell recruitment, cell containment, cell adherence, diffusion of nutrients to the cells, delivery of growth fac-tors, and cell behavior An ideal scaffold provides for uniform dis-tribution of cells throughout its three-dimensional lattice, facilitates efficient diffusion of biochemical molecules, and undergoes degrada-tion at the same rate that it can be replaced by host tissue The latter characteristic dictates the species-specific suitability of a scaffold Certain scaffolds can be impreg-nated with expression vectors,
there-by creating gene-activated matrices (GAMs) In addition to fulfilling the previously mentioned scaffold char-acteristics, GAMs can provide for nonviral direct gene transfer, thereby avoiding the immunologic risks of
Figure 2 Two basic approaches of gene therapy exist Ex vivo gene therapy consists of
cell harvest and expansion in culture, in vitro transduction or transfection of the isolated cells using the appropriate vectors, and reimplantation of the transduced cells into the patient In vivo gene therapy consists of direct injection of the vectors into the patient.
Cell harvest and culture
In vitro transduction
Virus
Reimplantation
of transduced cells
Direct injection
of vector
Trang 6viral vectors Host-tissue scaffolds
are attractive because they obviate
the need for a foreign body, may
contain responsive cells, and
poten-tially can be vascularized However,
donor-site morbidity and lack of
available tissue are prohibitive
fac-tors Naturally derived polymers are
attractive because they may be
remodeled by host cells to provide
space for growing tissue Collagen
menisci constructed from bovine
Achilles tendon type I collagen were
implanted into eight patients, with
encouraging results at second-look
arthroscopy and 2-year clinical
follow-up.15 The drawbacks of
natu-rally derived polymers are ensuring
pathogen removal and potential
lim-ited supply
Synthetic polymers, in contrast,
can be mass-produced and
de-signed specifically for a particular
application Their molecular
com-position can be modified to affect
scaffold-cell interactions and
scaf-fold degradation The presence of a
foreign body, however, may result
in an inflammatory reaction, to the
detriment of tissue growth, and
increase the risk of infection
Syn-thetic polymers have been used to
create composite phalanges by
seeding bovine chondrocytes and
tenocytes onto sheets of PGA and
suturing these structures to bovine
periosteum wrapped around cova-lently linked PLLA and PGA.16 The composite structures were subcuta-neously implanted into athymic mice to facilitate growth No gene transfer was used After 20 weeks, the composite tissue resembled the gross and histologic appearance of human phalanges
The science of polymer chemistry governing the porosity, cell- and growth factor–binding characteris-tics, and degradation kinematics of synthetic polymers is beyond the scope of this article It must be noted, however, that, given each scaffold’s inherent advantages and disadvan-tages, the scaffold for a given tissue engineering application must be cho-sen carefully
Specific Tissues Bone
The existence of bone-inducing growth factors has long been rec-ognized.17 These bone-inducing growth factors, termed bone mor-phogenetic proteins (BMPs), are members of the transforming growth factor-β(TGF-β) superfamily Recom-binant human BMP-2 (rhBMP-2)18 has been implanted directly on var-ious carriers and can heal critically sized bone defects in animal
mod-els.19 Likewise, other BMPs, such
as BMP-320and BMP-7 (osteogenic protein-1),21have proved to be effica-cious in animal models To achieve sustained BMP delivery, investiga-tors have developed both in vivo and ex vivo gene therapy approaches using BMPs Direct adenovirus-mediated approaches delivering BMP-222,23 and BMP-9,24 as well as
a direct GAM-mediated approach delivering BMP-4,25 have been reported Ex vivo approaches to deliver BMP-2 have used rodent bone marrow stromal cell lines,26,27 primary rodent bone marrow stro-mal cells,28,29primary rodent skele-tal muscle–derived cells,12,29primary human skeletal muscle–derived cells,30primary rabbit articular chon-drocytes,29 primary rabbit perios-teal cells (BMP-7),31 and primary rabbit skin fibroblasts.29 For clin-ical applications, primary autolo-gous cells (cells isolated from the host) are preferable to cell culture lines, which are allogeneic or xeno-geneic and may have tumorigenic and immunogenic potential The direct, in vivo gene therapy approach using BMPs has been ap-plied in rodent spine fusion mod-els,22,24 whereas the ex vivo ap-proach has been applied to healing critically sized rodent bone de-fects.13,26,28 In the spine fusion
Table 3
Categories of Scaffolds
Host tissue Responsive cells Donor site morbidity
(e.g., meniscus, skeletal muscle flap) Viability and potential vascularity Limitation on amount of tissue
No foreign body Naturally derived polymers Remodeling potential Pathogen removal must be assured (e.g., collagen, hyaluronic acid) No donor site morbidity Potential limited supply
Synthetic polymers Mass production Foreign body (infection risk)
(e.g., PLLA, PGA, PLGA) Custom designed Possible inflammatory reaction Injectable synthetic polymers Mass production Foreign body (infection risk)
(e.g., alginate, PEO) Can fill complex spaces In vivo chemical reaction
Trang 7directly injected into the paraspinal
musculature,22,24resulting in
para-spinal bone formation present at 3
weeks after injection In the ex vivo
approaches, the transduced cells
were seeded onto collagen sponges13
or demineralized bone matrix26,28as
scaffolds, resulting in improved
radiographic healing of both
calvar-ial13and long bone defects.26,28
A novel osteoinductive protein
termed LIM mineralization
protein-1 (LMP-protein-1) has been used in an ex
vivo gene therapy model for spine
fusions.32 Because LMP-1 is an
intracellular signaling molecule
(regulated by BMP-6), the use of
LMP-1 requires gene transfer Bone
marrow stromal cells transfected
with LMP-1, seeded onto a scaffold
of nonosteogenic devitalized bone
matrix, and implanted into the
paraspinal area of rats resulted in a
100% rate of spine fusion compared
with no fusion in the controls.32
Which cell type to use in ex vivo
gene transfer of osteogenic proteins
remains unresolved The ideal cell
type is expendable, easily isolated
from the patient, and amenable to
efficient transduction and protein
secretion; possesses
osteocompe-tence17; and has favorable survival
characteristics when reimplanted
into the patient Bone marrow
stro-mal cells26,28,29 and skeletal
mus-cle–derived cells12-14,29both possess
osteocompetence and have been
used successfully in ex vivo
osteo-genic protein gene transfer
Fi-nally, as mentioned previously, the
development of composite
pha-langes may be possible without
gene therapy using a combination
of cell transplantation and polymer
scaffolds.16
Growth plate disorders are
an-other possible skeletal gene therapy
application In a rabbit physeal
injury model, direct
adenovirus-mediated gene transfer of
insulin-like growth factor-1 (IGF-1) was
shown to inhibit subsequent
phy-direct BMP-2 gene transfer promoted premature physeal closure and sub-sequent growth disturbance.33 These results warrant further investigation into the use of IGF-1 gene transfer to inhibit physeal closure after physeal fractures and into the development
of physiodesis using BMP-2 gene transfer
Articular Structures
Patients with a variety of articu-lar disorders are potential candi-dates for gene therapy and tissue engineering applications Innova-tive approaches for treating arthri-tis, chondral and osteochondral de-fects, meniscal tears, and ligament injuries currently are being investi-gated
Arthritis
Arthritis was the first nonlethal disease for which a human gene therapy trial was approved and undertaken.34 This trial consisted of the ex vivo transfer of the inter-leukin-1 (IL-1) receptor antagonist protein (IRAP or IL-1RA) to syno-viocytes obtained from six post-menopausal women with severe rheumatoid arthritis A retroviral vector was used to transduce the synoviocytes with IRAP, a protein that inhibits the arthritogenic cy-tokine IL-1 The transduced syn-oviocytes were injected into the patients’ metacarpophalangeal (MCP) joints 1 week before sched-uled MCP joint arthroplasty At the time of MCP joint arthroplasty, the joints, synovium, and synovial fluid were harvested for analysis The trial was designed to establish feasi-bility and safety but not necessarily efficacy No adverse reactions in the patients have been reported to date, and lifelong clinical follow-up continues
Animal studies have established that direct adenoviral-mediated gene transfer of a soluble IL-1 recep-tor and a soluble tumor necrosis
matrix degradation in rabbit knees with antigen-induced arthritis.35 Combining multiple antiarthritic proteins in gene therapy applica-tions may lead to additive or syner-gistic effects
Cartilage
Different approaches have been applied to chondral and osteochon-dral defects RhBMP-2 regulates the behavior of costochondral growth plate chondrocytes36and maintains the phenotype of articular chon-drocytes in cell culture.37 Collagen sponges impregnated with rhBMP-2 improve the healing of rabbit full-thickness cartilage defects.38 The feasibility of using articular chon-drocytes in ex vivo BMP-2 gene transfer has been established.16 The effects of in vitro gene transfer of IGF-1, BMP-2, and TGF-β to rabbit articular chondrocytes have recently been investigated.39 Gene transfer
of BMP-2 was a potent stimulus for proteoglycan synthesis in the pres-ence of IL-1 Gene transfer of IGF-1 was a strong stimulus for collagen and noncollagenous protein synthe-sis Other factors, such as BMP-7, cartilage growth and differentiation factors, and various transcription factors, also may play a role carti-lage healing.40
Cell transplantation of autolo-gous chondrocytes injected into chondral defects and covered with
a periosteal flap has shown some promise clinically, especially for femoral condyle lesions.41 Cell transplantation using various natu-rally derived scaffolds, synthetic scaffolds, injectable scaffolds, and autologous scaffolds42 is being in-vestigated in animal models.43
In addition to other tissues, bio-reactor vessels may hold promise for tissue engineered cartilage Bioreactor vessels are cell culture containers specially designed to facilitate manipulation of the cells’ environment Environmental
Trang 8fac-tors, such as culture medium flow
and mechanical stress, can be
ma-nipulated to influence cell behavior
and tissue growth Bioreactor
ves-sels can be used both to engineer
cartilage for possible implantation
and provide a controlled in vitro
environment for the study of
chon-drogenesis.44 Size limitations of
bioreactor vessels are the current
restraints to clinical application
The optimal combination of growth
factor, delivery method, cells, and
scaffold to heal cartilage injuries is
the subject of ongoing investigation
Meniscus
The possibility of creating in
vitro a custom replacement
menis-cus using scaffolds,15 cells, and/or
gene therapy for subsequent in vivo
implantation is intriguing
Alterna-tively, meniscal cells might be
mod-ulated in vivo using gene therapy to
promote healing of certain injuries
Meniscal cells are amenable to gene
transfer of both marker genes and
various growth factor genes using
either in vivo or ex vivo gene
ther-apy, with gene expression persisting
for up to 6 weeks.45-47 Successful ex
vivo gene transfer has been
accom-plished using either myoblasts46,47
or meniscal cells.45 Implanted
me-niscal scaffolds constructed of
bo-vine collagen appear to be replaced
by host tissue mimicking a human
meniscus.15 Research is ongoing
into the preferred growth factors to
promote meniscal healing,
tech-niques to improve long-term gene
expression, and the optimal scaffold
needed to create new menisci
Ligaments
Gene therapy techniques also are
being applied to ligaments
β-Galactosidase (lacZ) gene transfer to
ligament has proved to be feasible in
animal models by either the in vivo
or ex vivo approach, using both
ade-novirus as well as retrovirus Ex
vivo gene transfer to ligaments has
been successfully achieved using either ligament fibroblasts48,49 or skeletal muscle myoblasts.49 Many growth factors, such as basic fibro-blast growth factor, platelet-derived growth factor (PDGF), vascular endothelial growth factor, IGF-1 and -2, TGF-β, and BMP-12, may play roles in ligament healing Data sug-gest that PDGF stimulates cell divi-sion and migration, whereas TGF-β and the IGFs promote extracellular matrix synthesis.50 Direct gene transfer of PDGF-β using a viral-liposome conjugate vector into rat patellar ligament resulted in initial improved angiogenesis and subse-quent enhanced extracellular matrix synthesis.51 Studies to improve liga-ment-bone healing using BMP-12 and to engineer ligament grafts in vitro are ongoing
Intervertebral Disk
Intervertebral disk nucleus pul-posus cells reside in an immuno-privileged site, which makes them potentially attractive targets for gene therapy approaches Direct adenovirus-mediated gene transfer
of lacZ into rabbit nucleus pulposus
cells in an in vivo model resulted in persistent gene expression for at least 12 weeks.52 A similar approach has been used to transfer the human TGF-β1 gene to rabbit nucleus pul-posus cells in vivo.53 The cells were harvested 1 week later and in vitro assays were performed TGF-β1 gene transfer resulted in a 30-fold increase in active TGF-β1, a fivefold increase in total TGF-β1, and a 100%
increase in proteoglycan synthesis
The results suggest that gene ther-apy to treat degenerative disk dis-ease warrants further investigation
Skeletal Muscle
Both in vivo and ex vivo forms of gene therapy to skeletal muscle for various types of inherited diseases, such as Duchenne muscular dystro-phy, have been investigated for
many years.54 In clinical trials, autol-ogous myoblast transplantation for Duchenne muscular dystrophy has proved to be safe,55although myo-blast survival is often suboptimal for inherited muscle diseases.54 Additionally, ongoing research into novel growth factors that might facilitate gene therapy approaches
to expedite the healing of acquired muscle injury has many potentially far-reaching applications IGF-1, basic fibroblast growth factor, and nerve growth factor have been shown in rodent models to improve muscle healing and strength (fast-twitch or tetanic strength) after con-tusion, laceration, and strain.56-58 However, myoblast transplantation combined with ex vivo gene therapy
is an even more attractive approach that can be used to deliver appropri-ate growth factors and responsive cells for acquired, traumatic muscle injuries
Summary
The biologic era of orthopaedic surgery promises to change the care
of musculoskeletal disorders in this century Extensive laboratory in-vestigations and preliminary clinical investigations have established the feasibility and promise of gene ther-apy and tissue engineering Re-search must continue to further un-derstanding of cytokines, gene delivery vectors, MSCs, and scaf-folds Pertinent issues, such as the optimal growth factors or genes, the timing and control of growth factor delivery, the optimal target cell, and the effectiveness of various scaf-folds, must be addressed before widespread clinical use can occur Most important, early clinical trials must establish patient safety before clinical efficacy is sought Respon-sible and innovative investigation will lead orthopaedic surgery into this new biologic era
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