TISSUE ENGINEERING
IN TENDON AND LIGAMENT
Michele Favata
BE-553
Spring 2001
INTRODUCTION
Soft tissue injuries account for 45% of the almost 33 million musculoskeletal
injuries that occur in the United States each year.1 Normal healing of these injuries is
typically characterized by formation of scar tissue, which is less organized than normal
tissue and exhibits inferior mechanical properties, even long after the initial injury has
occurred. Because tendons and ligaments play a major role in normal joint movement and
stability, such injuries and their pattern of poor healing can lead to degenerative diseases
of the joint. With the dawn of tissue engineering, however, has come the hope that we
might be able to improve upon this natural healing.
Tissue engineering has been defined as “an interdisciplinary field that applies the
principles of engineering and the life sciences toward the development of biological
substitutes that restore, maintain, or improve tissue function.”2 Researchers have tried
many approaches toward this goal, including the implantation of cells, scaffolds, DNA,
and proteins or protein fragments, and they have met with varying degrees of success. In
this paper, I will review recent advances in the engineering of tendon and ligament
repairs. I will also provide a brief description of tendon and ligament structure, as well as
a summary of the healing process as it is believed to proceed in these connective tissues.
BACKGROUND
Tendons and ligaments are strong, ropelike structures composed of dense
connective tissue. All connective tissues consist of cells surrounded by a matrix (referred
to as the extracellular matrix, or ECM). In tendons and ligaments, the primary cell type is
the fibroblast, and the main ECM component is collagen. The ECM also contains noncollagenous proteins, including the class of large, aggregating molecules called
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proteoglycans. The primary function of collagen is to lend tensile strength to the tendon
or ligament, whereas the proteoglycans serve mainly to resist compressive forces.
The process of tendon and ligament healing is a complex one. There are a
multitude of biochemical factors involved, the roles of which have only begun to be
elucidated. Furthermore, not all tendons and ligaments heal in the same way; their healing
properties tend to be site-dependent. The anterior cruciate ligament of the knee, for
example, has exhibited far less healing ability than ligaments in other locations. This sitedependency is likely due to a number of factors, such as cellular environment and
nutrition. All of these factors need to be taken into consideration by the tissue engineer.
Healing of ligaments and tendons can be separated into four phases – hemorrhage,
inflammation, proliferation and remodeling/maturation.3 The hemorrhagic phase is
characterized by the formation of a blood clot. Platelets become trapped in the clot, and
subsequently release biochemical molecules referred to as growth factors that attract
white blood cells to the wound. Specifically, platelets have been shown to release platelet
derived growth factor (PDGF), transforming growth factor-beta (TGF-), and epidermal
growth factor (EGF). The attracted cells, in turn, release additional factors that attract
other types of cells to the site of injury.
In the inflammatory phase, macrophages, a specialized type of white blood cell
that destroys necrotic tissue, predominate. The macrophages release more growth factors,
including TGF- and PDGF, and basic fibroblast growth factor (bFGF). As a result,
fibroblasts and other cells enter the wounded area. By the third day, the wound contains
platelets, various white blood cells, and mesenchymal stem cells (a type of pluripotent
stem cell found in bone marrow).
2
The proliferative phase is marked by an increase in the number of fibroblasts,
which begin to synthesize both collagenous and non-collagenous proteins. Capillary buds
also begin to form at this time. The fourth and final phase, remodeling and maturation,
involves a gradual decrease in the number of cells in the wound, as well as an increase in
collagen fibril diameter and collagen cross-links. Both of these events have been linked to
an increase in the strength of healed tendons and ligaments. Tendon and ligament healing
is not complete for at least a year, at which point the tensile strength is still inferior to that
of uninjured tissue. Despite a return to normal collagen content by 12-14 weeks, the
mechanical properties remain inferior because of decreased collagen fibril diameter,
decreased number of crosslinks and altered proteoglycan profiles.3
TISSUE ENGINEERING AND CONNECTIVE TISSUE INJURIES
Tissue engineering offers the appealing potential to make considerable
improvements upon the outcome of connective tissue injuries. To date, a large percentage
of the investigations in this area have focused upon defining the roles of growth factors.
The biochemical environment of wounded tissue is different from that of normal tissue.
Cells express different growth factors and different growth factors receptors, indicating
that the healing process entails many interactions and influences among the cells
involved. The variety of growth factors is extensive, and the responses they can induce
can seem equally daunting to an investigator whose goal is to understand them well.
Growth factors can cause cells to differentiate, proliferate, and migrate. They are involved
in the inflammatory response and matrix turnover, and can also interact with each other,
in either a synergistic or antagonistic fashion. To further complicate things, the response
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of cells to growth factors can be highly dose-specific, as well as site-specific, and can
even depend upon the time of application.†
Despite the overwhelming number of variables before them, however, researchers
have set out to understand the roles of these small but vital molecules, and some trends
have begun to emerge. Studies have shown that injured tendons and ligaments treated
with growth factors have improved structural properties when compared with untreated,
injured controls.3 Both in vitro and in vivo studies have been done.
The majority of in vitro studies describe experiments in which exogenous growth
factors are added to cell or tissue cultures. Because of the many variables involved, (such
as cell source, serum concentration, passage number, growth factor dosage, etc.), a
meaningful comparison of the results can be difficult. Certain patterns, however, have
appeared, and are summarized in the table below5:
Activity Enhanced
Growth Factor
Angiogenesis
bFGF, VEGF
Cell migration/division
PDGF, IGF, bFGF
ECM synthesis
TGF-, IGF
Differentiation
BMP-12
Anti-inflammatory
IL-1Ra, IL-10
Legend:
bFGF = basic fibroblast growth factor
BMP-12 = bone morphogenic protein-12
IGF = insulin-like growth factor
IL-10 = interleukin-10
IL-1Ra = interleukin-1 receptor antagonist
PDGF = platelet-derived growth factor
TGF- = transforming growth factor-
VEGF = vascular endothelial growth factor
A number of in vivo investigations have been done as well, and have generally
followed one of two strategies: detection of growth factors and their receptors in normal
or healing tissue, or application of exogenous growth factors to injured tissue.3 Multiple
†
In one study, the effect of PDGF on an injured rat ligament improved if the factor was applied
4
studies have shown that there is an increase in the expression of certain growth factors,
including PDGF and TGF-, during the first weeks after injury. Researchers have also
demonstrated the ability of these same two growth factors to enhance tendon and
ligament repair when applied to the site of injury.3,5 Improvements have been reported in
the strength, stiffness and breaking energy of tissue treated with PDGF or TGF-when
compared to untreated controls.3
From these studies and others, it is clear how growth factors might have potential
applications in tendon and ligament healing. Their delivery, however, is an obstacle, as it
is extremely difficult to maintain effective concentrations of necessary growth factors at
the site of an injury for extended periods of time. One approach to overcoming this
limitation is the use of controlled-release polymer matrices.6 Growth factors can be
encapsulated into such matrices or microspheres, which are then implanted at the site of
injury where they release their contents directly into the wound space. A more popular
approach, however, has been the use of gene transfer.
GENE THERAPY
Genes can be placed into cells ex vivo or in vivo, and can cause an increase in
expression of a protein, or suppress protein synthesis, in targeted cells. With this
technology, it might be possible to deliver multiple growth factors to cells, and to regulate
the extent and timing of their expression.
Genes can be introduced via viral vectors, liposomes, and “gene guns” (DNAcoated microprojectiles), each of which has advantages and disadvantages. While viruses
can be amplified easily, they can also elicit an immune response from the host. The use of
within 24 hours vs. 48.4
5
viral vectors also places constraints upon the size of the gene being transferred, and can
require that the target cells be actively dividing. In addition, there is also a small
possibility that the vector might integrate randomly with the host genome, which poses
the risk of tumor formation. Nonviral techniques, while free of several problems that
plague viral vectors, have had a history of fairly low transfer efficiency. A recent study,
however, has shown more promising results of nonviral gene transfer; a more detailed
description will follow.
Successful gene transfer to tendons and ligaments has been reported by several
researchers using various techniques. To establish successful gene transfer via virus, the
marker gene LacZ is often incorporated into the viral vector. The LacZ gene encodes a
harmless protein, -galactosidase, the levels of which can be monitored to determine the
extent and duration of gene transfer. Introduction of LacZ into patellar tendon has been
achieved in animal models, with gene expression lasting up to six weeks. Potential
therapeutic genes, including PDGF, have also been successfully transferred and expressed
in tendons.3
Recently, a series of experiments were conducted to study the potential of gene
therapy as a means of preventing adhesion formation in chicken digital flexor tendons.7
After injury, tendon repair is often hindered by the development of adhesions between the
healing tendon and the surrounding tissues. These adhesions occur as a result of injury or
surgical trauma. There have been several studies examining the effects of antiscarring
agents, such as steroids, for adhesion prevention following tendon. To date, however, no
approach has shown much promise.8
6
Using an adenoviral vector, Lou et al induced overexpression of pp125 focal
adhesion kinase (FAK), a protein they hypothesized might play a role in the formation of
adhesions. Their finding that the force necessary to flex the digits increased significantly
with pp125 FAK overexpression lends support to their hypothesis and suggests that
suppression of pp125 FAK synthesis might lessen adhesion formation. Their research
serves as an example of the potential for clinical applications of gene transfer.
In another study, the LacZ gene was successfully transferred into the medial
collateral ligaments of rabbits using both in vivo adenoviral and ex vivo retroviral
techniques.9 Expression of the gene in this study was unaffected by the presence of an
injury, and lasted longer when the gene was introduced via the in vivo adenoviral method.
In another ligament study, liposomes containing PDGF were injected directly into
injured rat ligament, and increased collagen deposition and blood vessel formation
resulted. The drawback of the particular nonviral method chosen by Nakamura et al,
however, are the difficulty in targeting specific cell types, and the low frequency of stable
transfer.10
A more recent article, however, reports what the authors believe to be the first
demonstration of high-efficiency gene transfer in vivo using nonviral techniques.11 In
their Methods, Goomer et al describe a novel, multi-step process to prepare liposomes for
gene delivery. They then inject these liposomes directly into canine flexor tendons during
a surgical procedure to repair injuries that had been inflicted experimentally. Six days
later, the tendons and surrounding tissues were harvested and examined for gene transfer
efficiency, which was found to approach 100% in the tendon and tendon sheath. The
endothelial cells of nearby blood vessels also exhibited a high level of marker gene
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expression. Thus, this study may provide a promising new strategy for overcoming the
inefficiencies that have been associated with nonviral gene transfer. In future studies, the
authors hope to improve upon their ability to target specific cell types using this method.
One of the goals of gene therapy is to have cells express a protein of interest at a
low, yet effective and nontoxic dose. It is possible that a narrow window exists between a
therapeutic effect, and an undesirable or even toxic one. TGF-, for example,
demonstrates the importance of maintaining this delicate balance well. Transient presence
of this growth factor at the wound site results in normal healing, whereas excessive
amounts cause scarring. One approach to controlling gene expression is through
modulation of the amount of vector delivered. This may not be sufficient, however, to
afford the fine level of control that clinical applications will demand.
In addition to precise and optimized dosage, gene therapy may also require the use
of other means to regulate the overexpression of genes, such as the use of antisense
oligodeoxynucleotides (ODNs). Antisense ODNs arrest protein synthesis by hybridizing
to a segment of mRNA, thereby preventing its translation into a protein. Though antisense
therapy offers precise and effective control of gene expression and a high level of
specificity, the instability of antisense compounds can reduce their potency and shorten
the duration of their affect.10 Nevertheless, this therapy was used successfully in a rabbit
ligament injury model, where the application of antisense decorin ODNs led to significant
improvements in wound strength.12
An alternative approach to regulating gene expression is the use of sense ODNs.10
These short DNA sequences bind to an activator protein that is necessary for transcription
of a specific gene, such as that containing the code for collagen molecules. With the
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associated activator protein bound, collagen synthesis would cease, which could be
desirable in the treatment of fibrotic diseases or prevention of excessive scar formation.
CELL METHODOLOGIES
In conjunction with continued research to develop gene therapies, investigators
have begun to explore the potential of cell-based therapies. Mesenchymal stem cells
(MSCs) have been shown to differentiate into several cells that play a vital role in the
healing process, including endothelial cells and fibroblasts. These cells secrete the ECM
components and growth factors that are essential to tendon and ligament repair. Using
bone marrow and periosteum as sources, researchers have extracted MSCs and observed
their effect in wounded tissue. In a recent study, the implantation of MSCs into injured
rabbit Achilles tendons significantly improved their structural properties.13
Cell-based treatments can also be combined with growth factor therapies. To
promote cell growth during culturing, for example, Young et al added FGF-2 to their
culture medium and produced larger numbers of MSCs than they had previously obtained
without use of the growth factor.13 In their undifferentiated state, MSCs may be
transplanted to a wound, hypothesizing that the cells in the wound environment will
furnish the biochemical and mechanical cues that will stimulate the MSCs to set out
along a differentiation pathway to the proper phenotype. Alternatively, as we begin to
gain a better understanding of these pathways, scientists may be able to influence the
timing and outcome of MSC differentiation, simply by adding the appropriate factor at
the right time.
There are a number of other factors to take into consideration when using cellbased therapies. An appropriate matrix must be selected for delivery of cells to the wound
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site, and it should be designed in such a manner as to promote optimal cellular activity1.
The ideal carrier matrix will initially be able to withstand the large forces present in the
injured area, but will also degrade at an appropriate rate in vivo. The rate of degradation is
crucial, as too rapid a pace could compromise the mechanical integrity of the repair, while
degradation that takes too long might shield the tendon or ligament from stress and
thereby have a detrimental affect on its remodeling.
The three-dimensional structure of the matrix is also important. It should be
porous so as to allow the infusion of nutrients, cells and growth factors, and to encourage
neovascularization. The matrix must also be nontoxic, and it should be possible to
package it in a way that permits easy handling and implantation. In the aforementioned
rabbit Achilles tendon study, cells were seeded onto tensed suture and consequently
assumed an elongated and fibroblastic morphology that, when compared to cells that had
been seeded in gel, more closely resembled actual tendon. Thus it is apparent that the
structure of the matrix can affect the activity of the cells that are seeded upon it. In the
future, researchers will need to develop appropriate injury models to test these cell and
matrix composites so that clinical applications can be realized.1.
CONCLUSION
In conclusion, there is an increasing wealth of experimental evidence indicating
that tendon and ligament healing can be enhanced through tissue engineering. Though
there is still a great deal left for us to learn, we have already proven an ability to improve
upon the natural healing process, and have begun to gain an understanding of the many
complex biochemical and mechanical interactions involved. Tendon and ligament injuries
can often result in prolonged disability. Though improved surgical repair techniques and
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post-operative therapies have helped, post-surgical scarring remains the most problematic
aspect of repair.8 By providing a better understanding of this deficient healing process and
a potential means to refine it, tissue engineering offers new hope to many patients for
whom there had previously been very little.
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mediated by recombinant adenovirus-induced tendon adhesion formation and epitenon cell
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8. Personal communication with Pedro K. Beredjiklian, Orthopaedic Hand Surgeon and
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12. Nakamura N, et al. Decorin antisense gene therapy improves functional healing of early
rabbit ligament scar with enhanced collagen fibrillogensis in vivo. Journal of Orthopaedic
Research 18:517-523, 2000.
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repair. Journal of Orthopaedic Research. 16:406-413,1998.
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osteogenic precursors from human bone marrow. Endocrinology. 138:4456-4462,1997.
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